Studies of drugs given before anaesthesia XXV: Medazepam, a new benzodiazepine. Medazepam, a new benzodiazepine, was studied as an oral preanaesthetic medication in adult women in a dose of 10 mg, and the findings compared with those obtained with diazepam 10 mg or a dummy capsule using a double-blind technique. In the doses used medazepam produced less drowsiness than diazepam and was no more anxiolytic than the placebo.


A simple method for computing acid-base state. Simple methods are described for computing the variables derived from the Siggaard-Andersen nomogram, where the in vitro buffer line is either established by direct measurement or calculated from measurements of pH, Pco2 and haemoglobin. Calculations are performed using the equation of the pH-log Pco2 buffer line, the Henderson-Hasselbalch equation, and the polynomials: BE=-38.402+1.8970 (SB)-0.013342 (SB)-2 m-equiv/litre; SL=-69.046+17.377 (pH40)-1.1121 (pH40)-2; SH=-123.30+31.357 (pH40)-2.0143 (pH40)-2. Then haemoglobin=7.5 (1+(S-SL)/(SH-SL)) G/100 ml, where S is the slope of the buffer line. Results are sufficiently accurate for clinical purposes.


The effect of non-depolarizing relaxants on plasma potassium. Plasma potassium concentrations were measured in four groups of unpremedicated patients in whom anesthesia was induced with thiopentone 5 mg/kg followed by tubocurarine 0.5 mg/kg, gallamine 2 mg/kg, pancuronium 0.1 mg/kg or AH 8165 1.25 mg/kg, prior to elective dental surgery. There was a small but consistent decrease in plasma potassium following the injection of pancuronium and, to a lesser extent, following gallamine. No changes were found during the first 10 min following tubocurrarine. With AH 8165 there was an early reduction in plasma potassium but the values returned to within normal limits by 5 min.


Acid pulmonary aspiration syndrome after antacids. A case report. This is a report of a patient who developed acid pulmonary aspiration syndrome following pulmonary aspiration of gastric contents at a pH of 3.5. The volume of the stomach contents was large and 15 ml of magnesium trisilicate was insufficient to prevent the effects of acid aspiration. After operation, considerable pulmonary shunting was demonstrable for several days. The patient was discharged home well, and a chest x-ray 2 months later showed no abnormality.


Pulmonary complications following smoke inhalation. Two patients with severe respiratory disturbance following burning accidents are described. It is suggested that smoke inhalation injuries may in future present a more frequent problem in the intensive therapy unit, and the mechanisms of the disorder and its treatment are discussed.


Exercise-induced asthma.

Exercise-induced asthma. A review of exercise-induced asthma is presented which describes work that has been carried out by the authors and by other investigators over recent years. The effect of exericse on lung function in asthmatic and normal subjects is compared. The influence of the type and severity of exercise on the response of the asthmatic is noted and the importance this has for interpretation of results. The effects of various drugs on exercise-induced asthma are considered in some detail. The clinical implications of the results of exercise tests in asthmatics, their relatives, and other subjects are considered in terms of the diagnosis and prognosis of asthma and its mode of inheritance. It is concluded that there is as yet no explantation for the mechanism of exercise-induced asthma, but it is a tool of potentially great value for research into the physiology and treatment of clinical asthma.


The physiological assessment of acid-base balance. Acid-base terminology including the sue of SI units is reviewed. The historical reasons why nomograms have been particularly used in acid-base work are discussed. The theoretical basis of the Henderson-Hasselbalch equation is considered. It is emphasized that the solubility of CO2 in plasma and the apparent first dissociation constant of carbonic acid are not chemical constants when applied to media of uncertain and varying composition such as blood plasma. The use of the Henderson-Hasselbalch equation in making hypothermia corrections for PCO2 is discussed. The Astrup system for the in vitro determination of blood gases and derived parameters is described and the theoretical weakness of the base excess concept stressed. A more clinically-oriented approach to the assessment of acid-base problems is presented. Measurement of blood [H+] and PCO2 are considered to be primary data which should be recorded on a chart with in vivo CO2-titration lines (see below). Clinical information and results of other laboratory investigations such as plasma bicarbonate, PO2,P50 are then to be considered together with the primary data. In order to interpret this combined information it is essential to take into account the known ventilatory response to metabolic acidosis and alkalosis, and the renal response to respiratory acidosis and alkalosis. The use is recommended of a chart showing the whole-body CO2-titration points obtained when patients with different initial levels of non-respiratory [H+] are ventilated. A number of examples are given of the use of this [H+] and PCO2 in vivo chart in the interpretation of acid-base data. The aetiology, prognosis and treatment of metabolic alkalosis is briefly reviewed. Treatment with intravenous acid is recommended for established cases. Attention is drawn to the possibility of iatrogenic production of metabolic alkalosis. Caution is expressed over the use of intravenous alkali in all but the severest cases of metabolic acidosis. The role of 2,3-diphosphoglycerate on tissue oxygenation is stressed and use of intravenous sodium phosphate as an alternative to intravenous bicarbonate is mentioned.


The effect of intravenous ritodrine on the acid-base status of the fetus during the second stage of labour. A progressive fetal respiratory acidosis occurs in the second stage of labour. A double-blind controlled trial showed that the intravenous infusion of Ritodrine abolished this.


Indicator dyes as probes of electrostatic potential changes on macromolecular surfaces. An indicator dye attached to an electrostatically charged macromolecular surface generally has a pK value (pKb') different from that of uncombined dye (pKf'). The question if changes in (pKb' - pKf'), designated as increment incrementpK, records changes in the electrostatic potential at the binding site has been examined in spectrophotometric and binding experiments, using the interaction of Chlorophenol Red and Phenol Red with human serum albumin and cationic micelles as examples. (1) In serum albumin solutions increment incrementpK is decreased by a reduction of pH. The decrease is correlated with the increase in positive charges on the protein molecule, and the response is attenuated by high ionic strength in accordance with electrostatic theory. (2) Opposite changes in binding affinity to serum albumin and increment incrementpK as a function of pH are observed; the binding of basic (bivalent anion) dye is more susceptible to a change in pH than in the acidic (univalent anion) form. (3) Preferential uptake of the basic as compared to the acidic form of dye is observed by binding to cetyltrimethylammonium chloride and cetylpyridinium chloride micelles (mu equals 0.033, [Cl-] equals 0.033 M). An increase in the ionic strength is accompanied by a positive value of increment incrementpK. The results are consonant with the view that the observed increment incrementpK values reflect changes in the electrostatic potential at the binding site with consequently little, if any, effect on the intrinsic pK. The extension of the method to measure changes in the electrostatic potential at binding sites on cell membranes is briefly discussed.


Catalysis of phosphoryl group transfer. The role of divalent metal ions in the hydrolysis of lactic acid O-phenyl phosphate and salicylic acid O-aryl phosphates. The spontaneous hydrolyses of lactic acid O-phenyl phosphate (I) and, to a lesser extent, 3-hydroxybutyric acid O-phenyl phosphate (II) have been investigated and compared with similar intramolecular and bimolecular reactions. Compared to bimolecular nucleophilic reactions, the reactivity of II is similar to other systems involving the formation of a six-membered ring intermediate, which suggests that the electrostatic barrier to attack of an anionic nucleophile on a phosphate diester anion is fully present in II. The reactivity of I, as compared to that of II, would suggest that at least a partial overcoming of the electrostatic barrier takes place upon closer approimation of the two reacting centers. The Mn-2+-catalyzed hydrolysis of I exhibits saturation kinetics, consistent with the enhanced reactivity of the metal ion-substrate complex. The binding constant for this complex, determined from kinetics, is in good agreement with that obtained by electron spin resonance (ESR) titration. It is argued that the complex of Mn-2+ with II, as observed by pulsed Fourier transform nuclear magnetic resonance (NMR) techniques, is a precursor to the complex of catalytic significance. The hydrolysis of I as catalyzed by a variety of divalent metal ions suggests an optimal metal ion size. The spontaneous and metal ion catalyzed hydrolyses of salicyclic acid O-aryl phosphates (IIIa-d) proceed through cyclic acyl phosphate intermediates after expulsion of phenol. Product studies on the parent compound have failed to detect phenyl phosphate as a product in either the spontaneous or metal ion catalyzed process. The dependence of the second-order rate constant for the metal-catalyzed hydrolysis on leaving group pKa, beta-1-g, decreases significantly relative to beta-1-g for the spontaneous hydrolysis. From the collective data a specific interation of the metal ion with a pentacovalent intermediate is inferred in the rate-determining step for esters I and III. The probable consequences of these mechanistic postulates for phosphoryl transfer reactions in biological systems are discussed.


Renilla luciferin as the substrate for calcium induced photoprotein bioluminescence. Assignment of luciferin tautomers in aequorin and mnemiopsin. A study was made of the effects of pH and protic and aprotic solvents on the spectral properties of Renilla (sea pansy) luciferin and a number of its analogs. The results have made possible the assignment of two tautomeric forms of Renilla luciferin, one which absorbs maximally at 435 nm and another which exhibits an absorption maximum at 454 nm. Furthermore the results provide an explanation for the visible absorption characteristics of the photoproteins aequorin (lambda-max 454 nm) and mnemiopsin (lambda-max 435 nm). In addition a Renilla-like luciferin can be extracted from both of these photoproteins. This luciferin produces light with Renilla luciferase, at a rate dependent upon the concentration of dissolved oxygen, and in other respects is indistinguishable from Renilla luciferin in this bioluminescent reaction. The results suggest that the native chromophore in both photoproteins is Renilla luciferin (or a nearly identical derivative). The results also suggest that a hydroperoxide intermediate probably exists in photoproteins, on energetic grounds, and to account for the oxygen concentration independency of the rate of photoprotein reactions. This hydroperoxide may be attached initially to an amino-acid side chain (possibly indolyl-OOH, imidazoyl-OOH, or -SOOH) rather than to the luciferin chromophore.


Essential and nonessential thiols of yeast hexokinase. Reactions with iodoacetate and iodoacetamide. The reaction of yeast hexokinase with iodoacetate or iodoacetamide has been investigated in detail, using pure hexodinase B. Of the four thiols in each subunit of the molecule, two (the "apparently essential thiols") are alkylated rapidly at 35 degrees, and the enzymic activity is lost in parallel with their reaction. The other two thiols react subsequently to completion, but at a very much slower rate. In the conditions use, no other uptake of the reagent occurs elsewhere during these thiol alkylations. Electrophoretically homogeneous kialkylated and tetraalkylated protein species are formed, in the two stages of the reaction. The inactivating reaction at 35 degrees with the apparently essential thiols is second order. The rate constant increases with increasing pH, in the range pH 7.0-8.5, in a manner consistent with control of the reaction by a group with pKa of approximately 10. The absolute (pH independent) rate constant is of the same order as that for a normal thiol in model compounds. The availability of the apparently essential thiols appears to be associated with some conformational change in the molecule in the monomer form: it declines at high ionic strengths, is maximal at intermediate values where the dimer first dissociates, but is lowered in the dimer at very low ionic strengths. The reaction also shows a sharp temperature dependence: the dimer at 30 degrees (in constrast to 35 degrees) shows no availability of the apparently essential thiols. A similar transition to a state permitting fast inactivation is found with pH, above pH 8.5. The reaction of the two apparently essential thiols is strongly inhibited by glucose. ATP and ADP, and their Mg complexes, protect significantly, but less effectively than does glucose. The affinities of these substrates at the active site of the enzyme are measured in this protection system. These various reactions appear to be of value for identifying the cysteine-containing regions that are involved in the active center or in its maintenance in the structure.


Superactivation of thermolysin by acylation with amino acid N-hydroxysuccinimide esters. Synthesis of a series of active N-hydroxysuccinimide esters of aliphatic and aromatic amino acids has yielded a new class of reagents for the covalent modification of proteolytic enzymes such as thermolysin. The activities of aliphatic acyl amino acid thermolysins are from 1.7 to 3.6 times greater than that of the native enzyme when hydrolyzing durylacryloyl-Gly-Leu-NH2, the substrate employed most widely. By comparison, the aromatic acylamino acid derivatives are "superactive," their activities being as much as 70-fold greater. Apparently, the aromatic character of the amino acid introduced is a critical variable in the determination of the functional response. The increased activity is completely restored to that of the native enzyme by deacylation with nucleophiles, such as hydroxylamine, and the rate of restoration of native activity is a function of the particular acyl group incorporated. Preliminary evidence regarding the chemical properties of the modified enzyme suggests that tyrosine, rather than lysine, histidine, or arginine, may be the residue modified. The functional consequences of successive modification with different reagents, moreover, indicate that each of them reacts with the same protein residue. The competitive inhibitors beta-phenyl-propionyl-Phe and Zn-2+ do not prevent modification with these active esters. Hence, the site(s) of their inhibitory action differ(s) from that at which modification occurs. The structure of the substrate is also a significant variable which determines the rate at which each acyl amino acid thermolysin hydrolyzes peptides. Depending on the particular substrate, the activity of aromatic derivatives can be as much as 400-fold greater than that of the native enzyme, and the resultant activity patterns can be ordered in a series characteristic for each enzyme derivative.


Hormonal regulation of fatty acid synthetase, acetyl-CoA carboxylase and fatty acid synthesis in mammalian adipose tissue and liver. The major objectives of this study were to define the roles of adrenal glucocorticoids and glucagon in the long-term regulation of fatty acid synthetase and acetyl-CoA carboxylase of mammalian adipose tissue and liver. Particular emphasis was given to elucidation of the mechanisms whereby these hormones produce their regulatory effects on enzymatic activity. To dissociate mental manipulation, nutritional conditions were ridgidly controlled in the experiments described. Administration of glucocorticoids to adult rats led to a marked reductionin activities of fatty acid synthetase and carboxylase in adipose in adipose tissue but no change occurred in liver. Adrenalectomy produced an increase in activities of these lipogenic enzymes in adipose tissure, but, again, no change was noted in liver. The decrease in enzymatic activities in adipose tissue with glucocorticoid administration correlated well with a decrease in fatty acid synthesis, determined in vivo by the 3-H2O method. The mechanisms whereby glucocorticoids led to a decrease in fatty acid synthetase activity were elucidated by the use of immunochemical techniques. Thus, the decrease in fatty acid synthetase activity observed in adipose tissue was shown to reflect a decrease in content of enzyme, and not a change in catalytic efficiency. The mechanism underlying the decrease in enzyme content is a decrease in synthesis of the enzyme. The relation of the effects of glucocorticoids to the effects of certain other hormones involved in regulation of lipogenesis was investigated in hypophysectomized and in diabetic animals. Thus, the observation that the glucocorticoid effect on synthetase and carboxylase occurred in adipose tissue of hypophysectomized rats indicated that alterations in levels of other pituitary-regulated hormones were not necessary for the effect. That glucocorticoids play some role in regulation of synthetase and carboxylase in liver, at lease in the diabetic state, was shown by the observation that the low activities of these enzymes in diabetic animals could be restored to normal by adrenalectomy. An even more pronounced restorative effect was apparent in adipose tissue of adrenalectomized, diabetic animals. Administration of glucagon during the refeeding of starved rats resulted in a marked reduction in the induction of fatty acid synthetase, acetyl-CoA carboxylase and in the rate of incorporation of 3-H from 3-H2O into fatty acids in liver, but no change in these parameters occurred in adipose tissue. Administration of theophylline resulted in intermediate reduction in liver. The mechanisms whereby glucagon led tto a decrease in fatty acid synthetase activity were elucidated by the use of immunochemical techniques. Thus, the changes in fatty acid synthetase activity were shown to reflect reductions in content of enzyme. The mechanism underlying these reductions in content is reduced synthesis of enzyme.


A simple and high yield purification of Crotalus adamanteus phospholipases A2. A new, high yield, procedure for the purification of the phospholipases A2 of Crotalus adamanteus venom is described. The procedure involves precipitation of the bulk of venom proteins with 50% isopropanol, precipitation of the enzymes from the isopranol soluble material with neodynium chloride, and final purification on DEAE cellulose. An overall yield of approximately 80% is achieved.


Affinity chromatography on hydroxyalkyl methacrylate gels. II. Isolation of thiol-containing protein and peptide using mercurial derivatives of gels. Absorption properties of hydroxyalkyl methacrylate gels containing various amounts of mercuri derivatives of p-acetaminophenoxyethyl methacrylates (Hg-APEMA) and methacrylanilide (Hg-MAA) were investigated by means of a reduced Ellman's agent (5,5'-dithiobis-2-nitrobenzoic acid) and papain. The optimum gel was used for the affinity chromatography of SH-protease from beans and for the isolation of a peptide with a free sulphhydryl group from the chymotryptic hydrolyzate of serum albumin.


Partial purification and characterization of the enzyme which converts precursor liver protein to factor X. A rat liver post-microsomal supernatant enzyme, which carries out an epigenetic conversion of a protein contained in liver microsomes to Factor X, has been partially purified 250-fold in 50% yield by a combination of salt fractionation and gel filtration. The crude enzyme is stable to freezing and thawing but unstable at 4 degrees C. However, the partially purified enzyme is more stable at 4 degrees C. It requires Ca2+ and HCO3 minus for optimum formation of Factor X activity. The supernatant enzyme is vitamin K dependent and exhibits its maximum rate of formation of Factor X between pH 8 and 8.5.


Comparative study on conformational stability and subunit interactions of two bacterial asparaginases. The denaturation and reconstitution of Erwinia carotovora and Escherichia coli L-asparaginases has been followed by optical rotatory dispersion, circular dichroism and analytical ultracentrifugation. Denaturation in urea results in dissociation of the native enzyme (mol. wt. 140 000 approx.) to produce unfolded subunits (mol. wt. 35 000 approx.); the Erwinia L-asparaginase subunits can be refolded by dilution or dialysis in alkaline conditions, pH 10.5, without aggregation to the active tetramer, to give a rather unstable solution of a monomer possibly in equilibrium with dimer. These alkaline-reconstituted subunits undergo a conformational change to a more ordered state in the presence of sodium dodecylsulphate, similar to those produced by the action of sodium dodecylsulphate on the native enzyme. If the denatured subunits are reconstituted in the pH range 5.0-7.5, the enzymically active tetramer is reformed in up to 80% yield, depending upon the conditions of temperature and concentration. Kinetic data for these various transitions suggest that dissociation is a rate-limiting step while conformational changes of the polypeptide chains are relatively much more rapid. The possible significance of these different rates of change to therapeutic considerations is discussed.


Binding of D-glucose to insulin.

Binding of D-glucose to insulin. Binding of D-glucose to insulin has been studied by equilibrium dialysis. The binding is not very specific and probably takes place in two steps. The average amount of glucose molecules bound per insulin molecule is eight, two molecules in the first and six during the second step of binding. The intrinsic binding constants for both steps are almost the same (6-10-2 M-minus 1 and 1-10-3 M-minus 1) which can be explained by assuming: (1) that after binding of the first two molecules a conformational change of insulin occurs which facilitates the binding of the next six molecules of D-glucose; or (2) that in the second step of binding the glucose binds to hydrophobic regions which are unmasked by dissociation of the insulin dimer. Using a three-dimensional model of the insulin molecule areas of the protein molecule where binding of glucose can occur were selected. The glucose-binding site very probably involves the area at the insulin surface where most of the invariant and modification-selective residues are present.


Mechanism of respiration-driven proton translocation in the inner mitochondrial membrane. Analysis of proton translocation associated with oxidation of endogenous ubiquinol. A study is presented of the kinetics and stoichiometry of fast proton translocation associated to aerobic oxidation of components of the mitochondrial respiratory chain. 1. Aerobic oxidation of ubiquinol and b cytochromes is accompanied in EDTA particles, obtained by sonication of beef-heart mitochondria, by synchronous proton uptake. 2. The rapid proton uptake associated to oxidation and b cytochromes is greatly stimulated by valinomycin plus K+, but is unaffected by carbonyl cyanide p-trifluoromethoxyphenylhydrazone. 3. 4 gion H+ are taken up per mol ubiquinol oxidized by oxygen. This H+/2e- ratio, measured in the rapid anaerobic-aerobic transition of the particles is unaffected by carbonyl cyanide p-trifluoromethoxyphenylhydrazone. 4. Intact mitochondria aerobic oxidation of oxygen-terminal electron carriers is accompanied by antimycin-insensitive synchronous proton release, oxidation of ubiquinol and reduction of b cytochromes. The amount of protons released is in excess with respect to the amount of ubiquinol oxidized. 5. It is concluded that electron flow along complex III, from ubiquinol to cytochrome c, is directly coupled to vectorial proton translocation. The present data suggest that there exist(s) between ubiquinol and cytochrome c one (or two) respiratory carrier(s), whose oxido-reduction is directly linked to effective transmembrane proton translocation.


Surface potential and the interaction of weakly acidic uncouplers of oxidative phosphorylation with liposomes and mitochondria. The pH dependence of the binding of weakly acidic uncouplers of oxidative phosphorylation to rat-liver mitochondria and liposomes is mainly determined by the pKa of the uncoupler molecule. The absorption and fluorescene excitation spectra of the anionic form of weakly acidic uncouplers of oxidative phosphorylation are red-shifted upon interaction with liposomal or mitochondrial membranes. The affinity for the liposomes, as deduced from the red shift, is independent of the degree of saturation of the fatty acid chains of different lecithins. The intensity of the spectra at one pH value is strongly dependent upon the surface charge of the liposomes. With positively charged liposomes the results obtained can be almost quantitatively explained with the Gouy-Chapman theory, but with negatively charged ones deviations are observed. At a particular pH, the divalent ion Ca-2+ stongly influences the intensity of the spectra in the presence of negatively charged liposomes, but has no effect with neutral liposomes. With mitochondrial membranes an effect of Ca-2+ similar to that with negatively charged liposomes is observed. Depletion of the phospholipids of the mitochondria and subsequent restoration of the mitochrondrial membrane with lecithin, strongly diminishes this effect, but restoration with negatively charged phospholipids does not influence it. From these observations it is concluded that the anionic form of the uncoupler molecule when bound to mitochondria is located within the partly negatively charged phospholiped moiety of the membrane, with its anionic group pointing to the aqueous solution.


Some biochemical and physiochemical properties of the potent uncoupler SF 6847 (3,5-di-tert-butyl-4-hydroxybenzylidenemalononitrile). Various physicochemical and biochemical properties of the most potent uncoupler of oxidative phosphorylation known to date 3,5-di-tert-butyl-4-hydroxybenzylidenemalononitrile (SF 6847), such as pH dependence of the uncoupling activity and binding to mitochondria, spectral properties in the presence of different types of liposomes, biopolymers and mitochondria, and effects on model membrane systems have been investigated. From the results, it is concluded that the uncoupler most likely is localized in the phospholipid part of the membrane.


Cytochrome c2 and reaction center of Rhodospeudomonas spheroides Ga. membranes. Extinction coefficients, content, half-reduction potentials, kinetics and electric field alterations. The reduced minus oxidized extinction coefficients (delta epsilon-red-ox) of reaction center P605 when in the chromatophore is about 20% smaller than in the detergent-isolated state. Presumably the coupling of the reaction center protein to the antenna bacteriochlorophylls and carotenoids causes this hypochromism. The chromatophore values for P605 are 19.5 mM- minus 1 times cm- minus 1 with the spectrophotometer on single beam mode at 605 nm, and 29.8 mM- minus 1 times cm- minus 1 on dual wavelength mode set at 605--540 nm. Cytochrome c2, which is not affected by detergent, has a delta epsilon-red-ox value at 550--540 nm of 19.0 mM- minus 1 times cm- minus 1.2. The total bacteriochlorophyll to reaction center bacteriochlorophyll protein (P) ratio is about 100: 1. The cytochrome c2: reaction center protein ratio approaches 2. In current French press chromatophore preparations, about 70% of the reaction centers are each associated on a rapid kinetic basis with two cytochrome c2 molecules (intact P-c2 units). The remaining reaction center proteins are not associated with cytochrome c2 on a kinetically viable bais and may be the result of damage incurred during mechanical rupture of the cells. 3...


Biochemical changes in Bifidobacterium bifidum var. pennsylvanicus after cell wall inhibition. VIII. Composition and metabolism of phospholipids at different stages and conditions of growth. 1. The phospholipid content and composition of Bifidobacterium bifidum var. pennsylvanicus is markedly influenced by the growth phase, the pH and the presence of human milk in the culture medium. 2. The lipid-phosphorus content of the cells increases during the first period of active growth, but decreases later. The lipid-phosphorus content of the cells in the stationary phase is at constant pH 5.5 about 45 percent of that at constant pH 6.8 and final pH 5.2. This difference is caused by a general reduction of all types of phospholipids. 3. Phosphatidylglycerol content decreases during growth, diphosphatidylglycerol increases in the first period, but decreases later and especially in the stationary phase by an increase of its lysoderivatives. The phosphogalactolipids rise during growth in the non-controlled pH-culture to 70 percent of the phospholipids in the stationary phase. When pH is constant at 6.8 and 5.5 glycerolphosphorylgalactosyldiglyceride remains at a constant level of about 20 percent during growth. At pH 6.8 glycerophosphorylmonogalactosylmonoglyceride increases to 24 percent during cultivation; at pH 5.5 this lipid contributes only a few percent. 4. Under all pH conditions, lack of human milk in the culture medium causes a marked increase of the lipid-phosphorus content of the cell, together with a high increase of the relative amounts of diphosphatidylglycerol and phosphatidylglycerol and a decline of the phosphogalactolipids. The same changes are observed after inhibition of cell wall synthesis by various antibiotics, but not after inhibition of protein synthesis.


Prevention of cold inactivation of 3-hydroxy-3-methyl-glutaryl coenzyme A reductase by NADPH. Solubilized 3-hydroxy-3-methylglutaryl coenzyme A reductase (EC 1.1.1.34) from rat liver microsomes has been reported to be reversibly inactivated by temperatures below 19 degrees C. Cold inactivation has now been found to be completely prevented by NADPH and by NADP+ at a concentration of 3 mM. NADPH, however, was more active than NADP+ at lower concentrations and prevented 50% of the cold inactivation at 0.2 mM, whereas a 1.1 mM NADPH+ without effect and the substrate 3-hydroxy-3-methylglutaryl coenzyme A prevented only 30% of the cold inactivation at a concentration 50 times greater than the Km value.


The permeation of organic acids through lecithin bilayers. Resemblance to diffusion in polymers. 1. The fluxes of aliphatic acids and their derivatives through black lipid membranes made of egg lecithin in decane were measured by means of a proton titration method. 2. Permeability coefficients were calculated and these were divided by the partition coefficient of the diffusing solute in different solvent systems: n-decane, olive oil, ether and octanol. The logarithms of the diffusion coefficients thus obtained were plotted against the logarithm of the molecular weight. The data could not be fitted to a single regression line in any solvent system. 3. When the logarithm of the diffusion coefficients were correlated to the logarithm of the molecular volume (equals molecular weight/ specific gravity) all the diffusants could be fitted to the same regression line, indicating that the molecular volume is a better index of molecular size and shape than the molecular weight. 4. Analysis of the experimental results assuming a model of diffusion through soft polymers (Lieb, W.R. and Stein, W. D. (1971) Current Topics in Membranes and Transport, vol. 2, pp. 1-39, Academic Press, New York) showed that decane and olive oil are not adequate model solvents for planar lecithin membranes but ether and octanol are good models. 5. The differential mass selectivity coefficient was found to be similar to that for soft polymers and biological membranes, i.e. greater than 3.0. 6. Water could be fitted by the same regression line, thus emphasizing the generality of passive transfer and implying that water crosses lipid membranes as single molecules.


The nature of the attachment of a regularly arranged surface protein to the outer membrane of an Acinetobacter sp. Acinetobacter 199A carries on the outer surface of its outer membrane a layer of regularly arranged protein subunits. The isolated surface protein assembles into the same regular array even in the absence of the underlying outer membrane. Cl- minus is required for this self-assembly. Evidence is presented that the interaction of the surface protein with the outer membrane involves the linking of a carboxyl group in the surface protein to a negatively charged group in the outer membrane protein, via a divalent cation. The surface protein could be detached from the outer membrane by the protein perturbant urea, by the chelating agent EDTA and by replacing Mg-2+ with Na+. It could not be detached by treatment with phospholipases A anc D or the detergents Tween 80 and sodium deoxycholate. The conditions favourable for reattachment of surface protein to the cell wall were the presence of divalent cations and a pH of 3-5. Conversion of carboxyl groups in the surface protein to amine with carbodiimide and ethylene diamine interfered with reattachment. The surface protein did not attach to isolated cell wall lipid or lipopolysaccharide.


Proteolytic activity associated with human erythrocyte membranes. Self-digestion of isolated human erythrocyte membranes. At least two kinds of enzymes are active in the proteolytic self-digestion of erythrocyte membranes. The specific activities of these enzymes do not decrease with repeated washings of purified stroma. The effects of a variety of inhibitors on the membrane preparation's capacity to digest 125-I-labelled casein, covalently linked to latex beads, have been examined. Pepstatin-inhibitable enzyme, active at low pH, digests the membrane extensively to small polypeptide fragments. Spectrin, located at the internal part of the membrane, is readily degraded. Diisopropylfluorophosphate-inhibitable enzyme, active at pH 8-9, has only limited digestive capacity. Some of the membrane components, such as the small molecular weight glycoproteins, are resistant to digestion. The restricted capacity of digestion is due to the membrane molecular arrangement; increased disaggregation removes the restriction and increases the activity. Spectrin is not digested unless the membrane topography is disrupted by NP-40 neutral detergent. These observations suggest that the enzymes active at basic pH are located external to the cell. Intact cells do possess a limited capacity to degrade 125-I-labelled casein when their surfaces are brought into contact with substrate-coated beads.


[Gamma-glutamyltransferase activity in the human red blood cell membrane (author's transl)]. A gamma-glutamyltransferase activity is found in the human red blood cell membrane. Membrane isolation was carried out according to the method of Dodge et al. (Dodge, J. T., Mitchell, C. and Hanahan, J. (1963) Arch. Biochem. Biophys. 100, 119-130) (modified) and proteins were solubilized either with 1% sodium deoxycholate or 5 mM EDTA or 10 mM of its disodium salt, under various conditions of time and temperature. The gamma-glutamyltransferase activity of the membrane preparations was investigated using two substrates, gamma-L-glutamyl-p-nitroanilide and gamma-L-glutamyl-alpha-naphthylamide. The specific enzymatic activities of the various preparations, expressed in m units per mg of protein, were found to have similar values under similar technical conditions. The chelating agents seem to allow a more specific isolation than the detergent. The presence of a gamma-glutamyltransferase activity in the erythrocyte membrane is discussed in relation to the membrane association of this enzyme in other tissues.


Hydrolytic and transglucosylation activities of a purified calf spleen beta-glucosidase. Certain properties of the transglucosylitic activity and the hydrolytic activity of a purified calf spleen beta-glucosidase (beta-D-glucoside glucohydrolase EC 3.2.1.21) were investigated. There was a stimulation of both activities by sodium taurocholate and "Gaucher's factor". The K-m values for 4-methylumbelliferyl-beta-D-glucoside and glucosylceramide as donors in the transglucosylation reaction were 2 mM and 0.075 mM, respectively. The K-m for ceramide as acceptor was 0.149 mM with both of these compounds. The ability of several glucoside to act as donors was examined. The capacity to catalyze this "transglucosylation" reaction is greatly diminished in spleen tissue samples from Gaucher's patients. The enzyme possesses the capacity to hyrolyze 4-methylumbellifery-beta-D-glucoside, p-nitrophenyl-beta-D-glucoside, glucosylsphingosine, glucosylceramide and deoxycorticosterol-beta-D-glucoside. It is postulated that a single enzyme protein may be responsible for both the hydrolytic and the transglucosylitic activities.


Purification of G-M-1-ganglioside and ceramide lactoside beta-galactosidase from rabbit brain. The major beta-galactosidase of rabbit brain has been purified over 400-fold. The enzyme converts G-M-1-ganglioside; Gal beta-1 yields 3 GalNAc beta-1 yields 4 (NANalpha-2 yields 3) Gal beta-1 yields 4 Glc yields ceramide (G-M-1) into Tay Sachs ganglioside GalNAc beta-1 yields 4 (NANalpha-2 yields 3) Gal beta-1 yields 4 Glc yields ceramide (G-M-2-ganglioside) and ceramide lactoside, Gal beta-1 yields 4 Glc yields ceramide (Gal-Glc-Cer) into glucocerebroside, Glc yields ceramide (Glc-Cer). The enzyme also hydrolyzes the synthetic substrates NPh-Gal and MeUmb-Gal. It is eluted as a single peak from Sephadex G-200 columns when natural and synthetic substrates were used and has an isoelectric point of 6.3. We were unable to resolve activity towards G-M-1-ganglioside and Gal-Glc-Cer by polyacrylamide electrophoresis in two buffer systems. With G-M-1 the pH optimum was 4.3 in acetate buffer and the K-m value 78 mu-M while with Gal-Glc-Cer, a pH optimum of 4.5 and a K-m of 17 mu-M were found. Hydrolysis of both natural and synthetic substrates was inhibited by gamma-D-galactonolactone, D-galactose and lactose. The data strongly suggest that a single beta-galactosidase hydrolyzes all the substrates tested.


Hexosaminidase C in Tay-Sachs and Sandhoff disease. 1. Hexosaminidase C has been purified from human placenta. Complete separation from hexosaminidases A and B was achieved. 2. The following properties of hexosaminidase C differ from those of the A and B isozymes. Presence in the supernatant rather than the lysosomes, neutral pH optimum, higher molecular weight, lack of activity on beta-N-acetylgalactosamine derivatives, and lack of immunological relationship. 3. Hexosaminidase C is active in patients deficient in hexosaminidases A and B and can be recognized by its characteristic electrophoretic mobility. It is concluded that the genetic origin of hexosaminidase C is probably different from that of hexosaminidases A and B.


Studies on the kinetics of glycosidases from chemically-induced rat colonic tumours and normal rat colon. K-m values of beta-N-acetylglucosaminidase (2-acetamido-2-deoxy-beta-D-glucoside acetamidodeoxyglucohydrolase EC 3.2.1.30), beta-N-acetylgalactosaminidase (EC 3.2.1.53), beta-galactosidase (beta-D-galactoside galactohydrolase EC 3.2.1.23) and alpha-L-fucosidase (alpha-L-fucoside fucohydrolase EC 3.2.1.51) of distal colonic tumours, induced in rats by 1,2-dimethylhydrazine, were found to be significantly different compared with the values for the enzymes of the colonic mucosa of the control and tumour-bearing animals and of the proximal colonic tumours. The inhibition kinetics data also showed a significant difference between the enzymes of the distal colon tumours and of other experimental tissues. The data on the effect of pH on enzyme kinetics (pK values) showed no significant difference in the catalytic groups of the active centres of enzymes from tumours and from the control colonic mucosa. Tumour beta-N-acetylglucosaminidase and beta-N-acetylgalactosaminidase compared with the enzymes from other experimental tissues were found to be different in their thermal inactivation kinetics. K-m values of 14 days old foetal intestinal beta-N-acetylglucosaminidase and beta-N-acetylgalactosaminidase were significantly different from the values obtained for the adult mucosal enzymes but were similar to those of the distal colonic tumour enzymes.


Properties and specificity of the major anionic trypsin-like enzyme in the keratinolytic larvae of the webbing clothes moth. The major form of the trypsin-like proteinases from the larvae of the webbing clothes moth Tineola bisselliella has been further purified and some of its properties investigated. It differs from bovine trypsin in several respects. It is anionic at neutral pH, is very stable at alkaline pH, has no requirement for calcium ions for this stability and is very sensitive to urea. It resembles vertebrate trypsins in its complete inhibition by diisopropylfluorophosphate, its pH optimum of 8.5 for hydrolysis of benzoyl-arginine p-nitroanilide and its cleavage specificity against glucagon and the beta-chain of S-carboxymethyl insulin.


Allantoin racemase: a new enzyme from Pseudomonas species. 1. Allantoin racemase is a novel enzyme which catalyzes the conversion of S(+)-and R(minus)-allantoin into the racemate. 2. The enzyme is present in Pseudomonas testosteroni, Pseudomonas putida and five biotypes of Pseudomonas fluorescens, but absent in a number of other Pseudomonas species. 3. The enzyme of Ps. testosteroni was purified 133-fold and exposes optimal activity at pH 8.0-8.2 and 50 degrees C. The enzyme is stable on heating for 15 min at 70 degrees C. 4. The enzyme appeared to be specific for the optical isomers of allantoin and no cofactors are involved in the reaction. 5. The optical aspecificity of allantoinase of Proteus rettgeri was reaffirmed.


Studies on matrix vesicles isolated from chick epiphyseal cartilage. Association of pyrophosphatase and ATPase activities with alkaline phosphatase. Fractions composed primarily of cells (Fraction I), membrane fragments (Fraction II) and matrix vesicles (Fraction III) were isolated from chick epiphyseal cartilage. The characteristics of the alkaline phosphatase (EC 3.1.3.1), pyrophosphatase (EC 3.6.1.1) and ATPase (EC 3.6.1.3) activities in the matrix vesicle fraction were studied in detail. Mg-2-+ was not absolutely essential to any of the activities, but at low levels was stimulatory in all cases. Higher concentrations inhibited both pyrophosphatase and ATPase activities. Both the stimulatory and inhibitory effects were pH-dependent. Ca-2-+ stimulated all activities weakly in the absence of Mg-2-+. However, when Mg-2-+ was present, Ca-2-+ was slightly inhibitory. Thus, none of the activities appear to have a requirement for Ca-2-+, and hence would not seem to be involved with active Ca-2-+ transport in the typical manner. The distribution of alkaline phosphatase, pyrophosphatase, and Mg-2-+ ATPase activities among the various cartilage fractions was identical, and concentrated primarily in the matrix vesicles. Conversely, the highest level of (Na-+ + K-+)-ATPase activity was found in the cell fraction. All activites showed nearly identical sensitivities to levamisole (4 - 10-3 M) which caused nearly complete inhibition of alkaline phosphatase and pyrophosphatase. About 10-15% of the ATPase activity was levamisole-insensitive. The data are consistent with the concept that the Mg-2-+-ATPase and pyrophosphatase activities of matrix vesicles stem from one enzyme, namely, alkaline phosphatase.


Acid phosphatase and adenosine triphosphatase activities in the cell wall of baker's yeast. In order to establish whether a specific adenosine triphosphatase is present in yeast cell wall, hydrolysis rates for p-nitrophenylphosphate (acid phosphatase activity) and for ATP (ATPase activity) were compared under various conditions. Rate determinations were made with both, intact cells and with preparations containing secreted enzymes from protoplasts. Acid phosphatase and ATPase activities had the same pH profile and were susceptible in the same way to the repression by orthophosphate and to the inhibition by 2-deoxyglucose. The Lineweaver-Burk plot shows biphasic kinetic behaviour for the hydrolysis of either p-nitrophenylphosphate or ATP. This suggests the existence of two enzymes with different affinities for the substrates, or one enzyme with at least two active sites. The two activities differ in thermostability and only one activity could be completely abolished by heat treatment. The thermostable enzyme activity had K-m values of 0.475 mM for p-nitrophenylphosphate, and 0.040 mM for ATP. ATP behaved as a partially competitive inhibitor of p-nitrophenylphosphate hydrolysis. Substrate competition studies showed that only a non-specific acid phosphatase is responsible for the hydrolysis of ATP.


Cyclic nucleotide phosphodiesterase in silkworm. Separation and characterization of multiple forms of the enzyme. The existence of two forms of cyclic AMP phosphodiesterase (3',5'-cyclic AMP 5'-nucleotidohydrolase, EC 3.1.4.17) was demonstrated in silkworm larvae by kinetic analysis and DEAE-cellulose column chromatography. The two forms of the enzyme (phosphodiesterase II and III) differ apparently in their characteristics from the previously reported cyclic nucleotide phosphodiesterase (phosphodiesterase I) of silkworm. The higher K-m form (phosphodiesterase II) has a molecular weight of approx. 50 000 and optimum pH of 7.8, and requires Mn-2-+ for maximum activity. The lower K-m form (phosphodiesterase III) has a molecular weight of approx. 97 000 and optimum pH of 7.2, and requires Mg-2-+ for maximum activity. Phosphodiesterase II and probably phosphodiesterase III are specific enzymes for the hydrolysis of cyclic AMP.


Purification of an alkaline ribonuclease from soluble fraction of beef brain. A ribonuclease (ribonucleate 3-pyrimidine-oligonucleotidohydrolase, EC 3.1.4.22) was purified 8300-fold from soluble fraction of beef brain and its properties were investigated. The enzyme is an endonuclease capable of hydrolyzing tRNA, rRNA, poly(C), but shows no activity towards poly(U), poly(A), and poly(G). The preparation is free of deoxyribonuclease, non-specific phosphodiesterase and phosphomonoesterase activity. The enzyme has a pH optimum of 7.6, is not heat stable, has a molecular weight of 25 000, and has a K-m of 134 mu rRNA and K-m of 1600 mug poly(C) per ml.


A Pseudomonas intracellular amylase with high activity on maltodextrins and cyclodextrins. An intracellular amylase from Pseudomonas MSl was purified 95-fold in a 14% yield by fractionation with (NH4) 2SO4, followed by chromatographies on CM-cellulose, hydroxylapatite and Sephadex G-200. On polyacrylamide gel electrophoresis at pH 8.3 it gave a single band and its molecular weight was determined to be 96 000 from its mobility on sodium dodecylsulfate gel electrophoresis. Its activity was maximal at pH 5.5 and 50 degrees C. This enzyme hydrolyzed maltotriose and maltotetraose faster than amylose, but did not hydrolyze maltose. It also rapidly hydrolyzed a series of cyclomaltodextrins. Amylopectin and glycogen, which have branched structures, were attacked much slower than amylose. The enzyme cleaved the substrates in an endo-wise fashion and produced almost equimolar amounts of glucose and maltose as the final products from various substrates. The enzyme mechanism is discussed on the basis of quantitative autoradiographic data. The K-m values for maltotriose, maltotetraose, cyclomaltohexaose, corn amylose and waxycorn amylopectin were 1.0, 1.4, 3.5, 3.3 and 13 - 10-minus 3 g/ml, respectively. The products of the enzyme had an alpha-configuration. Thus, this enzyme is a specific alpha-amylase.


The molecular weight of bovine lactoferrin. The molecular weight of bovine lactoferrin in a neutral solvent (e.g. 0.2 M NaC1, pH 7.8 20 degrees C) has been determined using high-speed sedimentation equilibrium at various concentrations and speeds. Under these conditions the protein behaved as a single thermodynamic component with a weight-average molecular weight [MW]C=0 = 93000 plus or minus 2000 and a second virial coefficient B = 4.3-10(-5) mol-ml(-1)-g(-2). This value of the molecular weight is in good agreement with values previously reported by Groves (J. Am. Chem. Soc. 82, 3345-3350, 1960) and Szuchet and Johnson (Eur. Polym. J. 2, 115-128, 1966); it is, however, in disagreement with the more recent value given by Castellino et al. (J. Biol. Chem. 245, 4269-4275, 1970). The possible reasons for this discrepancy are discussed.


The application of QAE-Sephadex for the purification of two staphylococcal enterotoxins. I. Purification of enterotoxin C2. A new method developed for purification of enterotoxin C2 from Staphylococcus aureus strain 361 consisted of four steps: batchwise adsorption from culture supernatant on QAE-Sephadex; gel filtration on Sephadex G-100; chromatography on QAE-Sephadex using a buffer of constant pH and molarity; and gel filtration using a volatile buffer of constant pH and molarity; and gel filtration using a volatile buffer as the eluting solvent. The purified enterotoxin appeared homogeneous by gel immunodiffusion, gel chromatography and in the analytical ultracentrifuge, although an apparent heterogeneity was noted on QAE-Sephadex chromatography and polyacrylamide disc electrophoresis at pH 4.5. The emetic dose, ED50, by intravenous route in cynomolgus monkeys was 0.04 mug/kg of animal weight. Upon treatment with sodium dodecylsulfate, beta-mercaptoethanol and urea, enterotoxin C2 separated into 3 bands in sodium dodecylsulfate-electrophoresis. One band mol. wt 29000, and two bands of lower molecular weight were so close that they moved as a single zone. After elution from gels, the zone of lower molecular weight were so close that they moved as a single zone. After elution from gels, the zone of lower molecular weight oligopeptides emerged as a single peak at the same position as untreated enterotoxin C2 during gel filtration with buffer lacking thiol and denaturant, and gave a reaction of complete identify to enterotoxin C2 in Ouchterlony immunodiffusion. The results suggest that enterotoxin C2 is a mixture composed of intact polypeptide chains, mol. wt 29000, and two fragments cleaved in the disulfide region of molecular weight of approx. 15400 and 12800 linked by the single disulfide bond in the toxin molecule. Amino acid analysis indicates that enterotoxin C2 consists of 255 amino acid residues.


Studies on phytohemagglutinins. XXIV. Isoelectric point and hybridization of the pea (Pisum sativum L.) isophytohemagglutinins. The isoelectric point of the two pea isophytohemagglutinins varies from pH 5.7 to pH 8.4 depending on the composition of the buffer used. Isoelectric focusing reveals three main molecular species with pI at pH 5.90, 6.35 and 7.00. Molecular species with pI at pH 5.9 and 7.0 correspond to the two pea isophytohemagglutinins which can be obtained by DEAE-cellulose chromatography (Entlicher, G., Kostír, J.V. and Kocurek, J. (1970) Biochim. Biophys. Acta 221, 272-281). A molecular species with pI at pH 6.35 is formed from the two pea isophytohemagglutinins by hybridization. According to the hybridization pattern and subunit composition of the pea isophytohemagglutinins the subunit composition AABB, AACC and AABC can be proposed for the three molecular species with respect to ionic properties of the subunits.


Haemoglobin Saki alpha 2 beta 2 14 Leu-Pro(a11) structure and function. The characterization of haemoglobin Saki alpha 2 beta 2 14 Leu-Pro(a11) is described. This new mutation is unique since it only induces modification of the stability of the molecule. In vitro precipitation of haemoglobin Saki upon heat or in the presence of chemicals is compared to the stability of haemoglobin A and haemoglobin S.


Interaction between proteins and detergents which contain a hydrocarbon chain longer than 16 carbon atoms. II. Difference spectra of various proteins in cetyldimethyl-benzylammonium chloride. The detergents which contain a hydrocarbon side chain longer than 16 cabron atoms were used as a perturbant for the study of protein structure. ta low concentration of cetyldimethylbenzylammonium chloride (CDBA) caused difference spectra for Ac-Trp-OEt and AC-Tyr-OEt. The delta e values at their difference maxima became constant above 30 mM of cetyldimethylbenzylammonium chloride, 1430 at 294 nm for Ac-Trp-OEt and 450 at 288 nm for Ac-Tyr-OEt. These delta e values are higher than any other delta e values resulting from solvent effects by such a remarkably low concentration of organic reagents described in the literature so far. The absence of denaturation blue shift in the difference spectra and the fact that the optical rotatory dispersion of the proteins examined in the present study was not changed significantly by cetyldimethylbenzylammonium chloride indicate that the secondary and tertiary structures of the proteins were not destroyed by cetyldimethylbenzylammonium chloride. These characteristics, together with small overlapping of their difference spectra at 288 and 294 nm were advantageous in the determination of tryptophan and tyrosine residues exposed in glucagon, insulin and alcohol dehydrogenase from yeast. No tyrosine residues in ribonuclease A was accessible to cetyldimethylbenzylammonium chloride. Unusual difference spectrum with a peak at 298 nm was observed for lysozyme which is known to contain tryptophan residues in special environments. Ovalbumin gave a novel unusual difference spectrum with a peak at 290 nm and a shoulder at 298 nm, showing the existence of unusual tryptophan and probably tyrosine residues in the molecule.


The effects of the trinitrophenylation of the amino groups of glucagon on its conformational properties and on its ability to activate rat liver adenylyl cyclase. Trinitrophenyl groups have been specifically introduced into the alpha- and/or the epsilon-NH2 groups of glucagon by reaction with trinitrobenzenesulfonic acid. Introduction of this group into the epsilon-NH2 position of the hormone leads to an apparant increase in the helical content as measured by circular dichroism, while substitution on the alpha-NH2 position causes little change in this property. The usefulness of the trinitrophenyl group for the study of intramolecular singlet excitation transfer from tryptophan is suggested. The pK and reactivity of the amino groups, as measured by the pH dependence of the rate of reaction with trinitrobenzenesulfonic acid, showed that the two amino groups of glucagon have similar properties to those of small model peptides. The trinitrophenyl-glucagon derivatives have little or no activity in stimulating adenylyl cylase of rat liver. By comparison with previously reported results, this demonstrates that the effect of chemical modifications of the amino group on the biological activity of glucagon depends critically on the group which is introduced.


Circular dichroism analysis of the secondary structure of Z protein and its complexes with bilirubin and other organic anions. Circular dichroism (CD) methods were employed to study the conformation of Z protein and characterize its complexes with bilirubin and other organic anions. Z protein-bilirubin complexes exhibited a spectrum with overlapping ellipticity bands of opposite sign in the bilirubin absorption region. These results were compared with those obtained with ligandin, the other major organic anion binding protein of liver. Secondary structural differences between the two proteins were easily demonstrated since ligandin is predominantly an alpha-helical protein and Z features mainly beta-structure. Furthermore, the optical activity pattern generated by bilirubin binding to Z was virtually a mirror image of that of the ligandin bilirubin system. CD experiments were designed to study the direct transfer of bilirubin between Z protein and ligandin, and it was shown that both proteins have almost equal affinities for bilirubin. The bilirubin on Z was readily displaced by oleic acid and displaced to a lesser extent by sulfobromophthalein,


The role of tryptophan residues and hydrophobic interaction in the binding of riboflavin in egg-yolk flavoprotein. Egg-yolk flavoprotein has 7.2 tryptophan residues exposed, while the apoprotein shows an apparent exposure of 80 percent of these (5.7 residues) with dimethylsulphoxide as the perturbant. In the apoprotein at pH 6.9 only 4 groups are perturbed to ethylene glycol, 3.2 to glycerol and 1.4 to sucrose. Diminishing estimates of exposure obtained with increasing molecular diameter of the perturbant suggests that part of indole chromophores of apoprotein are located in "crevices" of the protein molecule. The apoprotein was treated with 2-hydroxy-5-nitrobenzyl bromide, H2O2 and N-bromosuccinimide under conditions designed to accomplish modification of tryptophan residues. Five to six of the eight tryptophans present in the protein were modified. Under these conditions the apoprotein completely looses its capacity for binding riboflavin and the fluorescent intensity of the protein at 360 nm is quenched at the same time to about 80 percent of its initial value. The presence of nonpolar amino acid residues on the surface of the apoprotein suggested the importance of hydrophobic interactions as the dominant factor controlling the binding of riboflavin. The hydrophobic probes Indocyanine green and 4-benzoylamide-4-aminostilbene-2,2-disulphonic acid bound to the apoprotein giving equimolar complexes with dissocation constants, KD 6.5-10(-7) M and 1.8-10(-6) M, respectively, Addition of an equimolar amount of riboflavin quantitatively displaced these dyes from their complexes with apoprotein as shown by spectrophotometric and spectrofluorometric studies.


Comparative micelle structure. IV. The similarity between caprine alphas-casein and bovine alphas-3- casein. The major component of caprine (goat) alphas-casein has been isolated by DEAE-and CM-cellulose chromatography in buffers containing urea and 2-mercaptoethanol. The protein has a molecular weight of 25700 as determined by gel filtration on Sepharose 6B in guanidine hydrochloride. Its composition, Asp17, Thr14, Ser14, Glu45, Pro18, Gly4, Ala10, Cys2, Val12, Met4, Ile12, Leu12, Tyr11, Phe8, His5, Lys22, Arg6, Trp2 and 7 phosphate residues, is much closer to that of bovine alphas3-casein than to bovine alphas1-casein. The caprin alphas-casein is more easily precipitated with Ca2+ than bovine alphas3-casein at 37 degrees C, pH 6.8, which in turn is more easily precipitated than bovine alphas1-casein.


The subunit structure of Pseudomonas cytochrome oxidase. Pseudomonas cytochrome oxidase (EC 1.9.3.2) is composed of two subunits. Each subunit has a molecular weight of approx. 63000 and, according to the iron determination, contains two hemes. Cytochrome oxidase was subjected to various dissociation procedures to determine the stability of the dimeric structure. Progressive succinylation of 14 to 68% of the lysine residues of the enzyme increases the amount of the protein appearing  in the subunit form (S20,W approximately 4 S) from 18 to 92%. At a high degree of succinylation a component with a sedimentation coefficient of approx. 2 S appears. The subunits with sedimentation coefficients of approx. 4 S and 2 S are also formed when the pH is below 4 or above 11. The same molecular weight (63000) was found for these two components in sodium dodecylsulphate electrophoresis. No dissociation of cytochrome oxidase was observed in salt solutions like 3 M NaC1 and 1 M Na2SO4, or in 6 M urea. The slight decrease in the sedimentation coefficients in NaC1 solutions is partly explained by preferential hydratation of the protein.


Inhibition of pyruvate transport by fatty acids in isolated cells from rat small intestine. 1. A technique is described for the rapid separation of intestinal epithelial cells from the incubation medium by passage through a silicon-oil layer and collection in acid, in which their soluble constituents are released. 2. The inhibition by fatty acids of pyruvate oxidation is further studied. Measurement  of pyruvate transport in epithelial cells at 0 degree C showed that short- and medium-chain fatty acids as well as ricinoleate inhibit this transport. Propionate inhibits pyruvate transport by another mechanism than octanoate. 3. Differences between pyruvate propionate and octanoate transport across the epithelial cell membrane were obtained in efflux studies. These studies revealed that acetate, propionate, butyrate and high concentrations of bicarbonate readily stimulate the efflux of pyruvate, probably by anionic counter-transport. No effects were seen with octanoate and hexanoate. The data obtained in these efflux studies suggest that lipophilicity and the pKa values of the monocarboxylic acids determine the contribution of non-ionic diffusion to overall transport. 4. Saturation kinetics, competitive inhibition by short-chain fatty acids and counter-transport suggest a carrier-mediated transport of pyruvate.


Studies of normal and nephritic rat glomerular basement membrane. Solubilization of the normal glomerular basement membrane with various solvents revealed that the material is held together by hydrogen and disulfide linkages as well as ionic salt bridges which ionize at around pH 10.0. Pronase digestion indicated that differences in susceptibility to enzyme digestion exist between normal and nephritic membrane. Titration of a urea-insoluble material indicated that some alteration must have taken place in the association between various components of the nephritic basement membrane. Chemical analysis of alkali-solubilized fractions suggested that greater alkali susceptibility of the nephritic material may be present. A collagen-like material resembling both tendon and dog basement membrane collagen in its amino acid composition was isolated. It contained 10% hexose, but in addition to glucose and galactose, mannose was also detected. A glycopeptide fraction obtained by pronase and collagenase digestion has a carbohydrate composition similar to the collagen-like material above. These substances probably represent incompletely digested fragments of the basement membrane.


Chicken fumarase. II. Kinetic studies.

Chicken fumarase. II. Kinetic studies. The catalysis of the hydration of fumarate and deshydration of L - malate by chicken fumarase was measured spectrophotometrically over a range of substrate concentrations from 4 times 10(-3) M to 8 times 10(-5) M for fumarate and from 8 times 10(-2) M to 10(-3) M for L - malate. For the forward and reverse reactions, linear Lineweaver and Burk plots were obtained. The Michaelis constants and the maximum initial velocities for both substrates were determined and the Haldane relation was found to be obeyed. The effect of pH on activity was investigated over a pH range from 5.5 to 9.0 and the data indicate the presence, in the active site, of two ionizable groups, one in the acidic form and one in the basic form. The values of the ionization constants, determined for the enzyme - substrate complexes, agree closely with the ones obtained for the porcine enzyme. The mode of action of twenty-four structural analogs on the initial velocity of the dehydration of L-malate, by chicken fumarase was examined. From these studies, two regions positively charged appear necessary for the effective binding of the carboxylates of the substrates and competitive inhibitors to the active center. Moreover, the data suggest the presence of an additional group, in the catalytic site of chicken fumarase, that stabilizes the carbon-carbon double bond common to fumarate and its structural analogs. Finally, from the comparison of the kinetic properties of the chicken and pig fumarases, it may be concluded that the catalytic mechanism of the homologous enzymes are very similar, if not identical.


[Purification and characterization of two peroxidases from hard wheat]. Two peroxidases A and B were purified from a borate buffer extract (pH = 10,4) of durum wheat semolina (Triticum durum), var. Bidi 17, by chromatography on DEAE-cellulose, salting out by 3M ammonium sulphate and two chromatographies on CM-cellulose; specific activities of peroxidase A or B were increased 114 or 66 fold. Molecular weight, amino acid composition, absorption spectrum, pH optimum, thermal stability and KM values differentiate the two enzymes. Ion Ca++ was shown as an activator of both peroxidase activities; the presence of an inhibitor in the crude extract was demonstrated.


[Study of conformational transformations in serum albumin by the method of scanning microcalorimetry]. Thermal denaturation of serum albumin in the alkali pH region is investigated by the scanning microcalorimetry method. It is shown that at pH 8.5 a macromolecule undergoes a conformational transformation as a result of which its thermal characteristics are changed qualitatively. The form which corresponds to lower pH values is denatured according to the smaller than all or none greater than model with an intensive sorption of heat. The form corresponding to higher pH values is denatured noncooperatively without heat absorption but with an essential increase of heat capacity.


Studies on changes in DNA polymerase activity during the cell cycle in synchronized KB cells. We have demonstrated the presence of two DNA polymerases in KB cells and studied the variation of their activities in a synchronous cell population. During the cell cycle we observed in nuclei, only one DNA dependent DNA polymerase, the 3.4 S or minipolymerase, and similarly in the cytoplasm only one enzyme, the 8.3 S or maxipolymerase. The former shows preference for native DNA and the latter for denatured DNA. Their Mg++ and K+ requirements are different and their pH optima are 8.5 and 7 for nuclear polymerase and cytoplasmic polymerase respectively. The cytoplasmic polymerase activity remains stable from one cell cycle to the other with each cell reconstituting its stock at the start of the following cycle (G1 and early S phases). On the contrary nuclear activity decreases in G2, M and early G1, then increases to a maximum in the middle of the S phase. This fluctuation in enzyme activity could be due to degradation, transfer to the cytoplasm or the association of the enzyme with the chromatin and/or the nuclear membrane after completion of DNA synthesis. Our results do not permit us to choose between these three hypotheses. However their significance is discussed in the light of the results obtained by some authors who, on the contrary, have tended to minimise the role of the minipolymerase in DNA duplication, whereas we, from our findings, ascribe a preponderant role to this enzyme. The cytoplasmic maxipolymerase (8.3 S) may simply be a storage form of the enzyme from which minipolymerase can be formed as needed.


[Spectral-luminescent study of pH-dependent conformational changes in fibrinogen]. Results are presented of measurements of fluorescence parameters of fibrinogen at pH values of 2-12. It is stated that the stability zone of native conformation of fibrinogen molecule lies in the pH range of 5,5-8,5. In an acid medium the fibrinogen molecule twice (in the pH range of 5--3 and 3--2) undergoes structural rearrangement. In an alkali medium the structural rearrangement of fibrinogen is observed at pH greater than 9,5.


[Dextran glucosidase activity of the acid alpha-glucosidase from pig spleen]. Some properties of acid alpha-glucosidase from pig spleen are studied. Changes in the enzyme activity with respect to polymeric and oligomeric substrates were shown to take place under effects of temperature, pH, concentration of metal ions, urea treatment, trehalose inhibition. Dextran and glycogen were used as polymeric substrates, and disaccharides with different types of bonds were taken as oligomeric substrates. The data obtained confirm the hypothesis on separate regions of binding polymers and oligomers with the enzyme molecule.


[Isolation and some properties of exoribonuclease from the cell nuclei of the rat liver]. Exoribonuclease from rat liver cell nuclei was isolated and purified using selective extraction at pH 8.0, salting out with ammonium sulfate (30-50% saturation) and chromatography on DEAE-Sephadex A-50. The enzyme has the pH optimum 7.4-7.6, it requires Mg-2+. It catalyses the gradual removal of ribonucleoside-5'-monophosphates from synthetic polynucleotides and RNA; it preferably attacks single-stranded polynucleotides and is less efficient when hydrolysing poly-stranded helical polymers. It is supposed that exoribonuclease can participate in the destruction of non-informative regions in new-formed hyper-molecular RNA of cell nuclei.


[Interaction of aspartate transaminase from chicken heart cytosol with pyridoxal phosphate analogs]. The interactions were studied of the apoenzyme of aspartate aminotransferase from chicken heart cytosol with a variety of pyridoxal-P analogues. 2-Norpyridoxal-P, 2'-n-propylpyridoxal-P, 2'-isopropylpyridoxal-P, 6-methylpyridoxal-P, and 5'-methylpyridoxal-P were shown to display coenzyme activity. Estimated relative Vmax values of the complexes of apoenzyme with the above--mentioned analogues amounted respectively to 0.8; 0,2; 0,1; 0.1 and 0.1 (taking the Vmax value of the native holoenzyme as equal 1.0). The pH-dependence of reactivation rates of the apoenzyme with pyridoxal-P and pyridoxamine-P was evaluated. 3-Deoxypyridoxal-P, 3-0-methylpyridoxal-P, 2'-phenylpyridoxal-P, 5-nor-5-beta-carboxyvinylpyridoxal and 5-nor-5-beta-carboxyethylpyridoxal fail to activate the apoenzyme, but inhibit competitively the binding of pyridoxal-P to the protein; the estimated Ki values for these analoges were 2.4-10- minus 6; 3.1-10- minus 6; 3.5-10- minus 6; 7.2-10- minus 6 and 8.3-10- minus 6 M, respectively. It is of interest to compare reactivation effects of pyridoxal-P analogues for the apoenzymes of aspartate aminotransferases from chicken and from pig heart cytosol. Although the observed effects were fairly similar, it should be noted that the relative catalytic efficiencies of complexes of the chicken apoenzyme with pyridoxal-P analogues were much lower than those of complexes formed with the pig heart apoenzyme. It thus appears that of the two enzymes tested, the chicken heart aminotransferase makes more stringent demands with respect to structure of the coenzyme.


Studies on a circulating anticoagulant in systemic lupus erythematosus: evidence for inhibition of the function of activated plasma thromboplastin antecedent (factor XIa). Plasma proteins which interfere with blood coagulation have often been described in patients with systemic lupus erythematosus (SLE). The most frequent type interferes with the conversion of prothrombin to thrombin and thus prolongs the prothrombin time. Infrequently, SLE patients exhibit anticoagulants which appear to block the earlier stages of coagulation such as those involving factor VIII or the formation of activated factor XI (factor XIa). The anticoagulant reported here was studied by means of a sequential clotting system utilizing crude coagulation factors and was noted to interfere with the action of activated plasma thromboplastin antecedent (PTA) during the activation of factor IX. This anticoagulant was found in gamma-globulin-rich ethanol fractions of plasma. After gel filtration, it was found principally in fractions containing IgM globulins but also, to a lesser extent, in IgG-rich fractions. In this respect, it is similar to anticoagulants reported in certain other cases of SLE. Attempts to confirm the immunoglobulin nature of the anticoagulant by immunoabsorption were, however, inconclusive


Platelet storage at 22 degrees C: role of gas transport across plastic containers in maintenance of viability. Containers constructed of polyvinylchloride (PVC) are used for the storage of platelet concentrates (PC) for transfusion, At 22 degrees C, pH often falls to such low levels (pH is less that 6.0) that viability is lost. Far lesser degrees of pH fall are observed in bags constructed of polyethylene (PE). In this study, pH, PO2, PCO2, platelet count, lactate concentration, microscopic morphology, and viability after 51-chromium labeling were evaluated during storage at 22 degrees C under a variety of circumstances. The results indicate that (1) pH falls because of the generation of lactic acid by platelet glycolysis and, under some circumstances, the retention of CO2. (2) Rate of pH fall is, therefore, roughly proportional to the platelet count. (3) PE is more permeable to gases, thereby allowing CO2 escape from and easier O2 entry into the stored PC; the higher O2 tensions suppress glycolysis by the Pasteur effect. (4) Adequate agitation and container size are critical if the beneficial effect of PE is to be obtained. (5) In general, platelets stored in PE containers have excellent viability in vivo although CO2 escape can result in elevations in pH which are deleterious. (6) Storage in a 10% CO2 atmosphere prevents these deletrrious pH elevations without otherwise impairing platelet viability; (7) Results similar to those achieved with PE can be achieved with PVC if this material is made thinner to allow easier penetration of gases


The effect of carbon monoxide on red cell life span in sickle cell disease. Carbon monoxide at a concentration of 1000-2000 ppm was administered to sickle cell disease patients. In each of two patients, one 51Cr red cell survival study was carried out before CO administration, and a second study was initiated a few days before CO administration was started. In both, significant prolongation of red cell survival was observed, suggesting that the rheologic properties of sickle cells were favorably influenced in vivo. The administration of carbon monoxide is not recommended as a treatment for sickle cell disease. However, further trials would seem to be justified if conducted under carefully controlled conditions.


[Effect of nucleophiles on the conversion of choline esters under the action of cholinesterase]. It is shown that effect of alcohols ROH on the hydrolysis of butyryl-choline and acetylcholine by choline esterase (E. C. 3.1.1.8) is complex; it included the concurrent and the unconcurrent inhibitions, the interaction with the acylenzyme to form more reactive triple complex, and the acyl migration. By titrometric method it is found that proportion of the transacylation and hydrolysis rates depends on hydrophobity of R only being independent on its electronegativity.


[Some properties of beta-aspartate kinase from Micrococcus glutamicus-95 bacteria]. Kinetic and allosteric properties of beta-asparatatekinase from Micrococcus glutamicus-95 were studied. Coarse protein fraction, sedimented at 0.8 from saturated (NH4)2SO4, was used as an enzyme preparation. Curves of the dependency of the enzymatic reaction rate on substrate (L-aspartic acid and ATP) concentration were not found to be S-like. However, double reciprocal plotting of the data obtained revealed their deviation from the hyperbolic curve. The effect of amino acids (lysine, threonine, isoleucine, valine) on the activity of beta-aspartatekinase is studied. Lysine was shown to inhibit slightly beta-aspartatekinase, while threonine slightly activated it. Combined addition of both amino acids at a concentration of 1 mM resulted in the 50% inhibition. Isoleucine and valine activated beta-aspartatekinase and eliminated multivalent inhibitory effect of lysine and threonine. Interaction of isoleucine and lysine+threonine with beta-aspartatekinase was competitive with respect to L-aspartic acid and non-competitive in relation to ATP.


Dissociation of theophylline uptake and inotropic effect in myocardial tissue: influence of temperature, pH and calcium. 1. The myocardial uptake and the positive inotropic effect of theophylline (100 mug/ml; 0.56 mM) were studied in isolated electrically driven guinea-pig hearts perfused by the Langendorff technique under various extracellular conditions. [3H]-theophylline was used. 2. Variations in temperature, hydrogen ion and calcium ion concentrations of the perfusion media changed the time course and magnitude of the effect of theophylline on myocardial twitch tension but did not affect the time course and amount of theophylline uptake. 3. Under all conditions, the build-up of the positive inotropic effect of theophylline was about three times faster than the uptake of the drug into the heart. 4. Since no relationship could be found between theophylline uptake and inotropic effect, the cardiac positive inotropic response to theophylline is thought unlikely to be due mainly to an interaction of the drug with intracellular receptor sites but is assumed to occur via an effect of the drug on the sarcolemma, that is at a site which the drug reaches before it enters the intracellular space.


Role of IgG fractions with high isoelectric points in the thymol turbidity test in syphilis. Evidence for an increase in basic IgG in early syphilis. It is demonstrated that IgG fractions with isoelectric points above 7.6, isolated from pooled syphilitic sera, are able to elevate the thymol turbidity. The effect increases with increasing isoelectric points of the isolated fraction. IgG from individual syphilitic patients exerts a stronger effect on the thymol turbidity than normal IgG. It is concluded that elevated amounts of these basic immunoglobulins are present in the sera of syphilitic subjects.


Preference for basic IgG in early syphilis. By differential IgG measurements of sera of 27 patients with early infectious syphilis, it was found that infection with T. pallidium results in a preferential synthesis of IgG immunoglobulins characterized by high isoelectric points. The decrease in total IgG level observed after 6 weeks of treatment can to a large extent be accounted for by the decrease in basic IgG concentration. The relationship between the total and basic IgG levels and the number of T. pallidum present in the body during the three early stages of the disease studied is discussed.


Effect of pentazocine on the evoked potentials recorded in the primary somesthetic cortical areas of guinea pigs. The influence of pentazocine on the evoked potentials registered in the primary somesthetic cortical area in guinea pigs under pentobarbital anesthesia was studied. The stimulation was applied in the contralateral lip and the evoked cortical responses from different zones on the cortex stereotaxically localized were registered. Pentazocine (5-10 mg/kg i.v.) induced a significant increase in the latency of the evoked potentials as compared with controls. Since morphine does not modify these potentials, the effect of pentazocine is interpreted as being a direct one on the primary pain pathway.


The effects of axotomy on the development of the rat superior cervical ganglion. The effects of division of the postganglionic axons of the adrenergic neurones in the superior cervical ganglion of the rat were examined with regard to the total ganglionic protein content and tyrosine hydroxylase and DOPA decarboxylase activities. Axotomy before the twelfth postnatal day results in a marked atrophy of the ganglion and a reduction in the total enzyme content of the ganglion. Axotomy after postnatal day 21 results in the normal adult ganglion response with a large increase in the total protein content of the ganglion and only minor changes in the total enzyme content of the ganglion. Axotomy between the postnatal days 12 and 21 results in an intermediate response. Thus it can be concluded there is a critical period during the development of the ganglion during which the adrenergic neurones undergo a maturation governed by their contact with the peripheral target cell via axons. These results suggest a trophic role of the end organ on the adrenergic neurone.


The effect of hydrocortisone and adrenocorticotrophic hormone on monoamine oxidase and tyrosine hydroxylase in explant cultures of embryonic chick sympathetic ganglia. Sympathetic ganglia from 13- to 15-day-old embryonic chicks were cultured for up to 2 days in Leighton tubes. The influence of hydrocortisone and ACTH added to the culture medium on the enzymes monoamine oxidase (MAO) and tyrosine hydroxylase was studied. Hydrocortisone (5 times 10(-5)M) had no effect on tyrosine hydroxylase but increased MAO activity by up to 46 percent over control values under conditions of low or zero nerve growth factor (NGF) concentration. ACTH also increased ganglionic MAO activity, the effect again depending on NGF concentration. This time the maximal response (an increase of 50 percent over controls) was seen at high NGF concentrations. This response was similar to the effect of 1 mM dibutyryl cyclic AMP, and was blocked by 1 times 10-5 M propranolol and 10 muM prostaglandin E(1). ACTH only slightly increased tyrosine hydroxylase activity and this effect was due to a small (18 percent) increase in sympathetic neurone number. Guanosine 5-diphosphate (0.5 mM) was found to increase tyrosine hydroxylase activity by 57 percent and this effect was blocked by the presence of ACTH.


Seasonal variation in semen characteristics of White Leghorn, New Hampshire and indigenous chicken in Iraq. 1. The effects of season, temperature, humidity and light on semen volume, pH, concentration, motility and the proportions of dead and abnormal spermatozoa were investigated on White Leghorn, New Hampshire and Iraqi cockerels. 2. All semen characteristics, with the exception of pH, were significantly influenced by season. 3. The semen quality of the Iraqi breed was similar to that of the New Hampshire breed.


A hazard associated with improper connection of the Bain breathing circuit. A case of cardiac arrest is presented which was caused by improper connection of a modified Mapleson D circuit (Bain breathing circuit). Excessive enternal deadspace was created by interchanging the gas inflow line and the attachment for an airway pressure manometer. This resulted in a marked respiratory acidosis, clinically undetected until the concomitant hypoxia produced a severe cardiac depression and arrest. The sequence of events was reproduced in a dog under comparable anaesthetic conditions. In order to avoid this error it is recommended to permanently fuse the connecting piece placed in the circuit with the tubing leading to the airway pressure manometer of the respirator.


The effect of artificial ventilation on functional residual capacity and arterial oxygenation. II. Comparison of spontaneous respiration and artificial ventilation at similar arterial carbon dioxide tensions, tidal volumes and inspiratory gas flow rates. We have compared cardiac output, gas exchange and pulmonary mechanics during spontaneous breathing and artificial ventilaton under conditions which kept PaCO2 within the normal range and maintained constant tidal volume and inspired gas flow rate. In dogs anaesthetized with pentobarbitone and ventilated with air, artificial ventilation increased VD/VT but did not reduce Q angstrom, FRC, or CL. PaO2 increased and A-aDO2 decreased during aritificial ventilation, perhaps because of a small increase in Q angstrom and a small decrease in oxygen consumption. It appears that many of the reported deleterious effects of artificial ventilation may be due to the use of other anaesthetic agents and patterns of ventilation, and to changes in PaCO2.


Maternal and foetal effects of methoxyflurane anaesthesia in the pregnant ewe. Maternal and foetal cardiovascular, blood gas and acid-base changes were studied during 90 minutes of methoxyflurane anaesthesia. At 1.0 and 1.5 MAC anaesthesia, despite slight to moderate falls in maternal blood pressure, cardiac output and uterine blood flow, no serious foetal deterioration was seen. 2.0 MAC methoxyflurane was associated with marked falls in maternal blood pressure, cardiac output and uterine blood flow. Foetal hypoxaemia and a mixed respiratory and metabolic acidosis developed. Little foetal cardiovascular depression was seen with any level of anaesthesia. There was no direct effect of methoxyflurane on the uterine vasculature.


Doses of epinephrine causing arrhythmia during enflurane, methoxyflurane and halothane anaesthesia in dogs. The arrhythmogenic dosage of epinephrine, administered by constant intravenous infusion, was measured in five dogs during enflurane, methoxyflurane and halothane anaesthesia. While premature ventricular contractions were observed in only one of five dogs with enflurane and methoxyflurane, epinephrine-induced arrhythmias were seen in all animals during halothane anaesthesia. Epinephrine dosage and the resultant increase in mean arterial blood pressure at which arrhythmias occurred during halothane anaesthesia were significantly less (p less than 0.05) than with the other anaesthetics. These observations indicate that enflurane and methoxyflurane, as compared to halothane, possess relatively less arrhythmogenic potential as sensitizing agents in the presence of increased circulating catecholamines.


A kinetic study of the reaction of horseradish peroxidase with hydrogen peroxide. A kinetic study has been carried out over the pH range of 2.63-9.37 for the reaction of horseradish peroxidase with hydrogen peroxide to form compound I of th;e enzyme. Analysis of the results, indicates that there are two kinetic influencing, ionizable groups on the enzyme with pKa values of 3.2 and 3.9. Protonation of these groups results in a decrease in the rate of reaction of the enzyme with H2O2. A previous study of the kinetics of cyanide binding to horseradish peroxidase (Ellis, W.D. & Dunford, H.B.: Biochemistry 7, 2054-2062 (1968)) has been extended to down to pH 2.55, and analysis of these results also indicates the presence of two kinetically important ionizable groups on the enzyme with pKa values of 2.9 and 3.9.


Distribution of dipeptidase activity between lysosomes and soluble fraction of rat liver. Dipeptidase activity toward Arg-Phe, Arg-Gly, and Trp-Leu exhibited bimodal distribution in the lysosomal and soluble fractions of rat liver. The majority (50-70 percent) of the dipeptidase activity was present in the soluble fraction. Some evidence for a plasma membrane dipeptidase, which hydrolyzes Trp-Leu but not Arg-Phe or Arg-Gly, also was found. The lysosomal dipeptidase activity had a pH optimum of 6.0-7.0, and was activated by sulfhydryl reagents. Lysosomal localization for some of the dipeptidase activity was established with Triton WR-1339 fractionation and latency experiments.


Carboxylesterases (EC 3.1.1). The molecular sizes of chicken and pig liver carboxylesterases. The molecular size of pig liver carboxylesterase has been investigated under a variety of conditions of pH and ionic strength. From equilibrium and velocity sedimentation at pH 4.0 and pH 7.5, and from chromatography on Sephadex G-200,we conclude that the monomeric molecular weight is similar to 65,000 daltons and that the enzyme associates to form trimers. Association equilibrium constants for the monomer-trimer system were estimated to be 0.02 1-2 g-2 at pH 4 (concentration-dependent molecular weight data) and 2 times 10-5 1-2g-2 at pH 7.5 (frontal gel chromatographic results). These studies were aided by comparisons of the properties of the pig liver enzyme with those of chicken liver carboxylesterase, which is shown to exhibit the velocity and equilibrium sedimentation characteristics of a homogeneous protein with molecular weight similar to 65,000. Studies of pig and chicken liver carboxylesterases in 6 M guanidinium chloride, 0.1 M in beta-mercaptoethanol, support the proposition that the monomeric species of these enzymes have molecular weights of similar to 65,000. On polyacrylamide gel electrophoresis in SDS, there is no evidence for a major species of molecular weight less than similar to 65,000 for the pig enzyme, but ca. 50 percent of the chicken esterase is dissociated into two species of molecular weight similar to 30,000.


Carboxylesterases (EC 3.1.1). A comparison of some kinetic properties of horse, sheep, chicken, pig, and ox liver carboxylesterases. A comparative study of the kinetic be,avior of horse, sheep, chicken, pig, and ox liver carboxylesterases is reported. The enzymes exhibit similar specificites towards a series of phenyl esters in which the acyl group is varied, and towards a series of butyrate esters in which the alcohol group is varied. Non-Michaelis-Menten kinetics are exhibited by the horse enzyme in the hydrolysis of methyl and ethyl butyrates, and by the pig enzyme with ethyl butyrate. Each enzyme exhibits inhibition by one or more substrates. A simple scheme which accounts for both activation and inhibition is discussed. pH-k(cat) profiles for the horse and chicken liver carboxylesterase-catalyzed hydrolyses of phenyl butyrate demonstrate dependencies on pK(a)S of 4.75 and 5.0, respectively.


Photooxidation of dinitrophenylhistidine-200 human carbonic anhydrase B. Partial inactivation of tau-dinitrophenylhistidine-200 human carbonic anhydrase B, induced by visible light, followed first order kinetics (k(app) = 6.05 times 10-2 min-1). After 50 min the tau-dinitrophenylhistidine (tau-DNP-histidine) content decreased to a negligible level, but the illuminated enzyme retained, at pH 7.6, approximately 9.2 percent of the esterase activity of the native enzyme. The following lines of evidence suggest that the loss of activity results from the destruction of tau-DNP-histidine-200. (1) No significant loss of amino acid other than tau-DNP-histidine was detected after illumination. (2) The rate of loss of activity correlated well with the loss of tau-DNP-histidine. (3) In the photooxidized enzyme the DNP moiety was retained but had lost the characteristic sensitivity of tau-DNP-histidine to nucleophilic attack. Titration of the illuminated enzyme with acetazolamide indicated that the residual activity is an intrinsic property of the modified enzyme. The chromatographically purified photooxidized enzyme migrated as a single band on isoelectrofocusing in polyacylamide gel, and at pH 7.6 possessed 7.5 percent esterase activity relative to the native enzyme. By establishing effective destruction of histidine-200, it can be concluded that neither the pi N nor, as previously shown, the tau N of histidine-200 is critical for the catalysis.


The lipopolysaccharides of Neisseria gonorrhoeae colony types 1 and 4. The lipopolysaccharides (LPS) of strains of Neisseria gonorrhoeae grown in type 1 (T1) and 4 (T4) colony forms have been isolated. LPS from T4 colony type cells on mild hydrolysis gave a lipid A and a core oligosaccharide composed of 2-amino-2-deoxy-D-glucose, D-glucose, D-galactose, L-glycero-D-manno-heptose and 3-deoxy-D-manno-octuosonic acid that appeared to be common to all the strains examined. LPS from T1 colony type cells on mild hydrolysis gave a lipid A and high molecular weight O polysacc,arides which showed considerable differences in glycose composition for each strain examined. In those strains examined, T4 cells appear to produce a common "R" type LPS whereas T1 cells produce an "S" type LPS with structurally different O polysaccharide structures which probably account for serologically differentiated strains of N. gonorrhoeae.


The functional groups of the Mg-Ca ATPase from Escherichia coli. The influence of hydrogen ion concentration on binding and conversion of MgATP and CaATP by membrane bound and solubilized ATPase from Escherichia coli has been investigated. The reaction of enzyme (E), hydrogen ion (H+), and substrate (S) procedes according to the following scheme, where Me is the metal ion and P is the product(s). (See article for formular). Within experimental error, the results obtained with membrane-bound and solubilized ATPase are identical. Changing the concentration of Mg2+ ions or replacement of Mg2+ by Ca2+ ions alters the dissociation constants Kb, KHMeATP, and Ka'. The kinetics and experiments with group-specific inhibitors suggest that integrity for amino, imidazole, tyrosyl, carboxyl, and arginyl residues is required for activity of membrane-bound and solubilized E. coli ATPase.


Wheat embryo ribonucleates. V. Generation of N2--dimethylguanylate when 'fully sequenced' homogeneous species of transfer RNA are used as substrates for wheat embryo methyltransferases. When S-adenosly[methyl-14-C]methionine and various species of transfer RNA are used as substrates for wheat embryo methyltransferases, the principal site of guanylate-N-2 methylation can be shown to be a G-residue between the stems of the dihydrouridine and anticodon loops. This common site of guanylate-N-2 methylation is referred to as the interstem target site. 2. When the interstem target site is the non-terminal G-residue in a G-C-G-C sequence, as in the cases of Escherichia coli tRNA1-Leu, tRNA-Ile, and tRNA3-Ser, there is preponderant dimethylation to yield N-2-dimethylguanylate. 3. When the interstem target site is part of a U-C-G-U sequence, as in the case of E. coli tRNAf-Met, there is diminished dimethylation and correspondingly increased monomethylation to yield N-2-monomethylguanylate. 4. When the interstem target site is the non-terminal G-residue in an A-U-G-G sequence, as in the case of yeast tRNA-Asp, there is negligible dimethylation and almost exclusive monomethylation to yield N-2-monomethylguanylate. 5. The concerted way in which the primary, secondary, and tertiary structures of tRNA molecules might influence the efficacy of these methylations is the subject of a brief discussion. Attention is also focused on the evolutionary and molecular basis for the generally non-random distributions of methylated oligonucleotide sequences in ribosomal and transfer ribonucleates.


Specificity of gamma-glutamyl cyclotransferase. Using the phthaloyl method, 18 gamma-L-glutamyl peptides labelled with 14-C in the N-terminal position have been synthesized. The products were isolated by simple procedures using a Dowex-1 column or high voltage electrophoresis. The synthetic peptides contain minor impurities of the corresponding D-glutamyl isomers. The proportion of D-isomer was determined by the use of glutamic decarboxylase, or by a new method using digestion with purified gamma-glutamyl cyclotransferase and determination of the resulting 2-pyrrolidone-5-carboxylic acid (5-oxoproline). Evidence was obtained that gamma-glutamyl cyclotransferase acts only on the L-form of gamma-glutamyl substrates; the enzyme could, therefore, be used for preparation of gamma-D-glutamyl peptides from their racemic mixtures. The specificity of gamma-glutamyl cyclotransferase has been examined using pure enzyme prepared from pig liver, and extracts from tissues of rat and man. The basic structural requirement in substrates may be represented as gamma-L-glutamyl-NH--CHR--COOH. The amino acid linked to the gamma-glutamyl group must be in the L configuration.


Kinetics and reaction mechanism of the carbamylphosphate synthetase of a multienzyme aggregate from yeast. We have studied the kinetics and reaction mechanism of the carbamylphosphate synthetase of an enzyme aggregate functioning in the pyrimidine pathway of yeast. MG--ATP was found to be one of the substrates of the enzyme reaction which was activated by free Mg-2+ and inhibited by free ATP. Feedback inhibition by UTP was non-competitive with respect to both glutamine and bicarbonate. Potassium ions were essential for activity and could not be replaced by sodium. Glutamine could be replaced partially by ammonium ions as nitrogen donor. A bicarbonate-dependent cleavage of ATP was shown to take place in the absence of L-glutamine; L-glutamate was a competitive inhibitor of L-glutamine and the enzyme was shown to synthesize ATP when incubated with ADP and carbamyl phosphate. The reaction mechanism was found to involve sequential addition of the substrates bicarbonate and Mg--ATP and release of ADP, followed by addition of the third substrate glutamine. The purine nucleotide XMP had a pronounced activating effect on the enzyme, acting at a site different from that of UTP. Saturating levels of Mg--ATP eliminated this activation.


Apparent thiamin status of cattle and its relationship to polioencephalomalacia. The thiamin status (thiamin concentration in whole blood, plasma, and erythrocytes; erythrocyte transketolase activity) of normal cattle consuming varying diets did not differ from that of cattle with polioencephalomalacia or lead poisoning. Dairy cattle had higher ruminal content of thiamin and lower thiamin destroying activity than did beef cattle. Renal oxalosis was no more frequent in cattle which had polioencephalomalacia than in postnatal calves. In normal beef cattle, approximately 75% of total blood thiamin is in erythrocytes and the remainder in plasma.


Exercise-induced increase in the capacity of rat skeletal muscle to oxidize ketones. During and after strenuous prolonged exercise, sedentary individuals develop high blood levels of acetoacetate and beta-hydroxybutyrate whereas exercise-trained animals and human subjects do not. We have investigated the possibility that exercise training can increase the capacity of skeletal muscle to oxidize ketones. In this study we measured rates of D-beta[3-14-C]-hydroxybutyrate and [3-14-C]acetoacetate oxidation, and the levels of activity of the enzymes involved in the oxidation of ketones in homogenates of gastrocnemius muscles of exercise-trained and of untrained male rats. The trained animals had markedly lower blood ketone levels immediately and 60 min after a 90 min long bout of exercise than did the sedentary animals. The rates of D-beta-[13-14C]hydroxybutryate and [3-14-C]acetoacetate oxidation were twice as high in homogenates of muscles from the trained as compared to the sedentary rats. The increases in levels of activity in gastrocnemius muscle in response to the exercise program were: beta-hydroxybutyrate dehydrogenase threefold; 3-ketoacid CoA-transferase twofold; and acetoacetyl-CoA thiolase 55%. This exercise-induced increase in the capacity of skeletal muscle to oxidize ketones could play a role in preventing development of ketosis in the physically trained animal during and following prolonged strenuous exercise.


Patterns of action of glucoamylase isozymes from Aspergillus species on glycogen. Glucoamylase isozymes from black Aspergillus species have been freed of all traces of alpha-amylase by chromatography on Bio-Gel P-100, as evidenced by limited hydrolysis of oxidized amylose. Glucoamylase I retains its ability to hydrolyze rabbit-liver glycogen rapidly. By contrast, glucoamylase II hydrolyzes glycogen slowly, and addition of alpha-amylase to glucoamylase II does not enhance its activity toward glycogen. These results indicate that alpha-amylase is not involved in hydrolysis of glycogen by glucoamylase.