Mechanism of D-amphetamine inhibition of protein synthesis. At 1 h after intraperitoneal administration of D-amphetamine sulphate (15 mg/kg), rat brain polyribosomes show disaggregation accompanied by reduced capacity for in vitro peptide chain elongation. The direct action of amphetamine on cell-fine protein-synthesizing systems was therefore explored. When brain or liver polyribosomes from untreated rats were incubated with pH 5 enzyme, peptide chain elongation was not inhibited by the addition 4 mM amphetamine to the  medium. On the other hand, an initiation-dependent system consisting of rat liver of brain mRNA and wheat germ S-30 fraction showed inhibition of [3H]leucine incorporation by 50% when 4 mM amphetamine were added. The metabolites of amphetamine, p-hydroxyamphetamine and p-hydroxynorephedrine, had no inhibitory action in either system, but the potent neurotoxin p-chloroamphetamine was a more powerful inhibitor of initiation than amphetamine. By using [3H]amphetamine, it was shown that amphetamine binds to the 80-S ribosomes of the wheat germ system. This binding depended on the presence in the system of natural liver or brain mRNA or several synthetic mRNAs, but was not promoted by polyuridylic acid as the messenger. Significantly, polyuridylic acid-dependent polyphenylalanine synthesis by the wheat germ system was not inhibited by amphetamine or p-chloroamphetamine. Therefore, it was concluded that amphetamine inhibits protein synthesis by interfering with initiation through a step related to formation of the mRNA ribosome complex.


Model membrane studies of spin-label probes. Part 1. Mixed monolayers of 12-nitroxide stearic acid and myristic acid. Pure and mixed monomolecular films of a cell membrane spin label probe, 12-nitroxide stearic acid have been studied where myristic acid was selected as the host lipid. The behavior of 12-nitroxide stearic acid at the air water interface is understood in terms of two molecular configurations: erect (with only the carboxyl group in the interface) and bent (with both the carboxyl group and the oxazolidine ring in the interface). In mixed films both of these conformations play a role at high surface pressures. At low probe concentrations, 12-nitroxide stearic acid is primarily in an erect conformation, while at high probe concentrations the reverse is true. This particular host lipid appears capable of erecting the probe molecule with only small concentrations of myristic acid. In a condensed host lipid, the probe is partially immiscible, and segregates to form a heterogeneous film from which it is readily collapsed. The probe is seen to perturb the molecular packing in this mixed system and the perturbation to be dependent on both the molecular shape and nature of the probe.


Evidence for a sodium ion exchange carrier linked with glucose transport across the brush border of a flatworm (Hymenolepis diminuta, Cestoda). The manner in which the flatworm, Hymenolepis diminuta (Cestoda), regulates the transport of glucose and Na+ across the brush border was examined. While the presence of an unstirred region in the brush border may favor the reabsorption of leaked glucose, some leaked glucose was lost to the ambient medium. This loss was markedly enhanced by preloading the worms with glucose and by removing Na+ from the incubation medium. Since glucose and Na+ influxes are coupled, glucose leakage stimulated the influx of 22Na+. However, this 22Na+ influx was balanced by a simultaneous increased 22Na+ efflux. The presence of phlorizin inhibited both unidirectional fluxes of 22Na+ indicating that efflux of 22Na+ occurred by countertransport; countertransport of [14C] glucose appeared to be negligible. A model has been proposed in which the transport of glucose and compensating transfers of Na+ across the membrane occur via the same carrier.


Freeze-fracture electron microscopy of preexisting and nascent cell membrane in cleaving eggs of Xenopus laevis. During cell division in the Xenopus egg (diameter 1.25 mm) new cell membrane is formed in the furrow region (rate of growth approx 4-10(4) mum2/min). Freeze-fracture electron microscopy has produced the following data. Preexisting plasma membrane faces show a reversed polarity with respect to particle distribution, i.e. more particles are attached to the E-face (density 1600-2200 particles/mum2) than to the P-face (300 particles/mum2). A frequency histogram of 2331 measured intramembranous particles does not show a continuous range of sizes. The following sizes were very obvious: 95 A (12%), 125 A (30%) and 180 A (6%). At the tips of surface protrusions both the E- and the P- face are particle-free. Nascent cell membrane fracture faces are more difficult to obtain. The particle density is low (E-face 300-500 particles/mum2). Lowering the ambient temperature to 5 degrees C for approx. 5 mins does not change the normal particle pattern, but it improves the output in nascent membrane fracture faces. The fact that in the Xenopus egg preexisting and nascent membrane regions are continuous but nevertheless maintain their highly different particle densities is noteworthy. The freeze-fracture data are discussed in relation to, among other things, the known values of the specific resistances of these membrane regions.


Transport and metabolism of adenosine in human blood platelets. The uptake and metabolism of [14C]- or E[3H] adenosine have been studied in suspensions of washed platelets and in platelet rich plasma. The appearance of radioactivity in the platelets and the formation of radioactive adenosine metabolites have been used to determine the uptake. Adenosine is transported into human blood platelets by two different systems: a low Km system (9.8 muM) which is competitively inhibited by papaverine, and a high Km system (9.4 mM) which is competitively inhibited by adenine. Adenosine transported via the low Km system is probably directly incorporated into adenine nucleotides, while adenosine transported through the high Km system arrives unchanged inside the platelet and is then converted into inosine and hypoxanthine or incorporated into adenine nucleotides.


In vivo modification of plant membrane phospholipid composition. Tomato seedlings treated with ethanolamine showed altered phospholipid composition. The changes included altered acyl chain composition as well as changes in the relative amounts of the phospholipid classes. Specifically, there was an increase in phosphatidylethanolamine and phosphatidylserine with a concomitant decrease in phosphatidylcholine and no overall increase in phospholipids. Treatment with ethanolamine increased the relative amount of C18 acyl chains (especially 18 : 2) in phosphatidylethanolamine and phosphatidylcholine at the expense of 16 : 0 and 16 : 1. Acyl composition of other phospholipid classes were unchanged. Labeled ethanolamine was incorporated mostly into phosphatidylethanolamine and phosphatidylcholine. Ethanolamine-stimulated incorporation of labeled oleate was entirely into acyl chains and appeared only as 18 : 1 and 18 : 2. There was greater incorporation, but less conversion of 18 : 1 to 18 : 2 with choline. Stearate was incorporated but desaturated.


Ionic permeability of epithelial tissues. The overall permeability of epithelial tissues to solutes is generally determined by analyzing net or unidirectional transepithelial fluxes in response to transepithelial differences of concentration and/or electrical potential using relations that describe diffusional movements across a single membrane. If the solute is uncharged and diffusional movements are transcellular, the overall transepithelial permeability coefficient is determined by the permeabilities of the two limiting cell membranes combinded in series. However, if the solute is charged and the pathway for transepithelial movement involves diffusional flows across at least two membranes arranged in series (i.e. transcellular transport), the value of the overall transepithelial permeability coefficient determined using relations that describe ionic diffusion across a single membrane is not an accurate measure of the permeabilities of the two limiting membranes combined in series. Further, if ionic diffusion is transcellular, permeability coefficients determined from studies of transepithelial fluxes are not only quantitatively incorrect but can also result in grossly erroneous interpretations of changes in transepithelial permeabilities and faulty inferences regarding the route of transepithelial ionic diffusion.


The effect of temperature on the galactosyl- and sialyltransferases and on the ultrastructure of Golgi membranes. The effect of temperature on the activity of galactosyl- and sialyltransferases of rat liver Golgi membranes and the galactosyltransferase of serum has been studied. Arrhenius plots for three enzymes were different. Sharp breaks in the curves, indicative of phase transitions were observed for sialyltransferase (28 degree C) of Golgi and galactosyltransferase (34 degree C) of serum but not for galactosyltransferase of Golgi. The activation energy was greater above the break (above 28 degree C) than below for sialyltransferase of Golgi; The activation energy was lower (above 34 degree C) for galactosyltransferase of serum than below. Electron microscopic freeze replicas showed a patchy distribution of particles which increased as the temperature was raised accompanied by smooth areas. This was interpreted as representing lateral phase separation of the membrane components.


Transport of 2-deoxy-D-galactose in Saccharomyces fragilis. 2-Deoxy-D-galactose (dGal) transport in Saccharomyces fragilis is characterized by energy requirement and accumulation of the free sugar against a concentration gradient, indicating active transport. Besides free sugar dGal-1-phosphate, UDP-dGal and a trehalose-like derivative were found inside the cells. The accumulation of the phosphorylated derivatives was balanced by a concomitant decrease of ATP, orthophosphate and polyphosphates. With pulse labeling experiments it could be shown that the free sugar is transported into the cells. This conclusion was supported by several other experimental results, e.g. the lack of correlation between the sugar transport parameters and the dGal phosphorylation capacity, and the countertransport of free dGal evoked by galactose in the medium. The typical differences between this active transport mechanism and the transport-associated phosphorylation system, described previously, are discussed.


D-glucose uptake by a rat liver plasma membrane preparation. 1. Plasma membranes isolated from rat liver according to a modification of the method of Neville, D.M. ((1960) J. Biophys. Biochem. Cytol. 8, 413-422) were used as model to test current hypotheses on the mode of glucose uptake into the liver cell. Glucose uptake studies were performed by a filtration technique using labeled glucose analogues. 2. D-glucose Uptake by rat liver plasma membranes is characterized by features of simple diffusion, i.e. linearity of uptake, lack of stereospecificity, and by facilitated diffusion, i.e. temperature dependence, counterflow phenomenon and inhibition by phloretin. These findings confirm earlier studies on liver slices and perfused liver. 3. Binding studies on sonicated membranes provide evidence for a specific binding site or protein for D-glucose at the plasma membrane by isolating Tris-soluble membrane proteins which reveal a higher binding capacity than the unsonicated membrane. 4. These findings are interpreted as showing the presence of a "carrier" mediated transport system for D-glucose superimposed by free diffusion due to artificial disruption of the plasma membranes.


Studies on the mechanism of phosphorylation and transport of beta-galactosides by the lactose phosphotransferase system of Staphylococcus aureus. Kinetic investigations using tosyl galactosides as reversible dead-end inhibitors. Tosyl galactosides, previously shown to be potent reversible dead-end inhibitors of the membrane-bound Enzyme IIlac of the lactose phosphotransferase system of Staphylococcus aureus, were used for an investigation of the kinetic mechanism of the sugar phosphorylation/transport reaction catalyzed by this enzyme: phospho-Factor IIIlac&sugar Enzyme IIlac lead to Factor IIIlac&sugar phosphate. Inhibition of Enzyme IIlac was studied in three different systems. Washed membranes, and washed membranes in the presence of 0.1% Triton X-100 were used for phosphorylation experiments, and whole cells were used for transport studies. When washed membranes were used to supply Enzyme IIlac, inhibition of phosphorylation by tosyl galactoside was linear non-competitive against both the sugar and phospho-Factor IIIlac substrates, with an apparent Ki of about 0.5 mM. This Ki decreased with increasing Factor IIIlac concentration. In the presence of 0.1% Triton X-100, the phosphorylation reaction was stimulated; under these conditions the inhibition became strictly competitive against sugar, and completely uncompetitive against phospho-Factor IIIlac. Apparently washed membranes can catalyze phosphorylation both via a reaction sequence in which sugar binds first and via one in which phospho-Factor IIIlac binds first, but in the presence of 0.1% Triton the reaction does not occur by the former sequence. The inability of bound phospho-Factor IIIlac to hinder the binding of tosyl galactosides suggests that the initial binding sites of the two substrates of Enzyme IIlac are separated by at least the distance of the tosyl moiety. Radioactive methyl 6-O-(p-toluenesulfonyl) beta-galactoside was not converted into a phosphorylated product in the reaction mixtures, i.e. it is a true dead-end inhibitor. Inhibition of beta-galactoside transport into whole cells by tosyl galactosides was competitive, with an apparent Ki of 5-10 mM, an order of magnitude higher than the Ki for inhibition of phosphorylation by membrane preparations. This result suggest that a significant level of unphosphorylated phospho-Factor IIIlac is present inside the cells, or that cellular levels of this compound are considerably lower than those used for in vitro sugar phosphorylation assays. Radioactive tosyl galactoside inhibitor was not transported into whole cells.


Computer-simulated urea reflection coefficients in human red cells. Relative volume curves for human red cells were generated with a computer program to simulate the experimental results obtained by rapidly mixing red cells and urea. Known values for the permeability variables were used in the Kedem and Katchalsky (Biochim. Biophys. Acta (1958) 27, 229-246) solute and solvent flux equations. The different values used for the reflection coefficient, o, were 0.62, 0.75 and 1.0. The theoretical computer curves with o = 0.75 compared closely with the experimental stopped-flow curves.


Extracellular ascorbic acid in lung.

Extracellular ascorbic acid in lung. Fifty percent of the ascorbic acid content of sliced rat lung was released from the tissue to the media within a few minutes by either washing or incubating the slices with Krebs-phosphate solution. Measurement of the lactate dehydrogenase and potassium content of the medium after incubating lung slices for 5 min showed that about 20% of the cells were damaged by slicing. Sephadex chromatography of tissue extracts prepared from washed lung slices showed that none of the ascorbic acid in these slices were bound to protein. Also, metabolic poisons were shown to deplete the ascorbic acid content of washed lung slices. Approx. 57% of the lung ascorbic acid of guinea pigs that had been supplemented with ascorbic acid and 78% of the lung ascorbic acid of ascorbic acid-deficient guinea pigs were found in the medium when lung slices from these animals were incubated with Krebs-phosphate solution. These results were taken to indicate the presence of an extracellular pool of ascorbic acid in lung which is maintained even during scurvy.


Nitrogenase activity and respiration of cultures of Rhizobium spp. with special reference to concentrations of dissolved oxygen. Studies of nitrogenase in cultures of the cowpea rhizobia (Rhizobium spp.) strains 32H1 and CB756 are reported. Preliminary experiments established that, even when agar cultures were grown in air, suspensions of bacteria prepared anaerobically from them were most active at low concentrations of free dissolved O2. Consequently, assays for activity used low concentrations of O2, stabilized by adding the nodule pigment leghaemoglobin. In continuous, glutamine-limited cultures of 32H1, nitrogenase activity appeared only when the concentration of dissolved O2 in the cultures approached 1 muM. Lowering the glutamine concentration in the medium supplied to the culture from 2 to 1 mM halved the cell yield and nitrogenase activity was also diminished. Omitting succinate from the medium caused the concentration of dissolved O2 to rise and nitrogenase activity was lost. Upon restoration of the succinate supply, the O2 concentration immediately fell and nitrogenase was restored. The activity doubled in about 8 h, whereas the doubling time of this culture was 14 h. Sonic extracts of 32H1 cells from continuous cultures with active nitrogenase contained components reacting with antiserum against nitrogenase Mo-Fe protein from soybean bacteroids. Continuous cultures grown at higher O2 concentration, with only a trace of active nitrogenase, contained less of these antigens and they were not detected in highly aerobic cultures. Nitrogenase activity of a continuous culture was repressed by NH+4; the apparent half-life was about 90 min. Cells of 32H1 from a continuous culture growing at between 30 and 100 muM dissolved O2 possessed a protective mechanism which permitted respiration to increase following exposure to a rapid increase in O2 concentration from low levels (O2 shock). This effect disappeared as the O2 concentration for growth was reduced towards 1 muM.


Wheat germ agglutinin. Evidence for a genetic basis of multiple forms. Germ from hexaploid wheat (Triticum aestivum L.) contained three forms of agglutinin separable by ion-exchange column chromatography at pH 3.8, while germ from tetraploid wheat (Triticum turgidum L. (durum group)) contained only two such forms. The different number of forms, not due to protein modification occurring during the purification process, was demonstrable in commercial germ and in bran fractions containing germ from single wheat varieties. This evidence for a genetic basis of lectin multiple forms in wheat indicates the advisability of using genetically identified plant varieties in lectin research.


The mycelial cell wall of Penicillium charlesii G. Smith NRRL 1887. Mycelial cell wall preparations of Penicillium charlesii G. Smith have been shown to have a total neutral carbohydrate content of 69%. Glucosamine which comprises 12% of the wall is present principally as chitin. Lipids comprise 37.5% of the wall while polypeptides comprise 19.5%. No galactofuranose units are present in the wall of this organism which is known to produce an extracellular beta-1,5-galactofuranose containing polymer.


Quantitative microanalysis of cell walls of Saprolegnia diclina Humphrey and Tremella mesenterica Fries. Cell walls of the fungi Saprolegnia declina Humphrey and Tremella mesenterica Fries were analyzed quantitatively. Particular attention was paid to the hydrolysis and analysis of neutral sugars, amino sugars and amino acids. These components, together with total lipids, total uronic acids and the ashed residue, accounted for more than 90% by weight of the original dry cell wall preparation. There were substantial losses of amino acids during hydrolysis; however, analytical recovery approached 100% when total protein was calculated from the total nitrogen analysis. The analytical procedures were reproducible (+/- 3% for amino acids and amino sugars, and +/- 5-10% for other components) when applied to individual cell wall preparations. However, even under carefully standardized conditions, different cell wall preparations from the same species showed variable composition. Glucose was the predominant neutral sugar in the cell wall polymers of both species. The amino acid compositions were remarkable in that neither species contained detectable levels of cyst(e)ine. Hydroxyproline was detected in both species. The report from Tremella mesenterica is the first for this amino acid from the cell wall of a Basidiomycete.


The dissociation of insulin from human insulin antibodies. The dissociation of insulin from human insulin antibodies has been investigated using a technique that is rapid and does not require addition of excess unlabelled insulin. A slow (k1 = 2-10(-3) min-1) and a fast k2 = 4-10(-2) min-1) dissociating antibody component were identified in all studies. These have been shown to correspond, respectively, to the high and low affinity antibody components of equilibrium binding studies. The range of k1 and k2 values and their response to temperature change is small. Insulin resistance and stability of diabetes are not related to properties of antibody dissociation. Dissociation is faster in the presence of high (6-850 nM) insulin concentration due to increased binding to the fast dissociating component without change in the dissociation rate constants. When incubation time is increased beyond achievement of maximal binding there is a time-depent rise in binding to the slow dissociating component, with a concomitant fall in k1. The traditional concept that equilibrium is established at maximum binding requires further examination.


Glucocorticoid binding in the chicken liver cytosol. Characterization of five macromolecular binding components. The heterogeneity of the glucocorticoid binding in the chicken liver cytosol, previously suggested by the results obtained with crude preparations, was confirmed using different techniques such as stepwise ammonium sulfate precipitation, hydroxyapatite chromatography and gel filtration on Sephadex G-200. The latter method permitted the separation of five glucocorticoid binding macromolecules respectively named binders S1, S2, S3, S4 and S5, according to their decreasing apparent molecular size upon gel filtration. Apparent molecular weight, binding affinity, capacity and specificity of these five moieties were examined. In addition, S-aryl-transferase activity using glutathione as co-substrate was studied and found to coincide mostly with the fractions containing binder S4, which might represent an avian liver ligandin.


Regulation of the pyruvate dehydrogenase activity in the isolated perfused heart of guinea-pigs. The activity and the interconversion of the between the pyruvate after pyruvate should read: utilization in the perfused hearts and the pyruvate dehydrogenease complex has been measured in the isolated perfused working hearts of guinea-pigs. 1. The pyruvate dehydrogenase complex is transferred into the active form by high work, in anoxia, with 2,4-dinitrophenol and by perfusion without substrate. The rate of interconversion is faster in the perfused heart than in the homogenate. 2. The active form of the pyruvate dehydrogenase complex limits the pyruvate oxidation. There is a close correlation between the pyruvate utilization in the perfused hearts and the pyruvate dehydrogenase of the active form in the homogenates of the same hearts. 3. The "adenylate energy charge" of the cells is considered as the main regulating factor of the interconversion of the pyruvate dehydrogenase complex as seen in experiments with anoxia, dinitrophenol and high work. The inactivation of the pyruvate dehydrogenase complex by acetyl CoA can be overcome by decreasing ATP/ADP ratios.


Formation of mannosyl-lipids by an ectomannosyltransferase in suspensions of BALB/c fibroblasts. A mannosyltransferase has been detected in suspensions of BALB/c fibroblasts incubated with GDP-[14C] mannose. The experimental evidence indicates the cell surface as the most likely site for the enzyme. The transferase synthesizes both glycolipids and glycoproteins. The lipid compounds have properties suggestive of lipid-linked mono- and oligosaccharides which can function as intermediates in glycoprotein synthesis. The formation of these compounds by a cell surface enzyme suggests that lipid-linked intermediates may play an important role in the glycosylation of membrane components.


Acetylcholinesterase from the house-fly head. Molecular properties of soluble forms. 1. Polyacrylamide gel electrophoresis in Tris/glycine buffer (pH 8.3) revealed five forms of acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) in the 100 000 X g, 1-h supernatants of aqueous fly-head extracts from the DDT/S strain. Five other housefly strains (CSMA, Bayer 21/199, Cradson/P, Malathion/R and DDT/R)were shown qualitatively to have the same soluble forms of the enzyme. 2. Plots of the electrophoretic mobility versus polyacrylamide concentration indicated that the multiple forms constituted a size isomer family. From the retardation coefficients derived from these plots, molecular weight estimates were obtained; these suggested that the smallest active component was a form of approx. 80 000 daltons. The higher aggregates, however, did not appear as simple oligomers of this component. 3. Density gradient sedimentation supported the electrophoretic findings. The smallest active component, with a sedimentation coefficient of 5.3 S, was confirmed as a molecular species of acetylcholinesterase that has not previously been obtained from house-flies; higher aggregates gave sedimentation coefficients of 7.4, 7.8. 8.1, and 11.8 S. 4. Gel-filtration on calibrated Sephadex G-150 columns provided further evidence that the smallest active component was a form of about 80 000 daltons. 5. Autolysis converted much of the particulate enzyme and all of the soluble forms into a species of approx. 160 000 daltons indistinguishable from the native 7.4-S form. Both the autolysed enzyme and the native 7.4-S form were susceptible to cleavage by disulphide reducing agents, and released catalytically active subunits that corresponded to the 5.3-S form of 80 000 daltons. The data were compatible with a monomer-dimer relationship between the 5.3-S and 7.4-S forms. 6. The possibility is suggested that a form of molecular weight approx. 80 000 constitutes the "fundamental unit" of insect cholinesterase.


Acetylcholinesterase of the house-fly head. Affinity purification and subunit composition. 1. Acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) of house-fly head tissue was solubilized as a 7.4-S form by autolysis for 80-100 h at 25 degrees C and pH 8.0. 2. The autolysed enzyme was purified by affinity chromatography, firstly on Con-A-Sepharose and subsequently on m-trimethylammoniumaniline-Affi-Gel 202. This sequence permitted overall purification yields of approx. 50% of the solubilized enzyme. 3. The 7.4-S purified enzyme was essentially homogeneous on polyacrylamide gel electrophoresis, and its specific activity coincided with the highest previously reported for fly-head acetylcholinesterase. 4. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate and beta-mercaptoethanol revealed two major polypeptide components of molecular weight 82 000 and 59 000. Each of these polypeptides contained diisopropylphosphofluoridate-binding sites, as shown with [3H] diisopropylphosphofluoridate. 5. The results suggest a strong structural similarity between fly-head acetylcholinesterase and the purified electric eel enzyme.


Purification of urokinase by affinity chromatography. Commercially available urokinase (EC 3.4.99.26), though highly active, is still contaminated with unrelated proteins and degradation fragments of urokinase. Further purification of a urokinase preparation by chromatography on benzamidine-Sepharose is described. The final preparation consisted of two molecular forms of urokinase with molecular weights of respectively 31 000 and 54 000. The 54 000-dalton urokinase appears to be composed of two protein chains, one of which is the 31 000-dalton urokinase. A monospecific antiserum against urokinase was raised.


Preparation and characterization of two isozymes of choline acetyltransferase from squid head ganglia. Two isozymes of choline acetyltransferase (Acetyl-CoA:choline O-acetyltransferase, EC 2.3.1.6) have been isolated and purified from squid head ganglia. Each isozyme contains multiple isoelectric forms with isoelectric points ranging from pH 5.0 to 6.2. The isozymes differ in their affinities for cellulose phosphate on column chromatography, as well as in their heat stabilities and in their capacities to be activated by salt. Both isozymes are stabilized by sucrose and by sulfhydryl-protecting reagents such as mercaptoethanol and dithiothreitol.


A study of phosphoglycerate kinase in human erythrocytes. II. Kinetic properties. Kinetic studies on phosphoglycerate kinase (EC 2.7.2.3) were performed in the forward reaction leading from 1,3-diphosphoglycerate to 3-phosphoglycerate. Substrate activation was observed at fixed levels of ADP or Mg2+ and varying concentrations of 1,3-diphosphoglycerate. A biphasic curve was obtained in both linear and double reciprocal plots demonstrating two Km values (Km1 1.9 - 10(-6) and Km2 9.8 - 10(6) M). Michaelis-Menten-type kinetics were observed in both the linear and double reciprocal plots at fixed levels of 1,3-diphosphoglycerate of ADP and varying concentrations of Mg2+. Apparent Michaelis-Menten kinetics were observed in linear plots when conditions of fixed concentrations of 1,3-diphosphoglycerate or Mg2+ were maintained with varying concentrations of ADP. However, the double-reciprocal plots demonstrated biphasic curves with two Km values (Km1 1.7 - 10(-5) and Km2 1.0 - 10(-4)M). Apparent negative cooperativity was observed with respect to 1,3-diphosphoglycerate and ADP. Phosphoglycerate kinase activity was found to be inhibited by AMP and 2,3-diphosphoglycerate. Substrate activation by 1,3-diphosphoglycerate was maintained in the presence of AMP or 2,3-diphosphoglycerate but at a reduced level of enzyme activity. AMP was found to inhibit enzyme activity non-competitively with respect to 1,3-diphosphoglycerate, ADP and Mg2+. 2,3-Diphosphoglycerate inhibits phosphoglycerate kinase activity with respect to 1,3-diphosphoglycerate, ADP and Mg2+. 2,3-Diphosphoglycerate inhibits phosphoglycerate kinase activity non-competitively with respect of 1,3-diphosphoglycerate.


The oxidation of dithiothreitol by peroxidases and oxygen. Horseradish peroxidase (1.11.1.7), lactoperoxidase (1.11.1.7), and the fragment of cytochrome c known as microperoxidase have been shown to catalyze the oxidation of reduced dithiothreitol in an oxygen-consuming reaction. Evidence for horseradish peroxidase intermediates compound III and compound II has been observed, although ferroperoxidase was not identified during the course of the reaction. The stoichiometry has been extablished as 1 : 1 for oxygen consumed to dithiothreitol oxidized. Cysteine and glutathione have also been shown to be substrates for horseradish peroxidase oxidase reaction.


Purification and some properties of human erythrocyte hexokinase. 1. Human erythrocyte hexokinase (ADP:D-hexose 6-phosphotransferase, EC 2.7.1.1) was purified 50 000--100 000-fold with a final specific activity of about 25--50 units/mg protein using gel-filtration, ion-exchange chromatography and affinity chromagraphy. 2. After isoelectrofocusing ofthe preparation one major protein band could be detected besides a minor band. THe isoelectric point of the major protein band was found to be 4.7. 3. After purification the enzyme could be stabilized in a medium containing inorganic phosphate, glucose, glycerol and mercaptoethanol. 4. The molecular weight was determined by gel-filtration and was found to be 132 000+/-8000. 5. The enzyme shows a broad pH optimum ranging from 7.0 to 8.4. 6. The kinetic behavior of the purified enzyme at 37 degrees C was somewhat different from the normal Michaelis-Menten kinetics due to its instability. The affinity constants were 0.048--0.080 mM for glucose and 0.57--1.0 mM for Mg-ATP. 7. The enzyme was specific for Mg- ATP as the nucleotide substrate. Mg-UTP, Mg-ITP,Mg-GTP and Mg-CTP were not converted to corresponding diphosphates. Several hexoses could be phosphorylated by the enzyme. Mannose could be phosphorylated at the same rate as glucose, although the affinity for the enzyme was lower (5m=0.60mM). Much lower rates and lower affinities were found with 2-deoxy-D-glucose (5m=1.0mM), D(+)-glucosamine (5m=4.5 mM) and fructose (5m=10 mM). N-acetyl-D-glucosamine , galactose andsorbose were not phosphorylated at all.


Non-identical behaviour of the subunits of rabbit muscule creatine kinase. 1. The dimeric enzyme creatine kinase (ATP: creatine N-phospotransferase, EC 2.7.3.2) from rabbit muscle was reacted with three separate reagents, each of which specifically modifies one thiol group per subunit. 2. The reactions of the enzyme with these reagents (4-chloro-7-nitrobenzofurazan, 5,5'-dithiobis-(2-nitrobenzoic acid) and iodoacetate) all behave as normal second-order processes. This indicates that the thiol groups on the two subunits of the enzyme react at the same rate as each other in all three cases. 3. The effects of various ligands (Mg2+, ADP and creatine, and combinations of these) on the kinetics of the reactions were studied. In all cases the reactions behave as normal second-order processes. 4. In the presence of the ligand combination Mg2+ plus ADP plus creatine plus nitrate, which has been postulated to form a "transition state analogue" complex with the enzyme, the reactions of the thiol group show considerable deviation from second-order kinetics. This indicates that the thiol groups on the two subunits react at different rates from each other. A similar effect is also noted in the presence of the combination ADP plus creatine plus nitrate. 5. The binding of ADP to the enzyme (studied by equilibrium dialysis) is hyperbolic in the absence of other ligands or in the presence of Mg2+ or Mg2+ plus creatine. The dissociation constant is similar in all three cases. 6. In the presence of creatine plus nitrate (with or without Mg2+) the bindings of ADP to the enzyme is tightened considerably and the binding plots indicate the presence of either negative interactions between the subunits or two distinct types of binding sites. 7. Possible causes for the observed non-identical behaviour of the two subunits of the enzyme are discussed.


Modification of chymotrypsin with the specific cyanate reagent N-acetyl-p-cyanato-l-phenylalanine ethyl ester. The specific cyanate reagentN-acetyl-p-cyanato-L-phenylalanine ethyl ester (compound 1) was synthesized in an attempt to selectively modify the binding pocket of chymotrypsin (EC 3.4.21.1) while leaving the catalytic residues un touched. The reagent reacts with chymotrypsin to yield chiefly an inactive derivative 3a, with the active site Ser-195 carbamylated and the alpha-amino group, with the reagent 1 yields a modified enzyme(compound 4) with an additional carbamyl group on Ser-195.Neither derivative 3a nor 4reacts with diisopropylfluorophosphate under conditions where chymotrypsinogen is modified, indicating that Ser-195 is altered. Both derivatives 3a and 4 are retained on a 4-phenylbutylamine affinity colomn demonstrating that the substrate binding pocket is intact in both derivatives. The results indicate the potential value of aryl cyanates as protein reagents for the selective modification of nucleophilic sites. However, it is apparent that reaction at unreactive residues in the binding pocket of chymotrypsin with cyanates or similar reagents will require blockage of the more nucleophilic catalytic residues.


Purification and kinetic study of glyoxalase-I from rat liver, erythrocytes, brain and kidney. Glyoxalase-I (S-lactoyl-glutathione methylglyoxal-lyase (isomerizing), EC 4.4.1.5) was purified from rat liver, erythrocytes, brain and kidney using two different purification procedures. The similarities of the purification profiles, electrophoretic mobilities and kinetics suggest that a single major form of the enzyme exists in these tissues. The highest purification (9300-fold) of the erythrocyte enzyme gave nearly homogeneous protein, molecular weight 50 000, specific activity 2410 mumol/min per mg. Kinetic studies of the rat glyoxalase-I-catalyzed disproportionation of the hemimercaptals of GSH and aromatic or aliphatic alpha-ketoaldehydes revealed broad substrate specificity with V and Km values quite insensitive to the nature of the alpha-ketoaldehydes. Use of deuterated analogs of the alpha-ketoaldhydes methylglyoxal and phenylglyoxal showed that the intramolecular hydride migration is the rate-determining step.


Mammalian adenylosuccinate synthetase. Nucleotide monophosphate substrates and inhibitors. Guanylate cyclase (GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2) activity was examined in preparations from normal rat liver and a series of Morris hepatomas...


The separation of pyruvate-ferredoxin oxidoreductase from Clostridium pasteurianum into two enzymes catalyzing different reactions. The ferredoxin requiring cleavage of pyruvate to acetyl-CoA and CO2 is catalyzed by pyruvate ferredoxin oxidoreductase (pyruvate:ferredoxin oxidoreductase (CoA-acetylating):, EC 1.2.7.1). The same enzyme is thought to catalyze the reversal of this reaction, i.e. the synthesis of pyruvate from acetyl-CoA and CO2 in the presence of reduced ferredoxin. Evidence is presented that the forward and reverse reactions are catalyzed not by one, but by two proteins that are clearly separable by Sephadex G-200 gel filtration.


Magnetic circular dichroism of heme-isocyanide complex in aqueous media. The nature of the ethyl isocyanide complexes of ferri- and ferro-heme was investigated in terms of the magnetic circular dichroism (MCD) spectroscopy. The complex formation of heme with ethyl isocyanide was critically dependent on the solvent system, thus, on structures of heme before addition of the ligand. The MCD spectra of the heme ethyl isocyanide complexes revealed that: (1) The addition of ethyl isocyanide to both ferri- and ferro-heme in an aqueous solution gave respective high-spin heme complexes with no more than one ethyl isocyanide molecule per one heme. (2) The ferro-heme with ethyl isocyanide in an aqueous ethanol solution formed a typical monomeric low spin ferro-hemochrome with two ligands (ethyl isocyanide) on both sides of the heme plane. (3) In the presence of excess ethyl isocyanide in an aqueous ethanol solution the ferri-heme gave an MCD spectrum similar to that for the monomeric low spin ferro-hemochrone. Therefore, ethyl isocyanide was believed to reduce the ferri-heme in an aqueous ethanol solution. (4) The ferro-heme ethyl isocyanide complex in an aqueous solution offered a Faraday B term around 450 nm instead of an apparent Faraday A term observed for the ethyl isocyanide complex of the cytochrome P-450. These results are not consistent with the view that the ferro-heme ethyl isocyanide complex in an aqueous solution can be a heme chromophore model for the cytochrome P-450 ethyl isocyanide complex.


Effects of dietary molybdenum on the metabolism of copper and molybdenum in young cattle. On separate occasions young cattle were injected intrarumenally with 99Mo or 64Cu or intravenously with 64Cu. The metabolism of the isotopes were compared to evaluate metabolic changes associated with molybdenum-induced copper deficiency. Molybdenum-99 metabolism was the same in both controls and experimental subjects. Marked differences in plasma kinetics were observed following intrarumenal 64Cu injection, with experimental animals displaying earlier plasma appearance and maintaining higher plasma levels than did controls. Similarly, higher plasma levels, more rapid plasma reappearance and greater fecal excretion were observed following intravenous injection of 64Cu in the experimental animals than in controls. A mechanism involving abnormal ceruloplasmin synthesis associated with the molybdenum-copper interaction is proposed.


Aggregation of acridine orange: crystal structure of acridine orange tetrachlorozincate 2C17H19N3-2HCl-ZnCl2-CH3COOH. The crystal structure of the biological stain, "acridine orange," has been determined. This compound, when crystallized from ethanol, is shown to be a zinc chloride double salt of acridine orange, containing, in addition, acetic acid of crystallization. These additional components are residuals from the method of preparation of acridine orange. This complex, 2 acridine orange-2HCl-ZnCl2-CH3COOH, (2C17H19N3-2HCl-ZnCl2-CH3COOH) crystallizes in the monoclinic space group P21, a = 9.965 (2), b = 21.507 (6), c = 9.645 (2) A, beta = 113.98 degrees (2), V = 1888.7 (8) A3, FW = 800.0, Z = 2, DX = 1.41 g-cm-3, Dobs = 1.43 (9) g-cm-3. Three-dimensional diffraction data were collected with CuKalpha radiation, and the structure refined to R = 0.065 for 1885 observed reflections. In the crystal structure hydrogen bonds are formed, via the protonated nitrogen atom of the central rings of two acridine orange cations, to two chloride ions in a ZnCl42- tetrahedral grouping. These two acridine orange molecules are stacked in parallel planes, approximately 3.4 A apart, with the long axes of the ring systems inclined at 26.5 to each other. Thus an apparent dimerization of the acridine, orange is facilitated by the anions present, resulting in the complex studied. The two -N(CH3)2 groups of each acridine orange molecule are not protonated in this crystalline form. The mode of molecular packing found here may be relevant to models for the external stacking of acridine orange around a DNA molecule. The importance of removing any zinc salt from acridine orange preparations prior to aggregation studies is stressed.


X-ray photoelectron spectroscopy of copper(II), copper(I), and mixed valence systems. X-ray photoelectron spectroscopy using copper(II), copper(I) and the mixed valence Cu(II)/Cu(I) compounds was employed as a means  of studying electron transfer reactions in copper proteins. The X-ray photoelectron spectra of copper(II) compounds display characteristic satellites of both variable size and resolution. Some of these satellites could be assigned to specific ligand interactions. Unlike electron paramagnetic resonance spectroscopy, the X-ray photoelectron spectroscopic measurements of copper(I) compounds allowed the unequivocal assignment of this oxidation state. No satellites at all could be detected in the Cu(I) spectra. Furthermore, established mixed valence Cu(II)/Cu(I) complexes including Cu2SO3-CuSO3-2H2O and Cu4Cl5 (ethylenediamine)2 proved essentially a mixture of distinct portions of Cu(I) and Cu(II). This indicates that both oxidation states of copper survive in such complexes. In contrast, all Cu X-ray photoelectron signals of the more tentatively described mixed valence complexes Na2Cu3S3 and the mineral covellite, CuI4CuII2(S2)2S2, could be attributed exclusively to Cu(I). In view of the known binding of copper with sulfur in many copper proteins, it was of utmost importance to study the copper-sulfur interactions. We have demonstrated the absence of Cu(II) in CuS. This indicates strong metal-induced polarization of sulfur resulting in electron transfer to copper to yield Cu(I).


Paget's osteitis deformans: a carbonate-rich calcium alkali phosphate in the pre-malignant bone mineral and implications for lymphoma production by CO2. Comparison of powder diffraction patterns of unheated normal bone mineral with the damaged by Paget's Osteitis Deformans showed that due to a difference in the spacing of one weak inner reflection the possibility exists that carbonate-containing alpha-calcium sodium orthophosphate may be a minor constituent of the osteitic bone mineral. The chemical and physical properties of this substance make it a likely minor constituent of bone salt. The presence of this calcium alkali phosphate in the osteitic mineral was tentatively proposed after demonstrating the similarity of the diffraction patterns of synthetic mixtures of this substance with tricalcium phosphate hydrate to those of the osteitic bone salt.


Magnetic circular dichroism of protoporphyrin derivatives in the ultraviolet region. The MCD spectra of ferri- and ferro-heme in high and low spin states and zinc porphyrin for reference have been measured in the ultraviolet region. Zinc porphyrin offered an A term at 325 nm attributable to the N transition. A ferrous low-spin complex with CO or CN exhibited MCD spectra composed of B terms around 325 nm. This striking difference from that of zinc porphyrin in the ultraviolet region is noted from the fact that their Soret and visible MCD's gave A terms similar to those of zinc porphyrin. A ferric complex with high spin had MCD spectrum different from that of low spin, the former showing a negative C term at 287 nm and a B term at 339 nm, but the latter positive C terms at 275 nm and 320 nm. Some of these Faraday parameters were ascribed to charge-transfer transitions.


Reactions between nucleosides and platinum dimethylsulfoxide complexes. Nucleosides (Nuc) react with cis-[Pt(DMSO)2Cl2] in dimethysulfoxide (DMSO) solution, to produce trans-[Pt(DMSO)(Nuc)Cl2], which then isomerize to cis-[Pt(DMSO)(Nuc)Cl2]. They also displace chlorine from K[Pt(DMSO)Cl3] in water to form trans-[Pt(DMSO)(Nuc)Cl2]. K[Pt(inosine)Cl3] and DMSO give in water cis-[Pt(DMSO)(Inosine)Cl2]. The configuration assignment of the compounds was based on chemical reactions, NMR techniques, analytical results, conductivity measurements and comparison with pyridine analogues. Chemical shifts in NMR and coupling constants of the aromatic protons of the nucleosides with 195Pt were used for the assignment of the binding sites of the nucleosides.


Synthesis of the monomer and dimer peptide fragments of the Micrococcus lysodeikticus cell wall peptidoglycan. Three branched peptides, namely pentapeptide A and the two decapeptides B and C corresponding respectively to the monomer and the two dimer peptide fragments of the M. lysodeikticus cell wall peptidoglycan, were synthetized by adequate methods of peptide synthesis in homogeneous phase. The synthetized peptides showed the same electrophoretic and chromatographic behavior as the natural peptide fragments. Only decapeptide B was hydrolysed by the Myxobacter AL-1 protease by cleavage of the D-Ala-L-Ala bond and inversely, only decapeptide C was digested by the Streptomyces albus ML endopeptidase by cleavage of the D-Ala-epsilon-Lys linkage. In both enzymatic hydrolyses the pentapeptide monomer was formed.


Redox regulation of cytochrome P450cam mixed function oxidation by putidaredoxin and camphor ligation. A methylene group of camphor is hydroxylated by a P450 monoxygenase system containing in addition to the cytochrome and flavoprotein dehydrogenase a two-iron, two-sulfur redox protein termed putidaredoxin. The regulatory control of electron transport equilibrium occurs via ligation of substrate and redoxin to the cytochrome. The component interactions and electron flow are represented clearly and concisely by a free energy state diagram of the coupled binding and redox transfer reactions.


Varied effects of polyadenylic-polyuridylic acid on immune responses. The effect of the double-stranded synthetic polynucleotide, poly(A).poly(U), on the immune response of inbred mouse strains to multichain synthetic polypeptides was studied. Poly(A).poly(U) did not affect immune responses controlled by H-2 linked genes. Thus, when either (T,G)-A- -L or (Phe,G)-A--L were injected into high or low responder mice followed by administration of poly(A).poly(U) 24 h after immunization, no increase in the antibody titers was observed. In contrast, poly(A).poly(U) increased significantly the response to polyproline, which is controlled by a non H-2 linked gene, in low responder mice. However, the polyribonucleotide had no effect on the antibody titers of the SJL mice, the high responders to multichain polyproline. When poly(A).poly(U) was injected into DBA/1 mice following immunization with (Phe,G)-Pro- -L, the polynucleotide enhanced the low response to the Pro- -l region at the expense of the anti (Phe,G) response which is normally high in this mouse strain. In this case poly(A).poly(U) caused an intramolecular antigenic competition. The general conclusion of this study is that the chemical nature of the antigenic determinant plays an important role in determining the type of influence exerted by poly(A).poly(U).


A study on the receptor for a mycobacteriophage : phage phlei. From Mycobacterium phlei, glycolipid fractions have been isolated which inactivate phage Phlei. On the basis of the characteristics of the inactivation (specificity, kinetics, requirement for Ca++) typical of the phage-host cell system, it was concluded that these fractions contain the receptor sites for phage Phlei ; this conclusion was supported by electron microscopic studies. All the active fractions contain four kinds of components : fatty acids, glycerol, sugars (D-lyxose, 6-0-methyl-D-glucose, and low amounts of glucose and mannose), and water-soluble acids. These acids are isolated by degradation of the receptor fractions as oxalic and pyruvic acids. Variations of the ratio oxalic acid/pyruvic acid according to the mode of degradation and the absence of the peak characteristic of the protons of a pyruvic acid residue in the NMR spectrum, suggest that these acids might arise from the splitting of oxaloacetic acid. A tentative structure of the receptor is proposed, in many monoglycerides are linked through keto-acid to a polysaccharide core.


Grafting of enzymes on collagen films using Woodward's reagent "K" and a water-soluble carbodiimide derivative. Two new methods of activation were developed to graft enzymes on collegen films. They involved chemical modifications of surface groups of collagen either by Woodward's reagent "K" or by EDC, a water-soluble derivative of carbodiimide. EDC was a better coupling agent and a detailed study was conducted with this agent. It could be used either in a global method of activation and coupling, or in a two-step procedure of activation of collagen, followed by spontaneous coupling of enzyme. All enzymes tested were successfully bound: malate dehydrogenase, lactate dehydrogenase, aspartate aminotransferase, urease, creatine kinase, hexokinase. The influence on the yield of grafted enzyme, of pretreatment of films, time and temperature of EDC activation, concentration of EDC and enzyme, protecting agents was studied. Stability of enzyme activity on storage was greatly increased after grafting. A co-grafted dual system creatine kinase/heoxkinase, was achieved which exhibited a good efficiency. A striking renaturing process at 0-4degreesC after thermal denaturation, was observed with hexokinase.


Pig prothrombin: purification and properties. A procedure for the preparation of highly purified pig prothrombin is described. Compared to the initial clotting activity of the starting plasma, this protein was purified 776 times with a final yield of 8 per cent. The purified zymogen showed a specific activity of 1,460 NH units/mg of protein , a molecular weight of 65,000 as determined by SDS-polyacrylamide disc gel electroesis, E 1.0 mg/ml 1.0 cm, 280 nm = 1.45 at pH 7.0 and the following amino acid composition: Asx 51, Thr 38, Glx 62, Pro 23, Gly44, Ala 25, Half-Cys 30, Val 35, Met 3, Ile 30, Leu 32, Tyr 19, Phe 22, Lys 36, His 8, Arg 28, and Trp 13, which accounts for a minimum molecular weight of 59,370 (carbohydrates not computed). Alanine was found as the only N-terminal residue. Carboxypeptidases A and B failed to release any C-terminal residue. By hydrazinolysis however 0.4 mole of serine was released per mole of prothrombin. The activation of crude and chromatographed pig prothrombin was investigated.


Binding of nucleotides ATP and ADP to sarcoplasmic reticulm: study by rate of dialysis. Binding of the nucleotides ATP and ADP by preparations of sarcoplasmic reticulum was investigated by the method of flow dialysis. For ATP, experimental data could not be analyzed directly in terms of binding since a significant though small amount of hydrolysis could be observed even in presence of EDTA. ADP binding could be analyzed and gave a dissociation constant of 10-20 muM at neutral pH, and a stoichiometry of 0.35 - 0.45 per mole ATPase. The possible significance of this stoichiometry is discussed. Similar experiments were performed after ethoxyformylation of sarcoplasmic reticulum which inhibits the Ca2+ dependnet ATPase activity. The results confirmed the inhibition of ATP hydrolysis and pointed to a considerably reduced affinity for nucleotides. The method based on the measurement  of dialysis rates is convenient, and accurate enough to detect the effects of chemical modification on sarcoplasmic reticulum membranes.


[Specificity of thermolysin action on dog myoglobin]. The proteolytic specificity of thermolysin has been studied by quantitative analysis of an enzymic digest of dog myoglobin. Results confirm main specificities of thermolysin towards Phenylalanine, Isoleucine, Leucine or Tyrosine bonds; the influence of neighbourhood was also determined and the conclusions are in a good agreement with the known structure of the active site of thermolysin.


[Chemistry of urinary mannosides excreted in mannosidosis]. Mannose-rich oligosaccharides have been isolated from urines of 5 patients with mannosidosis. Their compositon and structure were determined. Three of them have been previously described by Norden et al: alpha p-Manp-(1 leads to 3) beta-d-Manp-(1 leads to 4) d-GlcNAcp; alpha-p-Manp-(1 leads to 2), alpha-d-Manp-(1 leads to 3) beta-d-Manp-(1 leads to 4) d-GlcNAc and alpha-d-Manp-(1 leads to 2) alpha-d-Manp-(1 leads to 2) alpha-d-Manp-(1 leads to 3) beta-d-Manp-(1 leads to 4) d-GlcNAcp, but the four others are new entities: alpha-d-Manp-(1 leads to 3) (alpha-d-Manp-(1 leads to 2) alpha-d-Manp-(1 leads to 2) alpha-d-Manp-(1 leads to 6) beta-d-Manp-(1 leads to 4) GlcNAcp; alpha-d-Manp-(1 leads to 2) alpha-d-Manp-(1 leads to 3) (alpha-d-Manp-(1 leads to 2) alpha-d-Manp-(1 leads to 6) beta-d-Manp-(1 leads to 4) GlcNAcp; alpha-d-Manp-(1 leads to 2), alpha-d-Man-(1 leads to 3) (alpha-d-Manp-(1 leads to 6) beta-d-Manp-(1 leads to 4) GlcNAp and alpha-d-Manp-(1 leads to 2) alpha-d-Manp-(1 leads to 3) (alpha-d-Manp-(1 leads to 6) beta-d-Manp-(1 leads to 4) GlcNAcp. These structures are related to the glycans of "oligomannosidic type" present in numerous glycoproteins. All possess a N-acetylglucosamine residue in terminal reducing position and reinforce the hypothesis of Kobata et al. and Montreuil et al. that catabolism of glycans N-glucosidically linked to the protein moiety begins by the aciton of a beta-endo-N-acetylglucosaminidase.


Enzymatic properties of a new type of lysozyme isolated from Asterias rubens : comparison with the Nephthys hombergii (annelid) and hen lysozymes. The enzymatic properties of Asterias rubens and Nephthys hombergii lysozymes were compared with those of hen egg-white lysozyme. The results allowed to conclude that the Asterias rubens lysozyme was representative of a new lysozyme type. The Nephthys hombergii lysozyme however could be classified among the hen type enzymes with a tendency to exhibit under certain conditions some properties attributed to the goose type lysozymes.


Electrophoretic studies of non-histone proteins from pig lymph nodes. Purified chromatin from pig lymph nodes has been prepared and used for the isolation of chromatin proteins and non-histine proteins. Saccharose nuclei from this tissue have been used for the preparation of nuclear phenol-soluble phosphoproteins. The isolated proteins have been compared by analytical electrophoresis in polyacrylamide gel in sodium dodecyl sulfate. Most chromatin non-histone proteins and nuclear phosphoproteins of pig lymph nodes have a molecular weight about 40 000 daltons and show a significant degree of homology of molecular range from 40 000 to 90 000 daltons. The phosphoproteins have been also analyzed by isoelectric focusing in polyacrylamide gel over the pH range of 3-10. They are distributed throughout the pI range of 5.8-9.0. The chromatin non-histone proteins and phosphoproteins are of acidic nature and contain 0,97 per cent tryptophan.


Iron stimulation of ferritin synthesis. II.-Involvement of polysomal kc1 extracts in the mechanism of stimulation. The iron stimulation of ferritin synthesis is a translational phenomenon. Iron acts through a modification of the microsomal apparatus as shown previously. In the present study we have shown that the factor(s) involved in this stimulation are present in the KC1 extracts from polysomes of iron-treated rats.


[New syntheses of S-adenosylhomocysteine and S-adenosylmethionine analogs]. With the aim of studying analogues of S adenosyl homocysteine and S adenosyl methionine as potential inhibitors of methyl-transferases, we describe the syntheses of such analogues, in which either the amino-acid chain is replaced by various aliphatic radicals of the N 6 amino group of adenine is substituted.


Differences in cell surface proteins of drug resistant and sensitive cell lines. Lactoperoxidase catalyzed iodination of cell surface proteins has been used to compare theophylline and ouabain resistant variants with the drug sensitive parental cell lines. In cells showing contact inhibition a high molecular weight protein was observed to be preferentially labelled. In contrast this was not observed for non contact inhibited cells. These results support a previously suggested correlation between growth regulation and the exposure of this cell surface protein.


Effects of recurrent postnatal stresses on hypothalamic concentrations of norepinephrine. The effect of recurrent postnatal stress on the norepinephrine content of hypothalamus has been studied. Repeated exposure to hunger or to stimulation of the  lateral nucleus of the hypothalamus served as stress-producing process. Regardless of their nature, recurrent stresses increased the norepinephrine content of the hypothalamus. The differences between controls and stressed animals were statistically significant.


Prostaglandin biosynthesis in the human umbilical cord. The content of prostaglandins (PGs) in homogenates of arteries, connective tissue from human umbilical cords, and the capacity for biosynthesis of PGs in these tissues were studied. Lipid extracts were prepared and subjected to chromatography on silicic acid columns. PGs were eluted by ethyl acetate after elution with ethyl acetate-benzene (1:9) and determined by bioassay. For studies of the content of PGs, the cords were immediately frozen after delivery and homogenized in cold ethanol. Cords taken at vaginal deliveries contained amounts equivalent to approximately 700 ng prostaglandin E2/g dry weight and cords taken at caesarean sections about 450 ng/g. After homogenization and incubation of arteries in potassium phosphate buffer at 37 degrees C for 30 min the PG content increased from about 80 to 200 ng/g. Addition of arachidonic acid increased the biosynthesis fivefold. Indomethacin inhibited it. Most of the smooth muscle stimulating activity of the homogenated cords was due to prostaglandin E compounds, but smooth muscle stimulating material similar to prostaglandin F1alpha and F2alpha was also present. Prostaglandin E2 was conclusively identified by gas chromatography -- mass spectrometry. The findings are compatible with the view that local biosynthesis of PGs is very active in the human umbilical cord after delivery.


Dental and craniofacial development in the normal and growth-retarded human fetus. Craniofacial and dental development were examined in 32 human fetuses, 22--42 weeks of gestational age, nine of which were classified as light-for-dates (LFD). Statistically significant differences were detected between the LFD and the normal group with respect to upper and lower jaw size and cranial base length but not in the case of neurocranial length, orbital height or deciduous molar crown size. The structures which are least affected by intrauterine growth retardation are those whose growth rate is normally either low throughout the second half of gestation or falls substantially during the 10 weeks prior to term.


Topography of lesions in newborn and infant brains following cardiac arrest and resuscitation. Damage to brain and hemispheres. The brain lesions produced by temporary arrest of circulation in a newborn and an 11-month-old infant are described. In the newborn, two periods of arrest occurred, one on the fifth day after birth and the second a few day before death on the sixth week. The older infant suffered a single episode of cardiac arrest at the age of 11 months and survived 8 days. In both cases, postmortem examination revealed lesions in spinal cord, in brain stem, and in cerebral hemispheres. This distribution of damage is compared with the patterns of injury produced experimentally by episodes of partial (hypoxia) and total (anoxia) asphyxia in subhuman primates. The coexistence of hemispheral and brain stem nuclear patterns of pathology indicates that both hypoxia and anoxia had occurred in the present cases.


Relations between birth weight and learning ability in young rabbits. Performance on visual discrimination learning was studied in 41 rabbit pups mainly in relation to their birth weight, but also as functions of their neonatal motor development and weight gain between birth and training time. Partial placental vascular ligation was performed on some fetuses to increase the number of small-for-gestational-age pups. No relation was found between learning ability and either birth weight or rate of motor development. Low birth weight showed some association with slightly delayed motor development, but resulted in no lasting motor handicap. Between two subgroups of low birth weight pups, those who showed substantial weight gain by training time learned faster than those who remained relatively small.


The effects of starvation, glucose infusion, and normal feeding, on muscle protein synthesis and catabolism in the newborn guinea pig. We determined the effects of feeding, starvation, and glucose infusion after starvation in newborn guinea pigs. We determined the rate of 14C-leucine incorporation into skeletal muscle (KS) as a measure of muscle protein synthesis and the rate of excretion of 3-methylhistidine as a measure of muscle myofibrillar protein catabolism (Kc). Fed newborns, who were in positive nitrogen balance, had the highest Ks and lowest Kc, while starved newborns had the lowest Ks and highest Kc. Infusing glucose after starvation decreased net protein catabolism and Kc, but did not increase Ks. The magnitude of change of Kc in response to starvation and glucose infusion was much greater than Ks. Changes in catabolic rate may influence net muscle protein balance to a greater degree than changes in synthetic rate.


A transportable sleep recording system.

A transportable sleep recording system. A system for monitoring sleep, which can be used in th ward without unduly disturbing either the patient or the medical and nursing staff, is described. The system records the electroencephalogram, electro-oculogram, the basal skin resistance and the galvanic skin resistance. In addition, the delta waves of the EEG are automatically counted and a histogram of their frequency plotted. The system allows a whole night's sleep recording to be compressed into just over a metre of paper.


An active probe for electrocardiography. In order to improve the signal to noise ratio during the recording of electrocardiographic maps from moving patients, the effect of using active electrodes with built-in amplifiers was investigated. Two kinds of electrodes using different technologies were developed: a miniature printed circuit and a thin film circuit. The first method was found to be suitable for the fabrication of small quantities in a normally equipped electronics workshop while the second, requiring highly specialised equipment, is intended for industrial manufacture in large quantities.


Science, statistics, and paired comparisons. A prologue on science and statistics focuses attention on the role of statistics in science. Statisticians must be trained as scientists and to meet the needs of science. Those needs surely involve the formulation, modification and verification of stochastic models designed to represent natural phenomena. The method of paired comparisons provides a simple experimental technique but one with a literature rich in model development.


Composite sampling.

Composite sampling. In sampling certain types of materials, such as bags of fertilizer, or subsampling large quantities of water, as might be done in investigating the density of plankton in certain environmental situations, it is customary to composite the samples. That is, several samples are drawn, mixed into a composite sample, and a sample taken from the composite is then used to determine relevant characteristics of the population. In this paper the results of Brown and Fisher [1972] are extended to more complex situations (e.g., more than one variance component present).


Design of experiments to estimate heritability when observations are available on parents and offspring. The design of experiments to estimate heritability when data are available on both parents and offspring and the offspring data have a hierarchical structure is considered. Univariate maximum likelihood (ML) estimation is discussed, and extensions to the multivariate case are outlined. The efficiency of ML estimation is evaluated in cases where simple regression estimators are available. Optimum designs for ML estimation are given when various strategies of selecting and mating are followed. The variance of the heritability estimate can be approximately halved relative to designs in which no selection of parents is done.


Models allowing for age-dependent survival rates for band-return data. Seber [1970] and Robson and Youngs [1971] developed a statistical model for a tag-recapture experiment on an exploited animal population in which banded or tagged individuals are released into a population each year, and the bands from harvested individuals are returned by hunters or fishermen in following years. Consideration of certain biological and ecologicw models for an experimental situation where data are recorded separately for birds released as adults and as young-of-the-year. In particular, the effect of different survival rates of young birds, and the effect of migration on reporting rates, are taken into account in the models presented in this study. Maximum likelihood estimators of parameters are derived under the different models, and procedures are developed to discriminate between alternative models as well as provide non-discriminant goodness-of-fit tests.


Mathematical models for nonparametric inferences from line transect data. A general mathematical theory of line transects is develoepd which supplies a framework for nonparametric density estimation based on either right angle or sighting distances. The probability of observing a point given its right angle distance (y) from the line is generalized to an arbitrary function g(y). Given only that g(O) = 1, it is shown there are nonparametric approaches to density estimation using the observed right angle distances. The model is then generalized to include sighting distances (r). Let f(y/r) be the conditional distribution of right angle distance given sighting distance. It is shown that nonparametric estimation based only on sighting distances requires we know the transformation of r given by f(O/r).


A statistical model of the dynamics of a mosquito vector (Culex tarsalis) population. A model of the dynamics of a mosquito Culex tarsalis is derived that includes the life states through which the mosquito proceeds. Transition probabilities from one state (egg, larva, pupa and adult) to another are derived and they depend on the duration of stay and mortality in each state. A formula is derived for the expected number of mosquitoes alive at any time during the spring or summer. This formula depends on the number of eggs oviposited and the transition probabilities. Data are used to estimate the parameters and to illustrate the usefulness of this model in examining the effect of changes in mosquito survival on the dynamics of the population.


Estimating the rate constants in a two-compartment stochastic model. A procedure is described for estimating the rate constants of a two-compartment stochastic model for which the covariance structure over time of the observations is known. The proposed estimation procedure, by incorporating the known (as a function of the parameters to be estimated) covariance structure of the observations, produces regular best asymptotically normal (RBAN) estimators for the parameters. In addition, the construction of approximate confidence intervals and regions for the parameters is made possible by identification of the asymptotic covariance matrix of the estimators. The explicit form of the inverse of the covariance matrix, which is required in the estimation procedure, is presented. The procedure is illustrated by application to real as well as simulated data, and a comparison is made to the widely used nonlinear least squares procedure, which does not account for correlations over time.


A test for randomness.

A test for randomness. A procedure is proposed for studying the hypothesis that a sequence of dichotomous random variables are independent and identically (randomly) distributed. The procedure is relatively sensitive to departures from randomness involving multiple clustering. An application to testing for Schwann cell disease is discussed. The distrubution of the sample coefficient of variation from an exponential distribution, a useful statistic in testing randomness for a continuous model, is also studied.


Regression analysis of the log odds ratio: a method for retrospective studies. Quantification of the dependence of the odds ratio on concomitant variables associated with each of several 2 X 2 tables, using a regression model proposed by Zelen [1971], is an important tool for retrospective studies in epidemiology, An "exact" analysis may be based on the conditional likelihood obtained by fixing all the marginal totals. A symptotically this approach is equivalent to use of an unconditional log-linear model. The method is used to reanalyze data reported by Kneale [1971] on the relationship between obstetric radiation and childhood cancer.


Power spectrum of the respiratory system. Spectrum analysis is proposed as a method for analyzing biologic time series data that arise in the study of respiration. A mechanical model of the respiratory system is used to provide a theoretical basis for the interpretation of the air flow spectrum in terms of a physically defined energy concept. We show that under certain conditions the flow variance is proportional to the mechanical work of breathing, and indicate how this new parameter is related to conventional measures of respiratory function. An example is presented to describe the computational procedure and to illustrate the graphical aspects of cross-spectrum analysis.


The analysis of ranked data derived from completely randomized factorial designs. This paper presents a method for the analysis of ranked data arising from completely randomized factorial designs. The procedure, which is an extension of the Kruskal-Wallis ranks test, allows for the calculation of interaction effects and linear contrasts. A Monte Carlo study of the convergence of the test and a worked example are presented.


Stability of population growth determined by 2 X 2 Leslie matrix with density-dependent elements. The matrix considered contains four elements, each a function of total number. A special case for which the matrix may be appropriate is when the population may be divided into juveniles and adults, and the survival rates and fecundity are the same for all members of each group. This is true, at least approximatley, for many species of birds. The conditions for stability are determined and are illustrated with a model based on the data collected on the Marley Wood, Oxford, population of the Great tit (Parus major L.). A reformulation of the conditons permits comparison with the conditions for a population with identical survival rates and fecundity for all age groups, i.e., a population without effective age structure. Conditions under which the age structure may be ignored when discussing stability are considered.


Expectations for inbreeding depression on self-fertilization of tetraploids. The contribution to the inbreeding depression from a digenic tetrasomic locus upon self-fertilization involves three genotypic interaction effects which may be thought of as a generalization of the dominance deviation for a diploid locus. It is shown how this contribution may be expressed in terms of these genotypic interaction effects, the gene frequencies and the number of generations of selfing.


Reanalysis of some baboon descent data.

Reanalysis of some baboon descent data. Wagner and Altmann [1973] recently reported an analysis of a set of baboon descent times, some of which were left censored due to varying arrival times at the observation site. Any transformation of the data which reverses order produced a set of right censored observations. In this way, the Kaplan-Meier [1958] estimate of the descent time distribution can be computed. Estimates of the mean and variance of the distribution are given, as well as the standard error of the estimate of the mean.


A generalization of a matrix occupancy problem. An extension of the so-called committee problem is discussed in terms of the occupancy of a certain matrix whose row totals are taken as fixed, and whose cell entries are constrained to lie between zero and defined maxima. A statistic is constructed as a function of the column totals and used to test the significance of a column effect, irrespective of row differences. Two special cases when the cell maxima are constant, first within rows and second within columns, yield a covariance matrix for the column totals whose inverse may be found explicitly, and this in turn leads to a relatively simple expression for the test statistic. Applications of this generalized problem to certain health research situations are discussed, and the adequacy of a X2 approximation to the statistic's null distribution is mentioned.


Effect of ultrasound on the permeability of D2O through cellulose membranes. The effect of ultrasound energy on the permeability of water through cellulose membranes was studied using deuterium oxide (D2O) as a tracer. The same membrane was used for control (no ultrasound) and ultrasound experiments in a sequential reversal design. Transfer of D2O to a 2% albumin solution was measured in a glass dializing cell with membrane area of 11 cm2. Ultrasonic energy was supplied for 45 min by a hydrosonic bath (Linden Laboratories) at an intensity of 0.18 watt/cm2, an energy level commonly used for therapeutic purposes. Statistical analysis showed a significant increase in water transfer due to the ultrasound treatment irrespective of the sequence of application of ultrasound. It was concluded that ultrasound energy increases water permeability through cellulose dializing membranes. Whether or not increased water permeability is the operating mechanism underlying the beneficial effects of ultrasonic bath therapy remains unanswered.


Effect of resorbable calcium aluminate ceramics on regulation of calcium and phosphorus in rats. Ions released from resorbable ceramics could be toxic to the animal. Experiments were designed to study the effect of implanting three different weights of porous resorbable calcium aluminate ceramics (0.172, 0.332, and 0.504 g) in rats for a total duration of 300 days. Gross and microscopic examination of heart, liver, kidneys, trachea with thyroid, and muscle adjacent to the implant did not show any pathological changes. Calcium and inorganic phosphate content of bone, serum and urine were not affected by the implants. Urine hydroxyproline excretion did not change in the animals implanted with ceramics. Animals implanted with 0.332 g of ceramics had a significantly higher serum alkaline phosphatase activity than the control animals. Resorption of calcium and depositon of inorganic phosphates in the implanted ceramics suggested that ions were being exchanged with the body fluids. Implantation of 0.172 to 0.332 g porous resorbable calcium aluminate ceramic was not toxic to the animal.


2,3-Diphosphoglycerate red cell concentration changes during the menstrual cycle in woman. The authors showed that 2,3-DPG red cell concentration (2,3-DPG) WAS SIGNIFICANTLY HIGHER DURING THE LAST WEEK Of the menstrual cycle of five women (1.08+/-0.09 mol-,p;Hb-1) (M +/- 1 SE) THAN DURING THE First part and the last but one week of the cycle (0.86+/-0.04 mol-molHb-1, p less than 0.01). PACO2 was significantly lower during the last week of the cycle (36.3+/-1.3 torr) than during the first part and the last but one week of the cycle(40.1+/-0.8 torr, p less than 0.001). This hypocapnia was due to transient hyperventilation in the second part of the menstrual cycle. 5 men were studied during 4 weeks to determinate control values. No change was observed. The 2,3-DGP mean value (1.10+/-0.01 mol-molGb-1) was not significantly different of the values observed in the women during the last week of the cycle. These results suggest that the 2,3-DPG increase during the last week of the menstrual cylce is problably due to a respiratory alkalosis induced by progesterone secretion.


A case of hemoglobin M Boston. New data about valency hybrids brought by isoelectrofocusing study. In Hemoglobins M, as the result of a mutation theheme iron of the abnormal chain is atabilized in an oxidized form unable to carry oxygen. In this paper, we discuss the case of hemoglobin M Boston characterizing this abnormal hemoglobin as valency hybrid by spectral studies and utilising this mutant as a marker, in isoelectricfocusing, we bring the arguments that the two intermediary bands of oxidation correspond to alpha2+beta2+2 and alpha2+2beta2+3 We also indicate that the abnormal spectrum of Hb M interferes with the estimation of methemoglobin, giving erroneous values. We have shown that isoelectric focusing and subsequent scanning give a definite idea about the amount of abnormal chain present.


Electrostatic free energy and shift of the phase transition for charged lipid membranes. For a charged membrane in an electrolyte solution the electrostatic free energy is derived treating the system as a diffuse double layer. The dependence of the energy on external parameters like surface charge density and temperature is obtained and the physical basis discussed. As an application the charges are shown to exert an electrostatic surface pressure on the lipid chain packing which leads to a shift in the phase transition of membranes. The results confirm the interpretation of experimental data as given by Träuble et al. in the accompanying paper.


Differential hydrogen ion titrations of the histidine residues in Helix pomatia haemocyanin. The properties of the histidine residues in Helix pomatia haemocyanin have been studied by differential hydrogen ion titrations. In oxy-and deoxyhaemocyanin 31 X 10(-5) histidine residues per g protein are titrated in contrast to 35 X 10(-5) residues in apohaemocyanin. The difference corresponds to a stoichiometry of one histidine residue per copper atom bound. Even in apohaemocyanin about 6 X 10(-5) histidine residues per  g protein are not titrated in their normal pH region. In the presence of sufficient calcium to displace the dissociation completely out of the titration region, the titration curve of apohaemocyanin could be linarized according to the model of Linderstrom--Lang. In oxy-and deoxyhaemocyanin, however, a distinct deviation from linearity was found under the same conditions. In the absence of calcium the effect of the dissociation adds up to this deviation. The electrostatic interaction factors were determined for the protein at 0.1 M KC1 and for the dissociation products: halves and tenths at 1.0 M KC1. The electrostatic interaction factor for the wholes and the halves are much smaller than the values calculated from the Linderstrom--Lang equation, using the radius of the equivalent sphere either obtained from electron microscopy or from the partial specific volume. This probably due to solvent penetration. For the tenths at 1.0 M KC1, this effect is small.


Ion binding by X-537A. Equilibrium and rate of complexation of Co(2+) in methanol. The reaction of X-537A (XH) with Co(2+) in methanol to form the complex CoX(+) has been studied fluorometrically to determine the equilibrium constant as a function of temperature. The effect of complexation on the proton NMR spectrum of the X-537A was studied to determine the kinetics of complex formation. Comparing the data for the reaction XM(+) leads to X(-) + M(2+) in methanol at 25 degrees for several M(2+) we find that the equilibrium constants increase in the order CoX(+) less than MnX(+) and span only a factor of 5 while the rate constants increase in the order NiX(+) less than CoX(+) less than MnX(+) and span a factor of more than 100.


Association kinetics with coupled diffusional flows. Special application to the lac repressor--operator system. The time development of the association of the lac repressor to the operator is considered in a model where the repressor is allowed to bind unspecifically to DNA and move along the DNA chain in a one-dimensional diffusion. The coupling to the three-dimensional diffusion outside the chain is introduced by letting the repressor associate and dissociate from the chain until it is finally bound to the operator. All distance correlations along the chain are included. The mean time of association is calculated and through a comparison with experimental data the molecular parameters are determined. The one-dimensional diffusion constant is found to be of the order of 10(-9) cm(2)s(-1). The model is sufficiently general to be applicable to other similar systems.


Thermodynamic and kinetic parameters of ion condensation to polynucleotides. Outer sphere complex formed by Mg++ions. The coupling of ion binding to the single strand helix-coil transition in poly (A) and poly (C) is used to obtain information about both processes by ion titration and field-jump relaxation methods. Characterisation of the field-jump relaxation in poly (C) at various concentrations of monovalent ions leads to the evaluation of a stability constant K = 71 M(-1) for the ion binding to the polymer. the rate constant of helix formation is found to be 1.3 X 10(7)s(-1), whereas the dissociation rate is 1.0 X 10(6)s(-1). Similar data are preseented for poly (A) and poly (dA). The interaction of Mg(++) and Ca(++) with poly (A) and poly (C) is measured by a titration method using the polymer absorbance for the indication of binding. The data can be represented by a model with independent binding "sites". The stability constants increase with decreasing salt concentration from 2.7 X 10(4) M(-1) at medium ionic strengths up to 2.7 X 10(7) M(-1) at low ionic strength. The number of ions bound per nucleotide residue is in the range 0.2 to 0.3. Relaxation time constants associated with Mg(++) binding are characterised over a broad range of Mg(++) concentrations form 5 muM to 500 muM. The observed concentration dependence supports the conclusion on the number of binding places inferred from equilibrium titrations. The rate of Mg(++) and Ca(++) association to the polymer is close to the limit of diffusion control kR =1 X 10(10) to 2 X 10(10) M(-1) s(-1)). This high rate demonstrates that Mg(++) and Ca(++) ions do not form inner-sphere complexes with the polynucleotides. Apparently the distance between two adjacent phosphates is too large for a simultaneous site binding of Mg(++) or Ca(++), and inner sphere complexation at a single phosphate seems to be too weak. The data support the view that the ions like Mg(++) and Ca(++) surround the polynucleotides in the form of a mobile ion cloud without site binding.


The measure-unit of evolution.

The measure-unit of evolution. A theoretical study on evolution has been carried out, with the aim of disproving some value judgements essentially represented by the idea that the degree of evolution can increase. Our conclusions are based upon the preliminary statement that efficiency of survival is directly related to regulative ability. On this ground our reasoning led us to conclude that: (i) actual fitness measure units derive from an anthropocentric bias, and they mainly evaluate similarity to man rather than some objective parameter; (ii) a complete and meaningful unit is, at present, impossible to achieve in practice; (iii) since the study of evolution is only descriptive, and since the evolutionary process is time dependent, every ecological dominant living today must be considered as the most fitted to its environment; (iv) the view we can have of evolution is simply a transection, so that many generalized phyletic trees are trivial and it is impossible to claim the persistence today of those "ancestor organisms" upon which such trees are constructed. Moreover, a functional definition of the term "organism" is given, following criteria drawn from bioenergetics and from biological hierarchization. The concluding step is the assemblage of a slightly heterodox model for evolution.


Evolution of C4 photosynthesis in response to changes in carbon and oxygen concentrations in the atmosphere through time. The hypothesis is presented that C4 photosynthesis has evolved at lease in part in response to reduced efficiency of C3 photosynthesis due to increased oxygen partial pressures in our recent atmosphere. Oxygen competition with carbon dioxide for the active site on ribulose diphosphate carboxylase was of little consequence when atmospheric levels of CO2 were high and those of O2 were low. However at the present low CO2 and high O2 partial pressures, adaptations for protecting the enzyme from oxygen have been selected.


Linkages in thermal copolymers of lysine. The thermal copolymerization of lysine with other alpha-amino acids has been studied further. The identity of the second amino acid influences various properties of the polymer obtained, including the proportion of alpha and epsilon linkages of lysine. A review of linkages in proteinoids indicated alpha and beta linkages for aspartic acid, alpha and gamma linkages for glutamic acid, alpha and epsilon linkages for lysine, and alpha linkages for other amino acids. Thermal proteinoids are thus more complex in types of linkage than are proteins.


A model for competing polymers leading to their spatial separation. A model for a prebiotic polymer synthesis in a gradient of monomer is presented. In the absence of mutations the synthesis of the polymer proceeds in the region where the monomer concentration is the highest. However if a favorable mutation occurs, the latter accumulates in the high concentration zone and the initial polymer is restricted to a poorer monomer concentration region.


Thermal polymerization of amino acids under various atmospheres or at low pressures. The kinds and proportions of amino acids formed in two simulated prebiotic experiments or detected in hydrolyzed extracts of three extraterrestrial samples were found to polymerize thermally under various atmospheres or at low pressures. Yields, tested properties, and amino acid compositions of the polymers were not influenced by the type of enveloping atmosphere, including two simulated prebiotic atmospheres and five pure gases. However, polyamino acids prepared at low pressure (0.02, 10(-4) atm) were obtained in appreciably greater yield than those synthesized at 1 atm; amino acid composition was somewhat influenced by low pressure. The results indicate that polyamino acids could have been formed thermally under a variety of possible prebiotic atmospheres and on planetary bodies of low atmospheric pressure.


Biological clocks: endogenous cycles synchronized by subtle geophysical rhythms. Organismic sensitivity to atmospheric electromagnetic forces, together with usually asymmetrical phase-response systems, have enabled a simple unified explanation of the biological clocks and their timed rhythms. The clocks, endogenous cycles synchronized by subtle geophysical Zeitgeber, are always accurate. The rhythms, freely phase labile, autophase in "constant conditions" to generate a host of overt frequencies.


Polycondensation of alpha-amino acids by pyrosulfuric acid. The thermal polycondensation of amino acids common to protein is promoted at 80 degrees C by pyrosulfuric acid. This is in contrast to the noncondensation at 100 degrees C in the presence of concentrated sulfuric acid. These results are in accord with an anhydride mechanism, as proposed earlier for copolycondensation promoted by polyphosphoric acid. The amino acid composition, molecular weight, near-homogeneity, are infrared absorption of the polymer formed are described. The potential significance of planetary pyrosulfuric acid is discussed.


The mode of interaction between the substrate and cell surface of the hydrocarbon-utilizing yeast Candida tropicalis. A method for the measurement of the affinity of the cell surface to hydrocarbons is described. The affinity was basically unaffected by different pH values and temperature as well as by the chain length of the substrate. The contact time required for saturation of the cell surface with substrate was 30 sec. Cells grown on glucose showed a 25% lower adsorption capacity compared to those grown on n-alkane. The glucose grown cells showed also a more distinguished dependence of the amount of adsorbed hydrocarbon on the quality of the emulsion. The interaction between the substrate and cell surface turned out to be an adsorption that did not involve an enzymatic reaction. These results led to the conclusion that a lipopolysaccharide moiety present at the cell surface is responsible for the affinity.


Pore diffusion model for a two-substrate enzymatic reaction: application to galactose oxidase immobilized on porous glass particles. An analysis of the pore diffusion model involving a two-substrate enzymatic reaction is presented. The resulting equations have been applied to the case of galactose oxidase catalyzed oxidation of galactose when the enzyme is immobilized on porous glass particles. The physical constants of the system were obtained by theoretical predictions and the enzyme concentration in the porous medium was derived from the experimental results. The calculations were performed with the assumption that the kinetic parameters of the enzyme remain unchanged upon immobilization. The theoretically calculated effectiveness factors were compared with the experimental effectiveness factors determined from the batch kinetic experiments and were found to be in agreement. The results are presented as effectiveness factor plots graphed as functions of bulk galactose and oxygen concentrations. The model was extended in order to study the effect of external mass transfer coefficients and pore enzyme concentrations on the effectiveness factors.


Immobilization of a cephalosporin acetylesterase by containment within an ultrafiltration device. A cephalosporin acetylesterase produced by Bacillus subtilis catalyzes the deacetylation of 7-aminocephalosporanic acid (7-ACA). Previous reports from our laboratory described the kinetic constants that characterize the reaction: Km = 2.8 X 10(-3)M, Kia acetate = 5 X 10(-2)M, and Kid deacetyl-7-ACA = 3.6 X 10(-2)M. These constants were used to predict the time course of the reaction using the following equation for dual competitive product inhibition. (see article) where St =mg/ml 7-ACA, At =mg/ml acetate, Dt =mg/ml deacetyl-7-ACA. The predicted time course closely matched the time course measured experimentally. The equation also was solved without the inhibition terms and the solution indicated that product inhibition caused about a 30% increase in the time required for complete (greater than 97%) hydrolysis of a 24 mg/ml 7-ACA solution. The esterase was immobilized by containment within an ultrafiltration device. With this technique the enzyme was reused 20 times over an 11 day span to deacetylate 7-ACA solutions containing 4 to 24 mg/ml 7-ACA. The specific activity after the 20th use was the same as the activity prior to the first use, indicating little enzyme inactiviation occurred.


A mathematical model for microbial growth under limitation by conservative substrates. A mathematical model is suggested for growth of microorganisms under limitation by "conservative" substrates such as inorganic ions or vitamins that are not broken down after uptake into the cells, but that wholely or partly remain available for production of biomass. The specific growth rate is expressed here as a function of the intracellular "concentration" of the limiting substrate, defined as the amount of substrate within the cells per unit of cell dry weight. In the model, the intracellular substrate is divided into two parts. One part is a "structural" substrate not available for further growth. The other part is an "excess" or "functional" substrate that is used for biomass production and is assumed to be converted into structural substrate proportionally to growth. The rate of growth is believed to be controlled by the intracellular concentration of excess substrate.


Computer assistance in multiparameter flow microphotometry of mammalian cells. Flow system technology enables the biological and medical experimenter to investigate the distribution spectra of various cellular characteristics separately or in parameter combination on the basis of ultrarapid single cell measurements. A typical rate of recognition is about 1000 to 5000 cells per second and the precision of measurements and their statistical relevance has been previously unobtainable. According to the approach of the multiparameter analysis and high data rate, computer assistance in flow system technology is given primary consideration. In this study three different kinds of software controlled modes in data acquisition are demonstrated: normal acquisition and linear accumulation of single parameters, spectra accumulation of two correlated parameters of each single cell and documentation as linear, two- or three-dimensional distribution pattern, and linear accumulation of two correlated parameters simultaneously with their actual signal-to-signal ratio. A first attempt to analyze distribution spectra was the application of the entropy of the structure routinely used in cybernetics. This function seems to be a measure for determining the degree of synchrony in an appropriate pretreated cell population. A special mathematical strategy has been applied to the linear spectra cellular DNA content, whose advantage is the quantitative extraction of the fractions concerning the various phases of the life cycle cells. The validity of this special curve fitting procedure has been proven on various experimental cell populations.


Microbiological leaching of a chalcopyrite concentrate by Thiobacillus ferrooxidans. The microbiological leaching of a chalcopyrite concentrate has been investigated using a pure strain of Thiobacillus ferrooxidans. The optimum leaching conditions regarding pH, temperature, and pulp density were found to be 2.3, 35 degrees C, and 22% respectively. The energy of activation was calculated to be 16.7 kcal/mol. During these experiments the maximum rate of copper dissolution was about 215 mg/liters/hr and the final copper concentration was as high as 55 g/liter. This latter value is in the range of copper concentrations which may be used for direct electrorecovery of copper. Jarosite formation was observed during the leaching of the chalcopyrite concentrate. When the leach residue was reground to expose new substrate surface, subsequent leaching resulted in copper extractions up to about 80%. On the basis of this experimental work, a flow sheet has been proposed for commercial scale biohydrometallurgical treatment of high-grade chalcopyrite materials.


Invertase biosynthesis by Saccharomyces carlsbergensis in batch and continuous cultures. The biosynthesis of invertase by Saccharomyces carlsbergensis LAM 1068 was studied in relation to its glucose effect at both unsteady and steady states of growth. Experimental correlations between the dilution rate and invertase specific activity (E/X) in chemostat cultures led to an optimum for the enzyme synthesis at a particular intermediate growth rate. The value of E/X increased from 1.1 (U/mg biomass) in batch cultures to 13 (U/mg biomass) in chemostat cultures. A mutant strain A3 showed the highest value for E/X = 25 (U/mg biomass) at high dilution rates where glucose repression was observed with the wild strain.


Dual control of invertase biosynthesis in chemostat culture. In a previous study on the chemostat culture of Saccharomyces carlsbergensis, maximum invertase specific activity was observed at an intermediate dilution rate. A possible regulation mechanism, assuming there are simultaneous effects of induction and repression on two sites of the operator loci for invertase formation, is proposed which might account for the observed curve of the dilution rate effect.


Effect of broad and narrow spectrum fluorescent light on blood constituents. Riboflavin in whole blood, G-6-PD activity in erythrocytes and amino acids in plasma of infants irradiated by broad and narrow spectrum blue fluorescent light were determined. Riboflavin was reduced by both types of light. Loss of G-6-PD activity during phototherapy with subsequent hemolysis was observed in some infants under each type of light source, both in vivo and in vitro. This phenomenon may be related to the indirect effect of riboflavin reduction and not to a direct effect upon the enzyme. The reduction of the plasma concentration of some amino acids observed under broad spectrum irradiation but not under pure blue light may not be of significance, but is of concern. These data indicate that all fluorescent light sources in current use for phototherapy produce biologic effects of importance beside the primary purpose of the treatment to reduce hyperbilirubinemia. It is apparent from the results of these studies that phototherapy light penetrates more deeply than the superficial skin to exert photochemical effects.