Removal of Mn from spinach chloroplasts by sodium cyanide and the binding of Mn2+ to Mn-depleted chloroplasts. Manganese and copper were released from spinach chloroplasts by NaCN-treatment, though iron was not affected. The Hill reaction activity was also inhibited by this treatment, but was partially recovered by the addition of either Mn2+ or Cu2+, but not of Fe3+. The interaction of Mn2+ with manganese-depleted chloroplasts by NaCN-treatment was studied using 54Mn2+. A Scatchard plot shows the high and low affinity binding sites of Mn2+ on NaCN-treated chloroplast membrane; high affinity binding being specific for NaCN-treated chloroplast with a binding constant, KH, of 1.9 X 10(5) M-1, and a maximum binding number, NH, of 0.0016 g-atom per mole of chlorophyll. The low binding site was also found on untreated chloroplasts; its binding constant, KL, being 1.2 X 10(4) M-1, and its maximum binding number, NL, of 0.0112 g-atom per mole oc chlorophyll at pH 8.2 NH was proportional to the degree of the removal of Mn by NaCN-treatment and was constant at pH 4--9. NL markedly increased at a high pH with a midpoint of pH 7.9 indicating the exposure of a new, similar binding site. Light illumination partially inhibited the binding of Mn2+. Within 1 min in the dark the binding reaction reached equilibrium in the absence of pyrophosphate, however, 20 min were required to transform into pyrophosphate-resistant form. The pH dependence of the binding of Mn2+ with pKa 7.2 and the ineffectiveness of p-chloromercuribenzoate suggest the possible ligand of Mn2+ is the imidazole nitrogen of the histidine residue.


Cobalt bovine superoxide dismutase. Reactivity of the cobalt chromophore in the copper-containing and in the copper-free enzyme. 1. The reactivity of the zinc site of bovine superoxide dismutase has been probed by observing optical and electron paramagnetic resonance changes, under several conditions, of the Co(II)-substituted protein. 2. Only in the absence of copper are the optical and electron paramagnetic resonance spectra of the cobalt chromophore appreciably affected by alkaline pH or by cyanide. With both reagents the reaction with the copper-containing protein appears to involve the water molecule bound to the copper and does not affect the magnetic coupling between copper and cobalt. 3. The reaction of cyanide with the copper-free Co(II) protein leads to a slow detachment of cobalt from the protein as pentacyanocobalt. An oxygen adduct forms in air, analogous to that described in Co(II) carbonic anhydrase (Haffner, P. H. and Coleman, J. E. (1975) J. Biol. Chem. 250, 996--1005.) 4. Acid titration modifies the Co(II) spectra in the same way in the Cu-containing and in the Cu-free protein and brings about uncoupling of the Co(II)--Cu(II) system. Protonation of histidine-61 on the zinc facing nitrogen is suggested. 5. H2O2 modifies the cobalt chromophore only in the presence of copper. Magnetic coupling between Cu(II) and Co(II) seems to be still present after H2O2 inactivation of the enzyme.


Enzymatic synthesis of two fucose-containing glycolipids with fucosyltransferases of human serum. Lacto-N-neotetraosylceramide incubated with human serum fucosyltransferase preparations gave rise to two fucoglycolipids. The faster migrating fucoglycolipid I on the basis of its thin-layer chromatographic mobility, susceptibility to alpha(1 leads to 2) fucosidase from Trichomonas foetus, radio-immunoprecipitation with Ulex europeus lectin and studies with Oh (Bombay) sera was identified as H-active glycolipid (H-I). The most probable structure of fucoglycolipid II should be that with fucose linked alpha(1 leads to 3) to N-acetylglucosamine. Lactosylceramide, ceramide trihexoside and globoside were not substrates for human serum fucosyltransferases. Lacto-N-neotetraosyl ceramide served as a fucose acceptor for all serum preparations tested while asialoganglioside was a substrate only when serum preparations containing H-gene dependent alpha-2-L-fucosyltransferase were used. With asialoganglioside only one radioactive reaction product was formed.


Properties of prostaglandin synthetase of rabbit kidney medulla. The formation in vitro of prostaglandins E2, D2, and F2alpha from arachidonic acid by rabbit kidney medulla homogenate or microsomal fraction is markedly affected by the composition of the incubation medium employed. Optimal biosynthesis is obtained in 0.1 M potassium phosphate buffer, with the optimum pH being 8.0--8.8. Under these conditions prostaglandin formation is linear up to arachidonic acid concentration of 30 muM. The initial rate of formation of prostaglandin E2 + prostaglandin D2 is 3--4 times higher than that of prostaglandin F2alpha. Reduced glutathione (1 mM) did not affect the biosynthesis by medulla homogenate and produced only small stimulation of the biosynthesis by microsomal powder. Hydroquinone produced a small stimulation at a low concentration of 0.005 mM, and a strong inhibition at concentrations of 0.1 mM or higher. Addition of bovine serum albumin (0.1%) reduced the microsomal biosynthesis of prostaglandins by approximately 80%. Addition of boiled homogenate or boiled 140 000 X g supernatant produced small stimulation of microsomal biosynthesis while 140 000 X g supernatant (not boiled) caused small inhibition which was not dose-related. It appears that rabbit kidney prostaglandin-synthetase converts arachidonic acid to prostaglandins E2 and F2alpha in comparable amounts, without apparent need for a cytoplasmic soluble cofactor or specific reducing agents.


Acetyl-CoA carboxylase during development of plastids in wild-type and mutant barley seedlings. A soluble acetyl-CoA carboxylase in homogenates of leaves from wild-type barley seedlings was studied. Centrifuging the homogenate at 150,000 X g did not reduce the total activity, but raised the specific activity. During chloroplast development in light-grown seedlings or during light-dependent greening of leaves grown in the dark, both the total activity of the carboxylase per plant and the specific activity per mg of protein in homogenates of the seedlings increased rapidly. The soluble leaf acetyl-CoA carboxylase was studied in a number of barley mutants with lesions in chloroplast development. In a group of three mutants light elicited an increase in acetyl-CoA carboxylase activity as in the wild-type. In two mutants light caused a decrease in activity. Dark-grown leaves of mutant albina-f17 contained levels of soluble acetyl-CoA carboxylase reached only in the light by the wild-type, whereas light-grown albina-f17 seedlings lacked carboxylase activities. The possibility is discussed that leaf cells contain two forms of acetyl-CoA carboxylase, one soluble with unknown location and a dissociable form located in the chloroplast.


Calf-spleen nicotinamide--adenine dinucleotide glycohydrolase. Solubilization purification and properties of the enzyme. NAD glycohydrolase of calf spleen was solubilized with pancreatic lipase and purified approximatively 800-fold to a specific activity of 7 units/mg of protein by successive DEAE-cellulose and carboxymethyl-cellulose chromatography. The purified enzyme has a molecular weight of 24,000 and is characterized by a double band on disc gel electrophoresis. Some kinetic properties of the NAD-glycohydrolase-catalyzed hydrolsis of NAD have been examined using a titrimetric assay for enzyme activity. The reaction is subject to inhibition be excess of substrate, which disappears at high ionic strength and low pH. At a pH below 5 the kinetic displays an apparent activation by substrate. The effects of pH (4.5-9.0) on the kinetic parameters do not reveal an essential ionizable group in the catalytic process.


N-Acetylbenzotriazole as a protein reagent. Specific behaviour towards delta-chymotrypsin. When N-[14C] acetylbenzotriazole, presented here as a new agent for the acetylation of proteins, reacted at pH 8 and 25 degrees C with delta-chymotrypsin, 15 amino groups (the epsilon-amino groups of lysing residues and the alpha-amino terminus of half-cystine-1) and two phenolic groups (those of the two exposed tyrosine residues) were acetylated with respective pseudo first-order constants of 0.056 +/- 0.003 and 0.15 +/- 0.03 min(-1). Surprisingly, in contrast with the acetic anhydride reaction, the alpha-amino group of Ile-16 was found to be not acetylated as shown by N-terminus determination and activity measurements: the modified delta-chymotrypsin (or acetylated delta-chymotrypsin) was fully active after neutral dialysis. Only a transient inactivation due to the incorporation of one [14C] acetyl group per mole of catalytic site was observed. The kinetic constant found for reactivation at pH 8.5 was 0.315 +/- 0.005 min(-1) at 25 degrees C. The enzyme-catalyzed hydrolysis of N-acetylbenzotriazole was described by a k(cat) value of 0.093 +/- 0.005 min(-1) at pH 7 and 25 degrees C. Circular dichroism changes observed at 230 nm during the reaction at pH 8.5, of acetylated delta-chymotrypsin with N-acetylbenzotriazole indicated a total conversion of the amount of enzyme molecules which were in the 'inactive' or 'alkaline' conformation at this pH, into the 'active' or 'neutral' one. Benzotriazole alone was unable to induce such a conformational change. The rate constant of the reverse structural process from the 'neutral' to the 'alkaline' conformation was 0.32 +/- 0.02 min(-1): identical to that of the deacetylation of the catalytic site. Thus, the unusual lack of acetylation of Ile-16 alpha-amino group during delta-chymotrypsin treatment with N-acetylbenzotriazole is interpreted as a stabilization of the enzyme 'neutral' conformation where the Ile-16 alpha-amino group is buried, thus inaccessible to the reagent. The properties of the delta-chymotrypsin modification using N-acetylbenzotriazole led to practical uses: direct spectrophotometric titration of chymotrypsin operational normality at pH 7 and rapid preparation of acetylated delta-chymotrypsin. As a protein reagent, N-acetylbenzotriazole is particularly interesting because of its reactivity towards amino and phenolic groups of amino acid residues, its stability at acid pH, i.e., k(hydrolysis=7.38 X 10(-3) min(-1) at 25 degrees C [Ravaux et al. (1971) Tetrahedron Letters, 4013-4015] and its aromaticity, responsible for optical properties.


Exo-beta-N-acetylmuramidase--a novel hexosaminidase. Production by Bacillus subtilis B, purification and characterization. Exo-beta-N-acetylmuramidase, or beta-2-acetamido-3-O-(D-1-carboxyethyl)-2-deoxy-D-glucoside acetamidodeoxyglucohydrolase, is produced by Bacillus subtilis B, growing in a succinate/peptone/salts medium, at the end of exponential growth and occurs partly in the medium and partly bound to the cells. A lysozyme digest of Micrococcus lysodeikticus cell walls, O-2-acetamido-2-deoxy-beta-D-glucopyranosyl-(1 leads to 4)-2-acetamido-3-O-(D-1-carboxyethyl)-2-deoxy-D-glucose and O-[2-acetamide-3-O-(D-1-carboxyethyl)-2-deoxy-beta-D-glucopyranosyl]-(1 leads to 4)-2-acetamido-2-deoxy-D-glucose in decreasing order of efficiency, induce the enzyme but O-2-acetamido-2-deoxy-beta-D-glucopyranosyl-(1 leads to 4)-2-acetamido-2-deoxy-D-glucose does not do so. The enzyme was purified from the growth medium, after removal of the cells by continuous centrifugation, by ammonium sulphate precipitation, continuous filtration through XM-300 membranes (to remove the high-molecular-weight material which renders the enzyme sedimentable in low-ionic-strength solutions), diafiltration through PM-30 membranes and ion-exchange chromatography on DEAE-Sephadex and CM-Sephadex. Two peaks of activity were obtained. Peak A was purified 1800-fold and was homogenous on polyacrylamide disc gel electrophoresis. A second heterogeneous fraction (peak B) was also collected. Exo-beta-N-acetylmuramidase is most stable at pH 8.0 and has a molecular weight of about 90000. The results of studies on its ability to attack several synthetic and natural substrates are given. The Km and V values for 4-methylumbelliferyl-2-acetamido-3-O-(D-1-carboxyethyl)-2-deoxy-beta-D-glucose and O-[2-acetamido-3-O-(D-1-carboxyethyl)-2-deoxy-beta-D-glucopyranosyl]-(1 leads to 4)-2-acetamido-2-deoxy-D-glucose are respectively 0.19 and 0.65 mM and 1.50 and 16.29 mumol min(-1) mg(-1). From these results and those of inhibition studies it is concluded that the enzyme is specific for substrates with non-reducing N-acetylmuramic acid end groups. Possible roles for this enzyme are discussed.


Studies on the active site of yeast hexokinase. Specific phosphorylation of a serine residue induced by D-xylose and ATPMg. Yeast hexokinase A(ATP:D-hexose 6-phosphotransferase) is inactivated when incubated in the presence of xylose and ATPMg, or in the presence of D-lyxose in a reaction medium in which ATPMg is being continuously regenerated (phosphoenolpyruvate and pyruvate kinase). The inactivation is due to the phorphorylation of the protein. A linear relationship was observed between the inactivation and the incorporation of 32P from [gamma-32P] ATP. All hexokinase and ATPase activity of the enzyme is lost when one phosphoryl group is incorporated per enzyme subunit (molecular weight 51,000). The phosphoryl group is covalently bound by a ester linkage with a serine residue of the protein.


The transport of S-adenosyl-L-methionine in isolated yeast vacuoles and spheroplasts. 1. The properties of S-adenosyl-L-methionine accumulating system for both vacuoles and spheroplasts are described. Yeast vacuoles were obtained by a modified metabolic lysis procedure from spheroplasts of Saccharomyces cerevisiae. 2. Isolated vacuoles accumulate S-adenosyl-L-methionine by means of a highly specific transport system as indicated by competition experiments with structural analogs of S-adenosyl-L-methionine. The S-adenosyl-L-methionine transport system shows saturation kinetics with an apparent Km of 68 muM in vacuoles and 11 muM in spheroplasts. 3. S-Adenosyl-L-methionine accumulation into vacuoles does not require glucose, phosphoenolpyruvic acid, ATP, ADP nor any other tri- or di-phosphorylated nucleotides. It is insensitive to azide and 2,4-dinitrophenol which strongly inhibit the glucose-dependent accumulation of S-adenosyl-L-methionine in spheroplasts. 4. The transport of S-adenosyl-L-methionine into vacuoles is optimal at pH 7.4 and is insensitive to nystatin while the uptake of S-adenosyl-L-methionine into spheroplasts is optimal at pH 5.0 and is strongly sensitive to nystatin. On this basis it has thus been possible to measure both the intracytoplasmic and the intravacuolar pool of S-adenosyl-L-methionine. 5. Our results indicate the existence of a highly specific S-adenosyl-L-methionine transport system in the vacuolar membrane which is clearly different from the one present in the plasma membrane of yeast cells.


Enzymic and molecular properties of base-plate parts of bacteriophage P22. Using 14C-labeled Salmonella bacterial cells as the substrate, the enzymic and molecular properties of the base-plate parts of phage P22 were studied. The base-plate part consisted of a single protein species which cleaved extensively the O-antigen of Salmonella typhimurium, Salmonelly schottmuellerie and with somewhat slower rate that of Salmonella typhi, releasing oligo-saccharide products with rhamnose at the reducing end. Much less cleavage was observed with a strain of S. typhimurium lysogenic for P22, and no significant reaction with Salmonella anatum, Salmonella newington and Salmonella minneapolis. The base-plate part enzyme was a very heat-stable protein and only 10-20% loss was observed after treatment at 85 degrees C for 5 min. The pH optimum of the enzyme was around 7.5, and the glycosidase activity was not influenced by the ionic strength (25-250 mM( of the medium or the presence of Mg2+. The molecular weight of the base-plate part was 320,000 by sedimentation equilibrium. Dodecylsulphate-acrylamide gel electrophoresis revealed a single band of molecular weight 77,000, indicating that a single base-plate part corresponds to a tetramer of identical subunits. Circular dichroism spectra of P22 base-plate parts showed a major contribution of beta structure. The protein was rich in acidic amino acids, glycine and serine.


Effects of alpha-adrenoceptor agonists and antagonists on pre- and postsynaptically located alpha-adrenoceptors. The effects of alpha adrenoceptor agonists and antagonists have been examined at pre- and post-synaptically located alpha-adrenoceptors in the pithed rat. The presynaptic receptors were those located at the cardiac sympathetic nerve terminals and the postsynaptic receptors were those present in vascular smooth muscle. Clonidine was approximately equipotent at pre- and post-synaptic alpha-adrenoceptors, whilst LSD and BAY-1470 were more active at the pre- than at post-synaptic sites. Oxymetazoline, naphazoline, methoxamine and phenylephrine were all much more active at the postsynaptic alpha-adrenoceptors. Phentolamine was the most potent antagonist at both pre- and post-synaptic alpha-adrenoceptors. Piperoxan, yohimbine and tolazoline were about 3-7X less potent than phentolamine at both sites. Thymoxamine was about 10X less potent than phentolamine at postsynaptic alpha-adrenoceptors but about 1000X less active at the presynaptic receptors. The differential actions of both agonsists and antagonists at pre- and post-synaptic alpha-adrenoceptors suggest that the receptors may be of different types.


Influence of methamphetamine on nigral and striatal tyrosine hydroxylase activity and on striatal dopamine levels. In previous reports, methamphetamine was shown to depress tyrosine hydroxylase (TH) activity in the rat corpus striatum. To evaluate further the mechanism of this decrease in TH activity, enzyme activity was measured in the rat corpus striatum and substantia nigra after repetitive and single-dose methamphetamine administration. Following repeated doses of methamphetamine, nigral TH activity decreased and reached 45% of controls at 12 hr and returned to normal at 60 hr. Striatal TH activity decreased to 40% of control at 36 hr and returned toward normal at 60 hr. When methamphetamine was administered every 6 hr for 30 hr and then discontinued, nigral TH activity returned toward control levels 4 days prior to recovery of striatal TH activity. Methamphetamine initially increased striatal dopamine levels at 6 hr (170% of control). Dopamine levels then decreased in parallel with striatal TH activity but failed to increase as the enzyme recovered. Concurrent administration of chlorpromazine with methamphetamine prevented the methamphetamine-induced decrease in nigral and striatal TH activity and striatal dopamine levels. The results indicate that the methamphetamine-induced depression of striatal and nigral TH activity may be related to increased stimulation of dopamine receptors in the striatum.


alpha-Adrenoceptors in the ventricular myocardium: clonidine, naphazoline and methoxamine as partial alpha-agonists exerting a competitive dualism in action to phenylephrine. Tha alpha-sympathomimetic agonists, clonidine, naphazoline, methoxamine, oxymetazoline and phenylephrine were used to further characterize the alpha-adrenoceptors mediating the positive inotropic effect in the isolated papillary muscle of the rabbit heart. The maximal inotropic effects of these amines were compared with the effect of isoprenaline and it was examined whether or not these amines compete for alpha-adrenoceptors. On the papillary muscle stimulated at 0.5 Hz, phenylephrine showed a high affinity (pD2 value=6.13) and produced the most pronounced intrinsic activity of the alpha-sympathomimetic amines. Therefore, the intrinsic activity of phenylephrine, in the presence of prindolol (3 X 10(-8) M), was used for comparison with those of the other alpha-agonists. Clonidine caused a positive inotropic effect: the intrinsic activity amounted to 0.32 of that of phenylephrine; the affinity was the highest among the amines tested (pD2 value=6.46); its effect was inhibited by 10(-6) M phentolamine. The affinity and the intrinsic activity of naphazoline were slightly lower than those of clonidine. Methoxamine showed a relatively high intrinsic activity (0.56) but the lowest affinity (4.68). Oxymetazoline did not cause any positive inotropic effect. Clonidine, naphazoline and oxymetazoline antagonized the positive inotropic effect of phenylephrine, mediated via the alpha-adrenocaptors in the presence of 3 X 10(-8) M prindolol, in a competitive manner. This observation suggests that these alpha-sympathomimetic amines compete with phenylephrine for the same receptor site. Thus the present results provide additional evidence for alpha-adrenoceptors mediating the positive inotropic actions of sympathomimetic amines in the rabbit papillary muscle.


Competitive oxidation of 6-hydroxydopamine by oxygen and hydrogen peroxide. Simultaneous oxygen electrode and conventional polarographic measurements show the net concentration of hydrogen peroxide produced by air oxidation of 6-hydroxydopamine is considerably less than that predicted from the known stoichiometry of the reaction. This is due to competitive oxidation of 6-hydroxydopamine by the generated hydrogen peroxide. The presence of ascorbic acid in this reaction also results in significant decreases of hydrogen peroxide under most conditions. The implications of these results to the molecular mechanism of 6-hydroxydopamine neurotoxicity are discussed.


Discriminative stimulus properties of benzodiazepines, barbiturates and pharmacologically related drugs; relation to some intrinsic and anticonvulsant effects. Using a food-reinforced two-lever operant method, rats (n = 12) were trained to discriminate chlordiazepoxide (5 mg/kg, p.o.) from solvent (p.o.). With rats trained thus as subjects, generalization experiments were done with various benzodiazepines, barbiturates and related compounds, and with two neuroleptic drugs. The ability of these drugs to induce a discriminative stimulus complex similar to that induced by chlordiazepoxide, was then compared with some intrinsic and anticonvulsant effects of the same drugs. It was found that the discriminative stimulus properties of benzodiazepines, barbiturates, and related compounds correlate with the ability of these drugs to induce ataxia, as well as with part of their anticonvulsant activity. However, the stimulus properties of these drugs, as defined by the procedure described, are based neither on their ataxia-inducing effect, nor on their general depressant or sedative action. It is concluded that these properties constitute a pharmacologically highly specific phenomenon.


Studies on the centrally mediated hypotensive activity of guanabenz. Prior studies demonstrated that guanabenz reduces systemic blood pressure by inhibiting central sympathetic outflow as well as by adrenergic neuron blockade. Potential mechanisms responsible for the reduction of efferent sympathetic activity were examined in the present series. Guanabenz failed to modify carotid sinus nerve activity in a perfused sinus preparation. It reduced sympathetic outflow, heart rate and blood pressure in debuffered cats indicating that its actions are not mediated primarily by baroreceptor mechanisms. alpha-Adrenergic blockade greatly attenuated the response suggesting that the central sympathoinhibitory effect of guanabenz results from alpha-adrenergic receptor activation. Only a high dose of the compound attenuated the increase in sympathetic nerve activity produced by stimulation of the posterior hypothalamus. These experiments lead to the overall conclusion that guanabenz acts primarily at sites which regulate the basal level of sympathetic outflow.


Modification and characterization of the permanent sympathectomy produced by the administration of guanethidine to newborn rats. The administration of guanethidine to newborn rats has been shown to produce a permanent sympathectomy with potential advantages over immunosympathectomy and 6-hydroxydopamine-induced chemical sympathectomy. In this paper, we report on a revised treatment regimen involving initiation of treatment (50 mg/kg/day) on day 7 after birth and continuing for 3 weeks. Animals treated by this protocol have a low mortality rate (approx. 10% above saline-treated controls) and no permanent growth deficit. Analysis of tyrosine hydroxylase activity in and light microscopic examination of superior cervical ganglia of the guanethidine-treated animals indicate complete destruction of sympathetic neurons by the end of the second week of treatment. During and after treatment there are no decreases in norepinephrine in whole brain of the treated animals. Norepinephrine levels in peripheral tissues are markedly reduced at both 9 and 16 weeks of age. Stimulation of vasomotor outflow produces no increase in blood pressure in guanethidine-treated rats at 9 or 26 weeks of age, indicating a complete and permanent functional denervation of the vasculature. The adrenal glands of the guanethidine-treated animals are not destroyed, but rather respond, apparently by transsynaptic induction, with increases in tyrosine hydroxylase and epinephrine content. Interestingly, despite the continued deprivation of a peripheral sympathetic nervous system in these animals. adrenal tyrosine hydroxylase and epinephrine levels return to control levels by 10 weeks of age. These data indicate that administration of guanethidine to newborn rats produces a very complete and permanent sympathectomy with significant advantages over immunosympathectomy and 6-hydroxydopamine-induced chemical sympathectomy.


Studies on the mechanism of the cardiovascualr effects of methyldopa. Oral administration of methyldopa (100 mg/kg, twice daily for 3 days) to mongrel dogs produced a significant decrease in blood pressure and heart rate. The drug treatment affected neither the resting venous tone nor the cardiac output. Thus, the hypotensive effect of the drug was predominantly due to a reduction in total peripheral resistance. Vasoconstrictor responses of the renal vasculature to sympathetic nerve stimulation were significantly impaired after methyldopa at all the frequencies, while mesenteric vasoconstrictor responses to sympathetic nerve stimulation were impaired only at the lower stimulation frequencies. In addition, methylnorepinephrine was a significantly less potent vasoconstrictor than norepinephrine in the renal vasculature, but was equipotent to norepinephrine in the mesentery. The finding of a reduction in the renal vascular resistance of methyldopa-treated dogs, with no such alteration in the mesenteric vascular resistance, is consistent with the nerve stimulation studies. Therefore, the results of the present investigation indicate that in addition to the existing evidence favoring a central site of action for methyldopa, the impairment of peripheral sympathetic neuronal function  is also of importance in accounting for the hemodynamic alterations observed following treatment with methyldopa.


Some adrenergic beta-blocking agents affecting lipolysis in human adipose tissue in vitro. Antagonistic actions of beta-blocking drugs on isoproterenol-induced lipolysis were studied in human omental adipose tissue. Competitive interaciton characterized by the following pA2 values was found: propranolol 8.7; trimepranol 8.7; practolol 7.1; H 35/25 6.0. The plot of pA2 values of these drugs for human versus rat adipose tissue is linear with slope 2.0 indicating a higher differentiation of beta-antagonist actions in human than in rat adipose tissue.


[Observation of bacteriophages in wine (author's transl)]. Electron microscopic examination of samples of Swiss wine, collected during the malolactic fermentation, revealed the presence of bacteriophages of three different morphological types. It is interesting to note that these phages have been found in a product whose pH is lower than 3.5.


Is glutamic acid the pyramidal tract neurotransmitter? Applied by microiontophoresis, 1-hydroxy-3-amino-pyrrolidone-2 (HA-966) antagonized excitation by glutamic acid but not by acetylcholine of neurones in the rat cuneate nucleus. HA-966 blocked the short latency excitation of cuneate neurones following stimulation of the pyramidal tract on 28 of 40 cells (70%). Thus, glutamate or a related amino-acid may be the neurotransmitter released by pyramidal tract neurones.


Effects of physalaemin, a vaso-active peptide from amphibian skin, on the excitability of an identifiable molluscan giant neurone of Achatina fulica Férussac. We examined effects of several vasoactive peptides (substance P, physalaemin, neurotensin, bradykinin, angiotensin etc.) on the excitability of molluscan giant neurones identified in the subesophageal ganglia of Achatina fulica Férussac. Of these peptides, only physalaemin showed a remarkable excitatory effect on a giant tonically autoactive neurone.


Graft-versus-host reaction and lymphoid organs in normally fed and protein-deprived rats. Lymph node graft-versus-host reaction (GVHR) induced by parental splenic lymphocytes inoculated into hind foot pads of F-1 hybrid rats is correlated with the state of the thymus and the spleen of the recipients. This may explain the depression of the reaction after protracted protein deprivation. Furthermore, GVHR provokes mainly in normal rats a reduction of thymus and spleen possibly due to a T-cell transfer to the grafted area.


Cortisone sensitive T-cells in Peyers' patches. Pretreatment of donor lymphoid cells with cortisone has been shown to depress the T-cell subpopulation responsible for cellular proliferation in the GVH reaction. A quantitative assay as well as the histological criteria of the GVH reaction have been used in this study to demonstrate the presence of cortisone-sensitive T-cells within the Peyer's patches as well as in the spleen and mesenteric lymph nodes in the rat.


[Detection and quantitative estimation of morphine in the urine of drug addicts by combined 2-dimensional thin-layer chromatography and spectrofluorometry]. Following extraction from urine and thin-layer bidimensional chromatography, the suspected spot of morphine is located by UV examination at 350 nm and eluted with methanol by means of "Eluchrom" apparatus. The dried residue is then subjected to spectrofluorimetric analysis, in definite conditions. This procedure can be used to confirm the identification of the alkaloid and to achieve its estimation. The sensitivity and the recoveries of various quantities of morphine added to urine were determined.


[Comparative evaluation of the antitoxic activity of central nervous system analeptics and their combinations in barbamyl poisoning]. Tests conducted on rats poisoned with increasingly lethal doses of sodium amytal demonstrated the antitoxic activity of an analeptic mixture to be superior to that of picrotoxin, strichnine, corasol and caffeine entering into its composition and also to the activity of bemegride. As to the degree of antitoxic activity in poisoning of mice with sodium amytal (one LD50) the CNS analeptics are arranged in the following descending oder of their action: analeptic mixture, picrotoxin, bemegride, corasol, strychnine. In poisoning with higher doses of sodium amytal (LD84) corasol, strychnine and caffeine are ineffective, the most productive being analeptic mixture and picrotoxin. Bemegride proves effective only in a single dose of 2.8 LD50 for intact animals.


[Effect of 1,4-benzodiazepine derivatives on the toxic action of oxygen under increased pressure]. The 1,4-benzodiazepine derivatives (diazepam, nitrazepm, lorazepam, clonazepam and two newly synthetized compounds of this series) increase the resistance of albino rats and rabbits to the toxic effect of oxygen under high pressure (7 and 5.5 atm respectively). The compounds under study are apt to avert over a long period the development of metabolic acidosis which appears following the action of high-pressure oxygen on the body.


[Effect of noninhalant anesthetics on the conduction of excitation in the ventrolateral columns of the cat spinal cord]. Acute tests on spinal cats were set up to enquire into the effect produced by sodium oxybutyrate, hexobarbital sodium and viadril used in anesthetic doses on the evoked responses in the ventrolateral columns of the spinal cord following stimulation of the skin and pelvic nerves with single and paired stimuli at an interval of 100 msec. Sodium oxybutyrate forces the amplitude of both the evoked responses after the paired stimulation below the initial level by 40-70 per cent. Hexobarbital sodium and viadril, without changing noticeably the amplitude of the first response, are instrumental in reducing the second one by as much as 30-40 per cent. The responses obtained on stimulation of the pelvic nerve are more sensitive to the test substances than are those evoked by the stimulation of the skin nerve.


[Neuromediator content in the synaptic vesicles of rat adrenergic nerves in some pharmacological actions]. Cytochemical electron microscopy was employed to study the content of neurotransmitters in the synaptic vesicles of adrenergic nerve fibers of Vas deferens of the rat following depletion of the meadiator's reserves by tyramine and subsequent accumulation of exogenous norepinephrine. Subject to investigation was also the effect of antidepressants (phthoracizine and imipramine) on the accumulation of exogenous norepinephrine in the synaptic vesicles. Phthoracizine and imipramine (1 and 10 gamma/ml) are shown to block the acculation of norepinephrine in the vesicles, when the mediator is introduced in a concentration of 0,5 gamma/ml, and not to impede this process, if the mediator's concentration is increased to 30 gamma/ml.


[Effect of some phenothiazine derivatives on kidney function]. Chlorathizin and Chlormorphathizin are shown to exercise in 24-hours long tests a substantial diuretic and saluretic action on rats and dogs. Fluorathizin, as well as imizine inhibited the urinary flow. Diprazine augmented the urinary output in dogs, but depressed it in rats. The new antidepressive drug azaphen did not have any marked effect on the urinary flow.


[Effect of psychotropic and anticonvulsive preparations on transport ATPase in the renal tubules of the guinea pig]. The effect of 4 groups of medicamentous agents, viz. neoruleptics, tricyclic antidepressants, somnifacients and antiepileptics - on the activity of the transport ATPase and p-nitrophenylphosphatase from the renal tubules of the guinea pig was studied. Used in high concentrations neuroleptics suppress almost completely the activity of the enzyme, but in small doses influence but little that of the p-nitrophenylphosphatase. The butyrophenone derivatives have a mild effect upon the activity of the p-nitrophenylphosphatase and in low concentrations they stimulate it. The effect of tricyclic antidepressants closely approaches the one produced by neuroleptics-phenothiazines. Lithium carbonate leaves the enzyme intact. Barbiturates and antiepileptic agents inhibit but feebly these enzymes.


Drugs and brain mitochondrial enzymatic activities during post-natal development in rat. The enzymatic activities of two mitochondrial enzymes, i.e. succinate dehydrogenase and NADH-cytochrome c reductase were investigated in the brain of rats at different stages of post-natal development. In addition, the effect of the pharmacological treatment with two drugs, nicergoline and bamethan, able to interact with the alpha or the beta receptors respectively, was evaluated. The results show that both the enzymatic activities rapidly increase in the first days of extra-uterine life, thus indicating an adaptation of mitochondrial oxidative processes to post-natal environmental conditions. The pharmacological treatment with the two drugs does not induce any changes in the enzymatic activities tested.


[Comparative characteristics of fluorasizine metabolism in different species of animals and man]. The process of phthoracizine (10-(beta-diethylaminopropiniol)-2-triphthormethylphenothiazine hydrochloride) transformation proceeding in various animal species and in man is similar in grosso modo to biotransformation of chlorpromazine and of compounds related to the latter, including sulphoxidation, dealkylation in the side chain nitrogen, hydroxylation, glucoronoconjugation and the formation of products with the side chain degradation. At the same time, one can note a difference which finds its expression in the development of a large quantity of the phthoracizine metabolites attended by destruction of the side chain, as well as of hydroxylated sulphoxidated products of its transformation. In rats, more than in rabbits and mice, the spectrum of the phthoracizine metabolites is closer to the products of its biotransformation passed with the human urine.


[Effect of analgestics and narcotics on the potentials of the posterior roots evoked by stimulation of the sensorimotor cortex]. Experiments set up on unanesthetized, curarized cats demonstrated that sodium oxybutyrate in doses of 75--200 mg/kg and nembutal in doses of 5--20 mg/kg prolong the duration of corticospinal posterior roots potentials (PRP). The initial effect of morphine and promedol in doses of 1--3 and 0.5--1 mg/kg, respectively was not only a longer duration (by 30--40 msec), but also a higher amplitude of the PRP, evoked by the stimulation of the sensomotor cortex. Phentanyl used in a dose range of 0.01 to 0.06 mg/kg did not produce any changes in the descending PRP. Anesthetics (sodium oxybutyrate, nembutal, ether) and analgesics (morphine, phenadon) inhibited the corticospinal PRP, when used in large doses.


[Effect of narcotic analgesics on the cortical control process of impulse transmission in the afferent pathways of the sciatic nerve]. The effect produced by narcotic analgetics with their intravenous administration on the process of cortical control over the transmission of impulses along specific routes of the sciatic nerve was studied. The conditioning stimulation of the cortex was effected by using a monopolar electrode through single electric impulses. The interval between conditioning and test (on sciatic nerve) impulses was of 80-120 ms. Morphine (1-2 mg/kg), promedol (trimeperidin) (1-2 mg/kg) and phentanyl (100 gamma/kg) potentiated the inhibition of evoked potentials in the nucleus gracilis and in VPL, observed upon stimulation of the cortex of optic lobuses. The intensification of inhibitory corticifugal mechanisms occurring under the effect of narcotic analgetics takes place both on the level of the medulla oblongata and of the thalamic one.


[Effect of adrenergic compounds on the brain potentials evoked by stimulation of the dental pulp]. Intraventriculare injections have brough evidence that alpha-adrenomimetic norepinephrine and beta-adrenomimetic isopropylnorepinephrine augment in the senso-motor region of the cortex the amplitude of primary responses evoked through a dental pulp stimulation. The alpha-adrenoblocking agents--dihydroergotoxin and phentolamine and the beta-edrenoblocking agent propranolol force down the amplitude of primary responses. The sphere of the drugs action is presumed to be the thalamic nuclei.


[Effect of izadrin, inderal and netalid on the periodic activity of the rabbit myometrium]. The contractility of an isolated rabbit uterus was studied. It was found that when used in low concentrations (1.10(-9)-1.10(-7) g/ml) isoprenaline, while stimulating the beta-adrenoreceptors of the uterus, inhibits its contractions, whereas in high concentrations (1.10(6)-1.10(-5) g/ml) it is capable of intensifying uterine contractions owing to stimulation of the uterine alpha-adrenoreceptors. Dihydroergotoxin (1.10(-6) g/ml), a blocking agent of alpha-adrenoreceptors, averts and eliminates the stimulating effect of isoprenaline on the uterus. Propranolol and netalide, blocking agents of the beta-adrenoreceptors, abolish the already developed depressing action of isoprenaline on the contractile activity of the uterus, but fail to prevent this effect with their preliminary application.


Studies of human transcortin at different pHs: circular dichroism, polymerisation and binding affinity. The transcortin we have used in this work is extremely pure. This was shown by the polymerisation observed at pH 4. This polymerisation is never observed with an impure form of transcortin [4]. Moreover, since it is known that the presence of cortisol in the binding site is an essential condition to the activity of purified transcortin [5], it appears that a correlation between the secondary structure and the biological activity of the transcortin exists. The results we have obtained are summarized below: (1) The inhibition of the transcortin binding capacity essentially takes place between pH 5 and 4. (2) A reorganisation of the structure of the protein moiety is observed between pH 6.5 and 5.9. (3) A decrease of the helicity ratio is observed between pH 5 and 4. It appears therefore that, in the limits of experimental accuracy of CD measurements to determine the amount of beta-structure, no appreciable change of binding activity is taking place after the appearance of a large percentage of beta-structure between pH 6.5 and 6. On the other hand, the sudden decrease of protein activity at low pH is likely to be correlated with the disappearance of a well-defined helical region. Other biochemical and physical experiments would be of course necessary, in order to precise this first observation of a structure-function relationship in transcortin.


Cryptorchidism in the subfertile male.

Cryptorchidism in the subfertile male. Certain conclusions may be drawn from the present review and presentation of new data concerning the unilaterally cryptorchid patient and possible subfertility: 1. The truly undescended testis--not the retractile testis of infancy--will not descend spontaneously after 1 year of age. 2. The seminal quality of the unilaterally cryptorchid patient is definitely impaired, although not rendered infertile, in a great majority of patients irrespective of the time of surgery. 3. The dystopic position of the maldescended testis appears to superimpose a second insult on what very likely may be an inherent abnormality in the cryptorchid testis, the latter accounting, perhaps, for its abnormal extrascrotal position. 4. Orchiopexy before the age of 5 seems advisable to ensure minimal histologic changes that may be secondary to the testis' increased exposure to elevated extrascrotal temperature. 5. The cryptorchid testis will be smaller in size irrespective of surgery, and usually correlates with significant testicular pathology. 6. Basal levels of gonadotropins, especially follicle-stimulating hormone, are likely to be elevated, but this does not necessarily imply overwhelming testicular damage. Androgen production should not be affected. 7. Surgical correction is advised when human chorionic gonadotropin stimulation fails to produce teticular descent, thereby defining the maldescended testis as not merely retractile but truly "crytorchid."


[Participation of the genetic apparatus in memory and learning phenomena]. The first series of experiments showed that inhibition of the proteins synthesis followed by memory disorders, was reflected in decrease of the synaptosomal proteins fraction which was identified as the cholinoreceptor protein. Another series of experiments: training of rats to use unpreferred paw, showed the system acetylcholine-acetylcholinesterase to be directly connected with memory phenomena, and the synthesis of this enzyme to be induced by genetic apparatus. The third series of experiments showed that feeding of rats with small doses of aminoacids for a long time leads to regular shifts in distribution of both the aminoacids and the monoamines. The aminoacids increasing the brain activity were found to activate the production of cycle-adenyl acid. Those aminoacids which inhibit the brain activity, decrease the production of cyclic-AMP. Tranquilizers which decrease the level of monoamines and inhibit the brain activity, also decrease the production of cyclic-AMP. Antidepressants inhibiting MAO and increasing the level of monoamines in the brain, activate the production of cyclic-AMP. As the monoamines act via cyclic-AMP and the latter participates in suppression of DNA, the mechanism of involvement of the genetic apparatus in regulation of memory phenomena and learning, becomes more apparent.


[Mechanisms of pial artery responses in hypo- and hypertension]. Simultaneous study of the pial arterial responses and quantitative changes of the systemic arterial pressure, as well as of the PO2, pH and PCO2 on the brain surface, showed no correlation between the values and time of changes of the above parameters in rabbits. The vascular responses are concluded not to be caused by the direct action of either the changes of the intravascular pressure, or of the mentioned metabolites on the arterial walls, but rather to be due to another, probably nervous, feedback mechanism.


[The chronotropic effect of maximal stress on the heart and its regulation]. The reflex bradycardia occurring at the maximal isometric heart strain produced by clamping of aorta was shown to be less intense if the heart rate prior to the experiment had been low. Vagotomy prevented or decreased the bradycardia. The beta-adrenoreceptor blockade with inderal significantly reduced the bradycardia, whereas the alpha-adrenoreceptor blockade with tropaphen increased it. The important role of relationship between adrenergic and cholinergic regulation of the heart in development of the bradycardia was discussed.


[The effect of atropine on acid formation in the stomach]. In dogs, the effect of atropine on gastric acid secretion induced by food, mechanical stimuli, and histamine involved a complete block of the acid secretion but left the gastric secretory effect of histamine unaffected. Atropine caused about a twofold increase in duration of histamine-evoked acid secretion and a rise of the intragastric acidity in all parts of the stomach.


Influence of the properties of skin allografts on the "graft survival promoting effect" of antilymphocyte serum. Changes in the properties of non-H-2 imcompatible skin allografts, resulting from treating the graft donors with whole-body irradiation, antilymphocyte serum, cyclophosphamide, hydrocortisone, amethopterin and azathioprine, affected differently their survival in the recipients pretreated with normal and antilymphocyte serum. While in NS--pretreated recipients the modified grafts showed the tendency for a prolonged survival, in ALS-pretreated recipients they survived for a shorter time than normal allografts. In ALS-pretreated recipients, also allografts from radiation chimaeras, whose blood-forming system was of the recipient's genotype, survived worse than normal allografts and grafts from the donor syngeneic radiation chimaeras. The findings indicate that a normal physiological state of the graft is necessary for the induction and working of the protective components of the allotrasplantation reaction involved in the graft survival promoting effect of ALS. The significance of passenger leucocytes and of the functional properties of the graft, especially of the reparation cellular and extracellular processes is discussed.


The effect of cimetidine, a new histamine H2-receptor antagonist, on meal-stimulated acid secretion, serum gastrin, and gastric emptying in patients with duodenal ulcer. Meal-stimulated acid secretion, measured by in vivo intragastric titration, was progressively inhibited by increasing oral doses of cimetidine (25 to 400 mg). Four hundred milligrams suppressed acid secretion by 73% for the first 3 hr after the meal, whereas it inhibited acid secretion by 94% during the 30-min period of maximal inhibition. The dose of cimetidine required to suppress acid secretion by 50% during the 30-min period of maximal inhibition was 25 mg. The duration of action of a 300-mg dose was at least 7 hr. Cimetidine was equally effective in inhibiting meal-stimulated acid secretion at two physiological intragastric pH levels (5.0 and 2.5). Cimetidine had no effect on serum gastrin concentration when intragastric pH was maintained at 5.0, but when pH was allowed to seek its own level, serum gastrin concentration was higher after cimetidine than after placebo. Cimetidine had no effect on gastric emptying. No side effects were noted in any patients.


Role of fat maldigestion in pathogenesis of steatorrhea in ileal resection. Fat digestion after two sequential test meals with and without cholestyramine. To clarify the role of fat maldigestion in the pathogenesis of steatorrhea in patients with ileal resection the total and aqueous phase concentrations of bile acid and fatty acid were characterized in 8 such patients (5 patients with small ileal resection, bile acid diarrhea, and steatorrhea less than 20 g per day; 3 patients with large ileal resection, fatty acid diarrhea, and steatorrhea greater than 20 g per day) as well as 4 healthy control subjects after a morning and an afternoon liquid test meal. The study was then repeated with cholestyramine, 4 g being administered before each meal to induce fat maldigestion. After a conventional test meal, patients with large resections and severe steatorrhea had significantly lower aqueous phase concentrations of bile acids (and fatty acids) than patients with smaller resections or control subjects, explained in part by intraluminal precipitation of about one-half of the bile acids during digestion. When cholestyramine was administered before the meal, aqueous phase bile acid concentrations decreased in all patients, including the normal control subjects; the degree of fat maldigestion induced in the patients with small resections (and the control subjects) became similar to that present after the conventional test meal in the patients with large resections. Because steatorrhea increased little in the patients with small resections when cholestyramine was administered continuously, the data suggest that fat maldigestion per se does not induce severe fat malabsorption in patients with sufficient anatomical reserve, because such patients can absorb fat efficiently by utilizing the distal small intestine. In patients with large ileal resections, severe steatorrhea is explained in part by the combination of fat maldigestion and decreased surface area. It is also speculated that the steatorrhea occurring in patients with small resections and relatively normal fat digestion during two test meals may be explained by impaired fat digestion which occurs during the final meal of the day, which is often the largest meal.


Evaluation of esophageal tests in the diagnosis of reflux esophagitis. The sensitivity and specificity of each of five esophageal tests used in the diagnosis of reflux esophagitis were compared with those of six combinations of two tests, one indicating esophagitis and the other indicating sphincter incompetence. The esophageal tests were performed in patients with reflux symptoms, chest pain, and esophagitis without reflux symptoms. Control data were obtained from normal subjects (negative control) and duodenal ulcer patients (positive control). The results indicate that the acid infusion test and esophageal biopsy combined with esophageal pH study after HC1 have similar sensitivity and greater specificity than any test alone. In normal subjects, the cumulative incidence of abnormalities with esophageal tests alone was 30%, but with combinations of two tests it was only 5%. The use of criteria (simultaneous esophagitis and sphincter incompetence) which establish the diagnosis of reflux esophagitis helps to resolve conflicting results obtained with single tests. The most sensitive and specific test combination for the diagnosis of reflux esophagitis appears to be esophageal biopsy with esophageal pH study after HC1.


[Investigations on the distribution, cause and prevention of early perinatal morbidity (author's transl)]. These investigations were designed to detect causes of early perinatal morbidity in order to develop methods to decrease the perinatal morbidity in our unit. From October 1972 to December 1974 the most important antenatal, intrapartum and postpartum data on 2210 deliveries were coded. In 98% of the cases continuous fetal monitoring was performed. To detect early perinatal morbidity the acid base balance of all deliveries was measured from the umbilical vessels and the Apgar rating was determined at 1,5 and 10 minutes. The data show that a further decrease of the incidence of acidosis and low apgar scores is only possible in a limited way. Of the 13% deliveries with pH values of/or less than 7.15 or Apgar scores of/or less than 6 at 1 minute, only 5% appeared to be avoidable in this retrospective study. The incidence of severe acidosis decreased from 2.3% in 1972 to 1.3% in 1974. A comparison of the years 1973 and 1974 showed an improvement. Apgar scores of 6 or less at one minute decreased from 16 to 10% and severe acidosis with pH values of less than 7.10 decreased from 2.7% to 1.8%. In vaginal operative deliveries, the incidence of severe acidosis was reduced from 8.5% to 1.1%.


Sugar and amino acid transport in animal cells. The molecular basis of intracellular metabolism of nutrients and its control is quite well understood in animal cells. Comparable knowledge about solute entry into cells is still lacking, as, in contrast to metabolism, no chemical reactions seem to be directly associated with the known nutrient transport. Nevertheless, translocations of sugars and amino acids across the plasma membrane are specific and controlled processes, biologically as well as chemically. Recent advances in techniques for isolation of plasma membranes have made it feasible to study transport properties of animal cells without the complications encoutered in viable cells. This approach has been applied to sugar and amino acid transport in plasma membranes of several tissues, and intact transport systems for D-glucose, D-fructose, neutral L-amino acids, and dipeptides have been demonstrated. This demonstration of intact transport systems in an in vitro setting accomplishes the first step in the direction of molecular isolation of transport systems. Furthermore, the information obtained about the transport mechanism catalyzed by some systems has settled controversies on active nutrient transport. For example, electrogenic cotransport of sodium and D-glucose or of sodium and neutral L-amino acids has been shown to form the basis for active, sodium-dependent absorption of these nutrients. A consequence of this type of mechanism is interaction between sugar and amino acid transport via the common charged cosubstrate sodium. Moreover, different types of transport systems for the same substrate have been demonstrated in the luminal and contraluminal regions of the plasma membrane of epithelial cells, which explains unidirectional transepithelial transport. The luminal membrane contains sodium-dependent, active transport systems, and the contraluminal membrane passive, facilitated diffusion systems. In vivo, the lower intracellular sodium potential would result in concentrative nutrient uptake from the lumen, but would not influence exit on the contraluminal side. Variations in the electrical components of the sodium potential, which have not been measured, may explain apparently contradicting results on active sugar and amino acid transport with various tissue preparations.


Diethylaminoethyl-cellulose-bacterial cell immunoadsorbent columns: preparation of serotype-specific globulin and immunofluorescent conjugates for Streptococcus mutans serotypes a and d. Diethylaminoethyl (DEAE)-cellulose was used as a support material for preparing bacterial cell columns. Pretreatment of the bacterial cells with formalin was essential in obtaining satisfactory adherence of the cells to DEAE-cellulose. Cross-reacting antibodies were removed from antibody preparations against strains of Streptococcus mutans serotypes a and d by adsorption on appropriate bacterial cell columns. S. mutans serotype d was further divided into two subtypes on the basis of immunofluorescent staining with conjugates of immunospecifically adsorbed immunoglobulin G. The DEAE-cellulose-bacterial cell columns were regenerated after use by desorbing the cross-reacting antibodies with low-pH buffer and were used repeatedly over and 18-month period with no detectable loss in effectiveness.


Kinetic studies of the alkaline mesentericopeptidase. Study on the active centre topography of alkaline mesentericopeptidase by means of bifunctional reversible inhibition. The inhibitory effect of alkylboronic acids H(CH2)nB(OH)2(n=2-8) and Ph(CH2)n-B(OH)2, (n=0-4), on the alkaline mesentericopeptidase-catalysed hydrolysis of synthetic substrates was studied. It was shown that alkylboronic acids act as bifunctional reversible inhibitors. The borate group interacts with an ionogenic group of the enzyme with a pKa of about 6.9-7.0. The latter is probably the catalytically active imidazole of the active centre. The hydrocarbon part of the molecule also takes part in the formation of the enzyme-inhibitor complex. The dependence of the degree of the enzyme-inhibitor complex formation upon the length of the side-chain of the inhibitor indicates the presence of two binding sites on the enzyme molecule.


Unusual behaviour of a protein: reversible quantitative precipitation of staphylococcal delta-haemolysin by low concentrations of some organic solvents. Staphylococcal delta-haemolysin is precipitated from watery solutions by low concentrations of chloroform. The effect on this precipitation of pH, kind and concentration of salts, and concentration of, and time of exposure to, chloroform have been investigated. Some but not all of the other solvents tested cause a similar precipitation. The haemolytic activity is not destroyed and under appropriate conditions precipitation is quantitative. The dry lysin will take up nearly double its weight of chloroform when exposed to the vapour. Some is bound strongly.


Deamidated active intermediates in the irreversible acid denaturation of ribonuclease-A. A study has been made on the changes in the enzymatic activity of Ribonuclease-A**-(RNase-A) exposed to highly acidic (pH less than 1) acqueous environment. Irreversible alterations of activity were observed when the protein was exposed to an acidic medium for a long period (20 to 60 h). Even prior to these changes in activity RNase-A was found to form intermediates which had very nearly the same activity as the native protein. The primary process in the acid denaturation of RNase-A was observed to be deamidation of the protein leading to the formation of active chromotographically distinct derivatives. The initial product of deamidation, a monodeamidated derivative, has been isolated by chromatography on Amberlite XE-64. This initial deamidation reaction proceeded with very high specificity. The subsequent deamidation reaction is comparatively slower, so that nearly 50% of the native protein could be converted to this derivative before any subsequent deamidation took place. This monodeamidated derivative has been designated RNase-Aa1. The conversion of RNase-A to RNase-Aa1 was not accompanied by any changes in the primary structure other than the observed deamidation. Apart from the differences in chromatographic and electrophoretic mobilities, RNase-Aa1 was found to have very nearly the same activity and physicochemical properties as the native enzyme. Significance of this specific and faster deamidation of RNase-A in this denaturing medium as well as the biological significance of such deamidation reactions of proteins are discussed.


Covalent coupling of bilirubin to albumin. A method for covalent coupling of bilirubin to albumin is described. Human serum albumin-bilirubin (1:1 complex) has been treated with water soluble carbodiimide in order to obtain covalent coupling of bilirubin to albumin. The reaction conditions have been varied with respect to pH, reaction time and concentration of reagent to obtain the optimal coupling. The prepared albumin-bilirubin compounds were investigated by spectrophotometry, gel filtration and gel electrophoresis to ascertain the covalent nature of the bond and to characterize the products further. Gel electrophoresis and gel filtration showed that a monomer fraction could be prepared, and this fraction was a suitable material for further studies.


Inactivation of trypsin and chymotrypsin with a photosensitive probe. The photosensitive inactivation of trypsin and chymotrypsin by 4-fluoro-3-nitrophenyl azide (FNPA) is described. A dark inhibition was observed at elevated probe concentrations, and was reversible. The enzymes were stable to photolysis in the absence of probe. Photolytic inactivation of trypsin and chymotrypsin with FNPA was found to be irreversible, and occurs in minutes at concentrations of FNPA where dark inhibition is negligible. The photoprobe was equally effective at pH 3 or pH 8. Nonspecific inactivation appears to be low, as evidenced by the stability of glucose oxidase and peroxidase to photolysis with FNPA.


Pathways of the eye's response to topical nitrogen mustard. We studied the effect of prior corneal herpes simplex infection with its resultant corneal hypesthesia on the irritative response of the rabbit eye to topical nitrogen mustard. Both the miosis and the breakdown of the blood-aqueous barrier that follow the application of topical nitrogen mustard were diminished in eyes infected three weeks previously with herpes simplex virus. Nonspecific corneal scarring did not affect the response. This suggests again that an axon reflex requiring intact sensory innervation mediates the response to nitrogen mustard. Pretreatment of normal (noninfected) rabbits with systemic H1 and H2 antihistamines, topical scopolamine hydrobromide, or topical and systemic corticosteroids was ineffective in blocking the miosis or increased protein in the aqueous humor following topical nitrogen mustard.


Intraocular pressure decrease in normal volunteers following timolol ophthalmic solution. Timolol ophthalmic solutions 0.5 per cent, 1.0 per cent, and 1.5 per cent lowered intraocular pressures significantly in normal human volunteers. Maximum lowering of the intraocular pressures was reached at two hours with the 0.5 per cent solution of timolol and at one hour with the 1.0 per cent and 1.5 per cent timolol ophthalmic solutions. The effect lasted the full seven hours of observations. No objective or subjective evidence of ocular irritation could be attributed to the drug. A single dose of timolol applied topically to the eyes of normal human volunteers had no effect on pupillary size, visual acuity, blood pressure, or pulse rate.


The adrenergic receptors of the intraocular muscles of the human eye. The adrenergic receptors in man were analyzed using isolated sphincter, dilator, and ciliary muscle strips, dissected from eyebank eyes. The dilator is mainly alpha, the sphincter both alpha and beta, and the ciliary muscle predominantly beta adrenergic.


pH-dependent temperature sensitivity of rat lens phosphofructokinase. Rat lens phosphofructokinase (PFK) has been found to be cold-labile at acidic pH, even in the presence of sulfate and inorganic phosphate, two known positive effectors. The inactivation appears to be an irreversible process, but can be prevented by including ATP in the incubating media. The enzyme is relatively stable at pH 8.2 incubated at 0 to 4 degrees, 25 degrees, or 37 degrees C. in the absence of the effectors, but is extremely thermolabile if the pH is lowered to 7.30 or lower. The thermolability is counteracted by many effectors, among them sulfate and ATP are the most effective. The physiologic significance of PFK instability and effector protection in the lens are discussed.


The fetal renin-angiotensin system in normal and hypertensive pregnancy. Plasma angiotensin II was measured in the umbilical cord arterial and/or venous blood of 54 babies delivered vaginally and in 12 delivered by elective lower segment cesarean section. Angiotensin II was also measured in the peripheral venous blood of 51 of the mothers. Mean angiotensin II levels at delivery were higher in the cord venous blood of infants born to hypertensive than to normotensive mothers. Cord venous angiotensin II levels were always higher than those of the mother and there was a strongly positive relationship between maternal and fetal angiotensin II. The duration of the second stage of labor was found significantly to affect fetal angiotensin II levels, prolonged labor being associated with high levels. When this was taken into account, there was a highly significant positive relationship between the body weight of the infants and cord venous angiotensin II levels. There was an inverse relationship between cord venous pH and angiotensin II levels. Babies delivered by lower segment cesarean section had much lower angiotensin II levels than those delivered vaginally. The levels were, however, twice as high as those in the nonpregnant adult.


Use of salazopyrin for prevention of peritoneal adhesions in rats. The effect of orally administered salazopyrin on the prevention of experimentally induced peritoneal adhesions in albino rats was investigated. Comparison of the findings with those for untreated rats and rats given penicillin for sulfadiazine indicated that salazopyrin has a preventive effect on the development of peritoneal adhesions. This effect is dose dependent and does not seem to be due merely to the antibacterial effect of the drug.


[Optimal dosage in peroral therapy of acne with vitamin A palmitate]. Successful oral therapy with vitamin A palmitate in acne vulgaris requires 150,000-200,000 I.U. daily for months. Side-effects were evaluated in 22 patients and in addition in 54 patients receiving 400,000 and 300,000 I.U. respectively for 3-4 weeks (SGPT, GGPT, Quick, electrophoresis, creatinine, bromosulfthaleine excretion). The same tests were done in 32 patients, who had received vitamin A palmitate 150,000-200,000 I.U. daily for at least half a year. Clinical experience and the presented data allow the following conclusions: There is no risk of liver impairement when 150,000-200,000 I.U. are given daily over extended periods. Doses over 300,000 I.U. are accompanied with liver impairement. During long-term treatment y-GT test should be performed regularly. Contraceptive advices are recommended.


Chemical modification of streptovaricin C I. 19-O-substituted damavaricin C. Damavaricin C, a degradative derivative of streptovaricin C, has reduced antibiotic activity relative to streptovaricin C. It has, however, a new phenolic hydroxyl group at the C-19 position of the naphthoquinone ring on which various groups can be substituted through an ether linkage. A series of 19-O-substituted derivatives of damavaricin C has been synthesized. The preparation of these derivatives, their in vitro antibacterial activities, in vitro inhibition of E. coli RNA polymerase, and lethal activity on the membrane mutants of E. coli are reported. It is believed that the original biological activity of damavaricin C is retained and that introduction of the functional groups at the C-19 position has increased the membrane diffusibility of the molecule.


Comparative in vitro activity of cephalosporins. The in vitro activity of cephalexin, cephaloridine, cephalothin, cephapirin, cefoxitin, cephamycin C, cepharadine and cefazolin was determined against 443 isolates of bacteria. At a concentration of 12.5 mug/ml, all of the cephalosporins inhibited more than 60% of the isolates of Klebsiella pneumoniae. At the same concentration, cephalexin, cephaloridine, cephalothin, cephapirin, cephamycin C and cefazolin inhibited more than 90% of isolates of proteus mirabilis. All of the cephalosporins except cephalothin and cephapirin inhibited over 60% of isolates of Escherichia coli at a concentration of 12.5 mug/ml. Cefoxitin was the most active cephalosporin against gram-negative bacilli. There was substantial differences in the activity of cephalosporins against gram-positive cocci. Cephaloridine was the most active cephalosporin against these organisms. There was considerable fluctuation in the proportion of isolates of gram-negative bacilli susceptible to these cephalosporins from year to year, but there was no evidence to suggest that the number of resistant isolates was increasing.


Oxygen dissociation curve for chorioallantoic capillary blood of chicken embryo. Oxygen dissociation curves for blood in the chorioallantoic capillary of chicken embryos were determined using a microphotometric apparatus made for measuring the reaction velocity of a red blood cell with oxygen and carbon monoxide. The modified Hill's equations expressing the dissociation curve during development were calculated by two methods. P50's at pH of 7.4 were found to be 60.0, 54.4, 46.2, 33.1, and 28.6 mmHg for 10, 12, 14, 16 and 18 days of incubation, respectively. Although the Bohr factor did not show a clear relation to age, the oxygen affinity and the oxygen capacity tended to increase with the lapse of days, and the power of heme-to-heme interaction, to decrease with age. The findings imply that there is a respiratory adaptation of embryos during development.


Failure of histamine antagonists to prevent hypoxic pulmonary vasoconstriction in dogs. The role of histamine as a mediator of hypoxic pulmonary vasoconstriction was examined in intact anesthetized dogs. Antagonism of histamine vasoconstrictor (H1) receptors with a classic antihistaminic drug (chlorpheniramine) failed to prevent or modify the pulmonary vascular responses to hypoxia (10% O2). Blockade of histamine vasodilator (H2) receptors with a newly synthesized blocking agent (metiamide) potentiated the vasoconstriction induced by hypoxia and prevented the normal increase in heart rate. Combined H1- and H2-receptor blockade also did not prevent or reduce the hypoxic pulmonary pressor response, although it did effectively abolish the cardiovascular actions of infused histamine. In other dogs, histamine infused (3.6 mug/kg per min) during hypoxia attenuated the pulmonary vasoconstriction induced by hypoxia. The results imply that, in the dog, histamine does not mediate hypoxic pulmonary vasoconstriction. However, histamine does appear to be released during hypoxia, and it may play a role in modulating the pulmonary vascular responses to hypoxia by opposing the hypoxia induced vasoconstriction. The results also imply that histamine may be responsible for the increase in heart rate during hypoxia.


Circulatory effects of prolonged hypoxia before and during antihistamine. Five chronically instrumented healthy dogs were exposed to a 5-day period of breathing 10% oxygen in a chamber. The response to hypoxia was found to be time dependent. During the first 24 h of hypoxia the circulatory response was characterized by increases in cardiac output, heart rate, pulmonary and systemic arterial blood pressures, and pulmonary vascular resistance. Systemic vascular resistance increased; left atrial pressure decreased. During the early part of hypoxia the animals became hypocapnic; the arterial blood pH rose significantly. During the rest of the hypoxic period cardiac output, heart rate, and arterial blood pH returned to the control values; pulmonary and systemic arterial pressures and pulmonary vascular resistance remained significantly elevated. Systemic vascular resistance rose; left atrial pressure remained below control. This response to hypoxia was not substantially modified when the experiment was repeated during the administration of the antihistamine promethazine, an H1-receptor blocking agent, in a dose which blocked the pulmonary vasoconstrictor response to small doses of exogenous histamine. The circulatory response to acute hypoxia in five anesthetized dogs was not modified by intravenous administration of metiamide, an H2-receptor blocking agent.


Dual contribution theory of regulation of CSF HCO3 in respiratory acidosis. Regulation of CSF HCO3-in respiratory acidosis was studied in light of the "dual contribution theory," which proposed that there were two sources for the CSF HCO3-increase: 1) HCO3-by diffusion from plasma and 2) HCO3-generated in the CNS and catalyzed by the local carbonic anhydrase (J. Appl. Physiol. 38: 504-512, 1975). In anesthetized dogs with an increase in Paco2 of 30 mmHg for 4 h the plasma HCO3 increased 2 meq/1 and CSF 6 meq/1. In combined respiratory and metabolic acidosis, plasma HCO3-did not increase but CSF HCO3-increased 6 meq/1. In combined acidosis and intraventricular injections of acetazolamide no increase in plasma or CSF HCO3-occurred. In combined respiratory acidosis and metabolic alkalosis and intraventricular acetazolamide, plasma HCO3-increased 15 meq/1 but CSF HCO3-increased 6 meq/1. Brain and CSF ammonia increased linearly and selectively with the increase in the relative contribution of CNS HCO3-increase. Therefore regulation of CSF HCO3-in respiratory acidosis depends on both components of the dual contribution theory, where each component can provide the total CSF HCO3-increase under appropriate experimental conditions. The control mechanism may be sensitive to changes in [H+] on the brain side of the blood-brain barrier.


Effects of continuous positive-pressure ventilation in experimental pulmonary edema. We compared the effects of continuous positive-pressure ventilation (CPPV), using 10 cmH2O positive end-expiratory pressure (PEEP), with intermittent positive-pressure ventilation (IPPV), on pulmonary extravascular water volume (PEWV) and lung function in dogs with pulmonary edema caused by elevated left atrial pressure and decreased colloid osmotic pressure. The PEWV was measured by gravimetric and double-isotope indicator dilution methods. Animals with high (22-33 mmHg), moderately elevated (12-20 mmHg), and normal (3-11 mmHg) left atrial pressures (Pla) were studied. The PEWV by both methods was significantly increased in the high and moderate Pla groups, the former greater than the latter (P less than 0.05). There was no difference in the PEWV between animals receiving CPPV and those receiving IPPV in both the high and moderately elevated Pla groups. However, in animals with high Pla, the Pao2 was significantly better maintained and the inflation pressure required to deliver a tidal volume of 12 ml/kg was significantly less with the use of CPPV than with IPPV. We conclude that in pulmonary edema associated with high Pla, PEEP does not reduce PEWV but does improve pulmonary function.


Evaluation of a dual-function pH and PCO2 in vivo sensor. A newly developed, dual-function pH and PCO2 sensor was evaluated in this study. The sensors were placed in the femoral arteries of dogs anesthetized with sodium pentobarbital. Comparisons were made between systemic arterial pH and PCO2 measured using the sensor and those measured from blood samples drawn at 15-min intervals over a 7-h period using a bench instrument. The mean pH of the bench instrument measurements was 7.43. The mean difference of the sensor measurements from the bench instrument measurements for 207 comparisons was 0.0003 pH +/- 0.061 SD. The mean PCO2 of the bench instrument measurements was 40 mmHg. The mean difference of the sensor measurements from those of the bench instrument for 212 comparisons was -1.43 mmHg +/- 5.17 SD. The sensors performed equally well in the presence of metabolic or respiratory acidosis and alkalosis. The dual-function sensors evaluated in this study are useful for trend monitoring of pH and PCO2 over at least a 7-h period without recalibration. With improvement in the consistency of sensor construction, these sensors will be reliable in vivo sensing devices for blood pH and PCO2 and thus valuable research and clinical instruments.


Polymer membrane sensors for continuous intravascular monitoring of blood pH. A new type of pH sensor suitable for chronic intra-vascular implantation by virtue of its small size, flexibility, and ruggedness was constructed and evaluated. The pH-sensitive element was a thin film of an elastromeric polymer made ion permselective to proteons by adding a lipophilic, specific H+-ion carrier. This was coated onto small diameter silver wires to form sensors. In preliminary trials in anesthetized dogs, the sensors permitted continuous, accurate in vivo blood pH measurement with rapid response (less than 0.1 s).


Temperature-induced changes in blood acid-base status: pH and PCO2 in a binary buffer. Equations for proton equilibria of a single-phase binary buffer system have been applied to temperature-induced changes in pH and PCO2 of separated dog plasma at constant carbon dioxide content. Predicted behaviour, measured as deltapH/deltaT and deltalog PCO2 /deltaT, and pH and PCO2 as a function of temperature (range 8-45 degrees C), are in reasonable agreement with theory. Theory predicts and data confirm that deltapH/delta T and deltalog PCO2/deltaT functions of temperature; no single "temperature correction factor" is applicable. Comparison of whole blood with binary buffer equations also shows acceptable agreement between theory and experiment. Blood and separated plasma show similar responses in deltapH/deltaT and deltalog PCO2/delta T when compared over identical temperature intervals. For blood or plasma with initial pH (AT 37.5 DEGREES C) values in the range 7.53-7.45 deltapH/delta T (u/ degrees C) values are -0.0139 (37.5-27.5 degrees C) and -0.0192 (19-7 degrees C); comparable deltalog PCO2/deltaT values are 0.0195 (37.5-27.5 degrees C) and 0.0240 (19-7 degrees C). The charge state of protein components in this system remains nearly constant as temperature varies.


Temperature-induced changes in blood acid-base status: Donnan rCl and red cell volume. The Donnan ratio for chloride ion (rCl) was determined for human red cells in plasma utilizing 36Cl. The effect of altered PCO2 and pH on rCl was followed in two ways. CO2 partial pressure was varied (1-1.5% CO2 in O2; pH range 7.1-7.9) at 37.5 degrees C (isothermal); PCO2 and pH were also changed by altering temperature (range 5-45 degrees C) at constant CO2 content (temperature induced). At pH 7.4 and 37.5 degrees C, rCl was 0.631 +/- 0.0269 (SE, N = 5); isothermal drcl/dpH = -0.306 +/- 0.0234. When measured under conditions of variable temperature at constant CO2 content (pH range 7.3-7.9), drcl/dpH = .018 +/- 0.0232, significantly different from isothermal response (P less than 0.001). Hematocrit (H) changes with pH for conditions of initial H(7.4) of 0.45, under these conditions were also determined: isothermal dH/dpH = -0.031 +/- 0.0019; temperature induced, -0.004 +/- 0.0009. Temperature change alone at constant carbon dioxide content produces no significant change in distribution of chloride ions or water between erythrocyte and plasma compartments.


Influence of pH on the rate of ribosomal ribonucleic acid synthesis during sporulation in Saccharomyces cerevisiae. The rate of synthesis of ribosomal RNA (rRNA) is much slower during sporulation than during vegetative growth of yeast. If sporulating cells are transferred from normal incubation conditions at pH 8.8 to the same medium adjusted to pH 7.0, the rate of rRNA synthesis increased to approach that observed in vegetative cells. The response to the pH change is quite rapid, occurring within 10 min. THE PH-dependent, rate-limiting step appears to be in the processing of 35S ribosomal precursor RNA to the final 26S and 18S RNA species. A similar pH effect also was found for the rate of protein synthesis. However, no change in respiration was observed when the pH was lowered. These results indicate that the observed differences in rate of rRNA synthesis in vegetative and sporulating cells are a consequence of pH and are not intrinsic to sporulation. The results also support the correlation between rRNA processing and protein synthesis.


Regulation of histidase synthesis in intergeneric hybrids of enteric bacteria. Regulation of the expression of the histidase coded by hutk of Klebsiella aerogenes in Salmonella typhimurium and in Escherichia coli and of the expression of the histidase coded by huts of S. typhimurium in E. coli was investigated. The hutk histidase was found to be sensitive to catabolite repression in K. aerogenes and in E. coli, but insensitive to catabolite repression in S. typhimurium; huts histidase has previously been shown to be catabolite sensitive in all three organisms. The expression of both hutk and huts histidase in E. coli was activated by nitrogen starvation. Apparently, the glutamine synthetase of E. coli may activate the formation of some glutamate- and ammonia-producing enzymes.


Role of polyadenylic acid in a deoxyribonucleic acid-membrane fraction extracted from pneumococci. After the addition of radioactive polyadenylic acid to cell suspensions of pneumocci, part of the radioactivity becomes associated with a deoxyribonucleic acid (DNA)-membrane fraction extracted from the cells. A variety of techniques show that a portion of this associated radioactivity may represent oligoadenylates complexed to DNA, probaby as part of a ribonucleic acid (RNA) component. Polyadenylic acid, which had previously been shown to enhance DNA synthesis in cell suspensions (Firshein and Benson, 1968), also enhances the extent of DNA synthesis by the DNA-membrane fraction in vitro under specific conditions of concentration and conformation. The mechanism of action of this enhancement may be related to the ability of oligoadenylates to increase the number of initiation sites for DNA replication by stimulating the production of an RNA primer, thus providing additional 3'-OH groups with which DNA polymerase can react.


Glutamate dehydrogenase: genetic mapping and isolation of regulatory mutants of Klebsiella aerogenes. The gene for glutamate dehydrogenase (gdhD) has been mapped in Klebsiella aerogenes by P1 transduction. It is linked to pyrF and trp with the order pyrF-trp-gdh. Complementation analysis using F' episomes from Escherichia coli suggests an analogous location in E. coli. Two mutants able to produce glutamate dehydrogenase in the presence of high levels of glutamine synthetase have been isolated. One, tightly linked to gdhD, shows normal repression control by glutamine synthetase but produces four times as much glutamate dehydrogenase activity as does the wild type under all conditions tested. The other revertant is not linked to gdhD or glnA.


Adenosine 5'-triphosphate synthesis energized by an artificially imposed membrane potential in membrane vesicles of Escherichia coli. Adenosine 5'-triphosphate (ATP) synthesis driven by an artificially imposed membrane potential in right-side-out membrane vesicles of Escherichia coli was investigated. Membrane vesicles prepared in the presence of adenosine diphosphate were loaded with K+ by incubation with 0.5 M potassium phosphate. Addition of valinomycin resulted in the synthesis of 0.2 to 0.3 nmol of ATP/mg of membrane protein, whereas no synthesis was observed after addition of nigericin. Addition of K+, dicyclohexylcarbodiimide, carbonylcyanide p-trifluoromethoxyphenylhydrazone, or azide to the assay buffer inhibited ATP synthesis. Adenosine diphosphate and Mg2+ were found to be required. Ca2+, which can replace Mg2+ for the hydrolytic activity of the Mg2+-adenosine triphosphatase (ATPase) (EC 3.6.1.3), could not replace Mg2+ in the synthetic reaction and, in fact, inhibited ATP synthesis even in the presence of Mg2+. Strain NR-70, a mutant lacking the Mg2+-ATPase, was unable to synthesize ATP using an artificially imposed membrane potential. Additionally, the Mg2+-ATPase was found to contain tightly bound ATP.


Regulation of enzyme formation in Klebsiella aerogenes by episomal glutamine synthetase of Escherichia coli. We studied the physiology of cells of Klebsiella aerogenes containing the structural gene for glutamine synthetase (glnA) of Escherichia coli on an episome. The E. coli glutamine synthetase functioned in cells of K. aerogenes in a manner similar to that of the K. aerogenes enzyme: it allowed the level of histidase to increase and that of glutamate dehydrogenase to decrease during nitrogen-limited growth. The phenotype of mutations in the glnA site was restored to normal by the introduction of the episomal glnA+ gene. These results are consistent with the hypothesis that glutamine synthetase regulates the function of its own structural gene.


Kinetic properties of Serratia marcescens adenosine 5'-diphosphate glucose pyrophosphorylase. The regulatory properties of partially purified adenosine 5'-diphosphate-(ADP) glucose pyrophosphorylase from two Serratia marcescens strains (ATCC 274 and ATCC 15365) have been studied. Slight or negligible activation by fructose-P2, pyridoxal-phosphate, or reduced nicotinamide adenine dinucleotide phosphate (NADPH) was observed. These compounds were previously shown to be potent activators of the ADPglucose pyrophosphorylases from the enterics, Salmonella typhimurium, Enterobacter aerogenes, Enterobacter cloacae, Citrobacter freundii, Escherichia aurescens, Shigella dysenteriae, and Escherichia coli. Phosphoenolpyruvate stimulated the rate of ADPglucose synthesis catalyzed by Serratia ADPglucose pyrophosphorylase about 1.5- to 2-fold but did not affect the S0.5 values (concentration of substrate required for 50% maximal stimulation) of the substrates, alpha-glucose-1-phosphate, and adenosine 5'-triphosphate. Adenosine 5'-monophosphate (AMP), a potent inhibitor of the enteric ADPglucose pyrophosphorylase, is an effective inhibitor of the S. marcescens enzyme. ADP also inhibits but is not as effective as AMP. Activators of the enteric enzyme counteract the inhibition caused by AMP. This is in contrast to what is observed for the S. marcescens enzyme. Neither phosphoenolpyruvate, fructose-diphosphate, pyridoxal-phosphate, NADPH, 3-phosphoglycerate, fructose-6-phosphate, nor pyruvate effect the inhibition caused by AMP. The properties of the S. marcescens HY strain and Serratia liquefaciens ADPglucose pyrophosphorylase were found to be similar to the above two S. marcescens enzymes with respect to activation and inhibition. These observations provide another example where the properties of an enzyme found in the genus Serratia have been found to be different from the properties of the same enzyme present in the enteric genera Escherichia, Salmonella, Shigella, Citrobacter, and Enterobacter.


Characterization of group B colicin-resistant mutants of Escherichia coli K-12: colicin resistance and the role of enterochelin. Nine classes of group B colicin-resistant mutants were examined to study the role of enterochelin in colicin resistance. Four of the mutants studied (cbt, exbC, exbB, and tonB) hypersecreted enterochelin. Enterochelin hypersecretion was apparently responsible for resistance of the exbC mutant to colicins G and H and for resistance of the exbB mutant to colicins G, H, Ia, Ib, S1, and V. All four mutants scored as colicin B tolerant, even in the absence of enterochelin synthesis. The mutants produced substantially increased amounts of two high-molecular-weight outer membrane polypeptides when grown under limiting iron conditions. The presence of these polypeptides was correlated with increased colicin B-neutralizing activity in the outer membrane preparations.


Metabolism of the reserve polysaccharide of Streptococcus mitior (mitis): is there a second alpha-1,4-glucan phosphorylase? The alpha-1,4-glucan phosphorylase (alpha-1,4-glucan: orthophosphate glucosyltransferase; EC 2.4.1.1) associated with the particulate cell fraction of Streptococcus mitior strain S3 was compared with the soluble maltodextrin phosphorylase that had been previously isolated from the same organism (Walker et al., 1969). The particulate enzyme was more sensitive to the glycogen content of the cell than the soluble euzyme; its activity was highest when the cells were grown under conditions favoring high glycogen storage. Substrate specificities of the two high activity towards endogenous glycogen, whereas low-molecular-weight maltodextrins were the preferred substrates for the soluble phosphorylase. The purification of the particulate phosphorylase included incubation of the particulate fraction in 160 mM sodium phosphate-10 mM sodium citrate-0.1% (wt/vol) Triton X-100 buffer (pH 6.7) and ion-exchange chromatography on diethylamino-ethyl- Sephadex A-50. The purified enzyme was fully soluble. The value for the purification factor was variable and depended on (i) the substrate used and (ii) whether the synthetic or the degradative reaction was being measured. The solubilization resulted in considerable changes in the properties of the phosphorylase: the pH optimum for activity was raised from 6.0 to 7.0-7.5 and the substrate specificity was altered. Consequently, the purified enzyme bore greater similarity to the soluble maltodextrin phosphorylase. The reported results are best explained in terms of a single phosphorylase, the specificity which is determind by its binding state in the cell. The enzyme acts as a glycogen phosphorylase in the particulate state and as a maltodextrin phosphorylase when soluble. The equilibrium between the two forms is related to the glycogen content of the cells.


Polygalacturonic acid trans-eliminase in the osmotic shock fluid of Erwinia rubrifaciens: characterization of the purified enzyme and its effect on plant cells. An endopolygalacturonic acid trans-eliminase (EC 4.2.2.2), released by osmotic shock of Erwinia rubrifaciens cells, has been purified to near homogeneity (3, 100-fold) by column chromatography on diethylaminoethyl-cellulose, phosphocellulose, and hydroxyapatite-cellulose followed by isoelectric focusing. It has a molecular weight of 41,000, s20,w of 3.09S, an isoelectric point of pH 6.25, pH optimum of 9.5, and a temperature optimum of 37 C and requires Ca2+ with an optimum concentration of 0.5 to 1.0 mM. Mg2+ could not substitute for Ca2+. Tyrosinyl residues seem essential for enzyme catalysis based on rapid inactivation by tetranitromethane. The enzyme prefers unmethylated polygalacturonic acid as the substrate, cleaving alpha-1,4-glycosidic linkages randomly to form unsaturated galacturonides at a Vmax of 1,166 mumol of product/min per mg of protein and a Km of 5 mg of polygalacturonic acid per ml. Over 90% of the enzyme activity is released from osmotically shocked E. rubrifaciens cells. Unlike E. rubrifaciens, trans-eliminase is not released from Erwinia carotovora cells by osmotic shock treatment, but enzyme activity is detected in the culture medium. The release of the enzyme is reduced fivefold by the addition of dibutyryl cyclic adenosine 5'-monophosphate. The hypersensitive reaction in tobacco leaves was induced within 60 min after injection of less than 1 mug of purified E. rubrifaciens trans-eliminase. Single cells of tobacco in suspension culture are readily killed by the enzyme, whereas tobacco protoplasts remain unaffected when treated in the same manner. These results indicate that endopolygalacturonic acid trans-eliminase is a constitutive enzyme possibly located in the periplasmic space of the E. rubrifaciens cell and releases enzyme into the culture medium in the presence of substrate. The release of the enzyme in tobacco tissue and the trans-eliminative cleavage of plant cell wall components may be steps leading to hypersensitivity of the tobacco tissue.


Purification of several bacteriolytic enzymes by affinity chromatography on lysozyme-lysate of Micrococcus lysodeikticus cell wall coupled with sepharose. Using lysozyme-lysate of Micrococcus lysodeikticus cell wall coupled with Sepharose, several bacteriolytic enzymes were purified from crude preparations of animal and microbial origin. Quail egg-white, human milk and salivary lysozymes [EC 3.2.1.17] were adsorbed onto the adsorbent at pH 5-7 and eluted with 2M NaCl at pH 10. By means of these treatments, lysozymes were purified 20-250 fold with activity recoveries of 60-80%, and the quail lysozyme thus purified was shown to be discelectrophoretically homogeneous. Some bacteriolytic enzymes of microbial origin were also highly purified by using this affinity adsorbent. A bacterial lysozyme from Bacillus sp. ML-208 showed high affinity for the ligand and was not eluted under the conditions mentioned above, but was recovered by elution with 2M guanidine-HCl at pH 5.8, resulting in a 500-fold increase in the specific activity. A Pseudomonas-lytic enzyme from Streptomyces sp. P-51 was easily released from the adsorbent by elution with 0.5M NaCl at pH 5.0. A staphylolytic F2 enzyme from S. griseus S-35 and a chitinase [EC 3.2.1.14] from yam, both of which were completely inert toward M. lysodeikticus cell wall, passed through the adsorbent column. A modified ligand, in which muramic acid and glucosamine residues were N,O-acetylated, failed to adsorb any of these animal and bacterial lysozymes. Some of the enzymatic properties and bacteriolytic action spectra of these purified enzymes are also described in this paper in comparison with those of hen egg-white lysozyme.


Enzymatic studies on a cellulase system of Trichoderma viride. III. Transglycosylation properties of two cellulase components of random type. Two highly purified cellulases [EC 3.2.1.4], II-A, and II-B, were obtained from the cellulase system of Trichoderma viride. Both cellulases split cellopentaose retaining the beta-configuration of the anomeric carbon atoms in the hydrolysis products at both pH 3.5 and 5.0. The Km values of cellulases II-A and II-B for cellotetraose were different, but their Vmax values were similar and those for cellooligosaccharides increased in parallel with chain length. Both cellulases produced predominantly cellobiose and glucose from various cellulosic substrates as well as from higher cellooligosaccharides. Cellulase II-A preferentially attacked the holoside linkage of rho-nitrophenyl beta-D-cellobioside, whereas cellulase II-B attacked mainly the aglycone linkage of this cellobioside. Both cellulases were found to catalyze the synthesis of cellotriose from rho-nitrophenyl beta-D-cellobioside by transfer of a glucosyl residue, possibly to cellobiose produced in the reaction mixture. They were also found to catalyze the rapid synthesis of cellotetraose from cellobiose, with accompanying formation of cellotriose and glucose, which seemed to be produced by secondary random hydrolysis of the cellotetraose produced. The capacity to synthesize cellotetraose from cellobiose appeared to be greater with cellulase II-B than with cellulase II-A.


Binding of N-acetyl-chitotriose to human lysozyme. The interaction of N-acetyl-chitotriose ((GlcNAc)3) with human lysozyme [EC 3.2.1.17] was studied at various pH values by measuring changes in the circular dichroic (CD) band at 294 or 255 nm and the data were compared with the results for hen and turkey lysozymes reported previously (Kuramitsu et al. (1974) J. Biochem.76, 671-683; Kuramitsu et al. (1975) J. Biochem. 77, 291-301). The pH dependence of the binding constant of (GlcNAc)3 to human lysozyme was different from those for hen and turkey lysozymes. The catalytic carboxyls of human lysozyme, Asp 52 and Glu 35, were not perturbed on binding of (GlcNAc)3. This is consistent with the previous findings that the macroscopic pK values of Asp 52 and Glu 35 of human lysozyme are 3.4 and 6.8 at 0.1 ionic strength and 25 degrees and were unchanged on complexing with (GlcNAc)3. An ionizable group with pK 4.5, which participates in the binding of (GlcNAc)3 to hen lysozyme and was assigned  as Asp 101, did not participate in the binding of the saccharide to human lysozyme. Between pH 9 and 11, the binding constants of (GlcNAc)3 to hen lysozyme remained unchanged, whereas perturbation of an ionizable group with pK 10.5 to 10.0 was observed for human lysozyme. This group may be Tyr 62 in the active-site cleft. The binding constants of (GlcNAc)3 to human lysozyme molecules having different microscopic protonation forms, with respect to the catalytic carboxyls, were estimated using the binding constants obtained in the present experiments and the microscopic ionization constants of the catalytic carboxyls obtained previously. All four species of human lysozyme had similar binding constants to (GlcNAc)3. This result is different from those for hen and turkey lysozymes.


Kinetics and equilibrium studies on autologous and heterologous recombinations of heavy and light chains of myeloma proteins. 1. The kinetics of the heterologous recombination reaction of alkylated H chains of a myeloma protein (Jo) with alkylated L chains of another myeloma protein (Ita) were studied by following changes with time in the circular dichroism at 235 nm and the results were compared with those for the autologous recombination of Jo-H chains with Jo-H chains reported previously (T. Azuma et al.(1975) J. Biochem. 77, 473-479 and the preceding paper). The heterologous reaction also followed second-order kinetics. The second-order rate constant (kapp) for heterologous recombination was about seven times smaller than that for autologous recombination at pH 5.5, while they were similar between pH 4.2 and 4.7.  2. The apparent association constants (Kapp) for the reaction, H2+L2=H2L2, were determined by measuring the ellipticities at 235 nm of mixtures of H and L chains in various ratios. The values of Kapp for the autologous and heterologous recombinations were both pH-dependent and changed from 10(6) M-1 at pH 3.9 to 108 M-1 at pH 4.3. Using these values of kapp and Kapp, the half-time for the dissociation of autologous H2L2 to H2 and L2 at pH 4.3 was estimated to be 80 hr.


Studies on phospholipases from Streptomyces. II. Purification and properties of Streptomyces hachijoensis phospholipase D. 1. Phospholipase D [EC 3.1.4.4] from Streptomyces hachijoensis was purified about 570-fold by column chromatography on DEAE-cellulose and Sephadex G-50 followed by isoelectric focusing. 2. The purified preparation was found to be homogeneous both by immunodiffusion and polyacrylamide disc gel electrophoresis. 3. The isoelectric point was found to be around pH 8.6 and the molecular weight was about 16,000. 4. The enzyme has maximal activity at pH 7.5 at 37 degrees. The optimal temperature is around 50 degrees at pH 7.5, using 20 min incubation. 5. The enzyme was stable at 50 degrees for 90 min. At neutral pH, between 6 and 8, the enzyme retained more than 95% of its activity on 24 hr incubation at 25 degrees. However, the enzyme lost 80% of its activity under the same conditions at pH 4.0. 6. The enzyme was stimulated slightly by Ca2+, Mn2+, and Co2+, and significantly by Triton X-100 and ethyl ether. It was inhibited by Sn2+, Fe2+, Fe3+, Al3+, EDTA, sodium dodecyl sulfate, sodium cholate, and cetylpyridinium chloride. 7. This phospholipase D hydrolyzes phosphatidylethanolamine, phosphatidylcholine, cardiolipin, sphingomyelin, phosphatidylserine, and lysophosphatidylcholine, liberating the corresponding bases. 8. The Km value was 4mM, determined with phosphatidylethanolamine as a substrate.