Characterization of the terminal stages of chlorophyll (ide) synthesis in etioplast membrane preparations. 1. Chlorophyll (ide) formation from protochlorophyll (ide) that is normally inactive was demonstrated in etioplast membranes isolated from maize and barlley plants, the process being dependent on intermittent illumination and the addition of NADPH. 2. The addition of NADPH to the membranes was shown to result in the conversion of inactive protochlorophyll (ide) absorbing at about 630 nm into a form(s) with light-absorption maxima at about 640 and 652 nm, both of which disappear when chlorophyll (ide) is formed on illumination. 3. The temperature-dependence of the activation process and its response to a variety of reagents were examined. From these, the conclusion is drawn that -SH groups are involved in the activation but in the active complex these are unavailable for reaction with -SH reagents. 4. Evidence is presented for the occurrence of glucose 6-phosphate dehydrogenase activity within etioplasts and the suggestion is made that the oxidative pentose phosphate pathway can provide the NADPH required for chlorophyll biosynthesis during the early stages of greening.


Succinate uptake and related proton movements in Escherichia coli K12. 1. The apparent Km values for succinate uptake by whole cells of Escherichia coli K12 depend on pH in the range 6.5-7.4.2. Uptake of succinate in lightly buffered medium is accompanied by proton uptake. 3. The apparent Km values for succinate uptake and for succinate-induced proton uptake are similar. 4. Approximately two protons enter the cell with each succinate molecule. 5. The pattern of inhibition of succinate uptake is similar to that of succinate-induced proton uptake. 6. Uptake of fumarate and malate, which share the succinate-transport system, is also accompanied by the uptake of approximately two protons per molecule of fumarate or malate. 7. Uptake of aspartate by the dicarboxylic acid-transport system is accompanied by the uptake of approximatley two protons per molecule of asparatate. 8. It is concluded that uptake of dicarboxylic acids by the dicarboxylic acid-transport system is obligatorily coupled to proton uptake such that succinate, malate and fumarate are taken up in electroneutral form and asparate is taken up in cationic form. 9. These results are consistent with, though they do not definitely prove, the energization of succinate uptake of the deltapH.


Evidence against the participation of the gamma-glutamyltransferase-gamma-glutamylcylclotransferase pathway in amino acid transport by rabbit erythrocytes. The GSH concentration of rabbit erythrocytes was monitored under conditions of large net transport of alanine, phenylalane and lysine in the absence of glucose. In no case was there an appreciable alteration in GSH concentration during amino acid uptake. It is suggested that the gamma-glutamyltransferase-gamma-glutamylcyclotransferase pathway does not participate in amino acid transport by these cells.


An investigation of protein conformation of cytochrome c by using cytochrome c insolubilized on to agarose gel. Cytochrome c insolubilized on to agarose gel was shown to be more resistant to denaturation and carboxymethylation than the soluble protein. These differences are discussed both with respect to the conformational changes that take place during denaturation of cytochrome c and with respect to the pH-dependent forms of carboxymethyl-cytochrome c.


Factors affecting the activity of citrate synthase of Acetobacter xylinum and its possible regulatory role. The citrate synthase activity of Acetobacter xylinum cells grown on glucose was the same as of cells grown on intermediates of the tricarboxylic acid cycle. The activity of citrate synthase in extracts is compatible with the overall rate of acetate oxidation in vivo. The enzyme was purified 47-fold from sonic extracts and its molecular weight was determined to be 280000 by gel filtration. It has an optimum activity at pH 8.4. Reaction rates with the purified enzyme were hyperbolic functions of both acetyl-CoA and oxaloacetate. The Km for acetyl-CoA is 18 mum and that for oxaloacetate 8.7 mum. The enzyme is inhibited by ATP according to classical kinetic patterns. This inhibition is competitive with respect to acetyl-CoA (Ki = 0.9 mM) and non-competitive with respect to oxaloacetate. It is not affected by changes in pH and ionic strength and is not relieved by an excess of Mg2+ ions. Unlike other Gram-negative bacteria, the A. xylinum enzyme is not inhibited by NADH, but is inhibited by high concentrations of NADPH. The activity of the enzyme varies with energy charge in a manner consistent with its role in energy metabolism. It is suggested that the flux through the tricarboxylic acid cycle in A. xylinum is regulated by modulation of citrate synthase activity in response to the energy state of the cells.


Bovine spleen cathepsin B1 and collagenolytic cathepsin. A comparative study of the properties of the two enzymes in the degradation of native collagen. Bovine spleen cathepsin B1 and collagenolytic cathepsin were separated by chromatography on Amberlite IRC-50 and collagenolytic cathepsin was partially purified by chromatography on DEAE-Sephadex (A-50). 2. Collagenolytic cathepsin degraded insoluble tendon collagen maximally at pH 3.5 and 28 degrees C; mainly alpha-chain components were released into solution. At 28 degrees C the telopeptides in soluble skin collagen were also cleaved to yield alpha-chain components. Collagenolytic cathepsin was thus similar to cathepsin B1 in its action against native collagen, but mixtures of these two enzymes exhibited a synergistic effect. 3. The addition of thiol-blocking compounds produced similar inhibition of collagenolytic cathepsin and cathepsin B1. The enzyme responded similarly to all other compounds tested except to 6-aminohexanoic acid, when collagenolytic cathepsin was slightly activated and cathepsin B1 was almost unaffected. 4. Leupeptin, which is a structural analogue of arginine-containing synthetic substrates, inhibited collagenolytic cathepsin as effectively as cathepsin B1. Collagenolytic cathepsin was shown to retain a low residual activity against alpha-N-benzoyl-DL-arginine p-nitroanilide during purification which was equivalent to 0.2% of the activity of cathepsin B1. 5. Cathepsin B1 and collagenolytic cathepsin could not be separated by affinity chromatography on organomercurial-Sepharose 4B. The two enzymes could be resolved on DEAE-Sephadex (A-50) and by isoelectric focusing in an Ampholine pH gradient. The pI of the major cathepsin B1 isoenzyme was 4.9 and the pI of collagenolytic cathepsin was 6.4. 6. From chromatography on Sephadex G-75 (superfine grade) the molecular weights were calculated to be 26000 for cathepsin B1 and 20000 for collagenolytic cathepsin. The difference in molecular weight was confirmed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis.


Gamma-glutamyltransferase of rat kidney. Simultaneous assay of the hydrolysis and transfer reactions with (glutamate-14C)glutathione. 1. The hydrolytic and transfer reactions catalysed by rat kidney-gamma-glutamyltransferase (EC 2.3.2.2) were studied in vitro with substrates [U-14C]glutamic acid-labelled glutathione and methionine. Initial-velocity patterns, isotope-exchange and binding studies were consistent with a branched non-sequential mechanism in which a gamma-glutamyl-enzyme intermediate may react either with water (hydrolysis) or with methionine (gamma-glutamyl transfer). 2. The Michaelis constant for glutathione in hydrolysis was 13.9 +/- 1.4 mum, for glutathione in transfer it was 113 +/- 15 muM and for methionine as substrate it was 4.7 +/- 0.7 mM. At substrate concentrations in the ranges of their respective Michaelis constants, the rate of transfer was about ten times higher than that of hydrolysis, but at concentrations of methionine approximating to the physiological (64 muM in rat plasma) the transfer is negligible. 3. The enzyme is reported to lie on the luminal surface of the proximal straight kidney tubule. In this situation, if the kinetic results obtained with the detergent-solubilized enzyme are relevant to the behavior of the enzyme in vivo, it appears likely that the main function of renal gamma-glutamyltransferase is not in amino acid transport, but rather to hydrolyse glutathione in the renal filtrate.


Equilibrium and kinetic studies of the aggregation of porphyrins in aqueous solution. An investigation of the behavior of protoporphyrin IX, deuteroporphyrin IX, haematoporphyrin IX and coproporphyrin III in aqueous solution revealed extensive and complex aggregation processes. Protoporphyrin appears to be highly aggregated under all conditions studied. At concentrations below 4 muM, aggregation of deutero-, haemato- and coproporphyrin is probably restricted to dimerization. At approx. 4muM each of these three porphyrins exhibits sharp changes in spectra consistent with a "micellization" process to form large aggregates of unknown size. This critical concentration increases with increasing temperature and pH, but is not very sensitive to variation in ionic strength. Temperature-jump kinetic studies on deuteroporphyrin also imply an initial dimerization process, the rate constants for which are comparable with those for various synthetic porphyrins, followed by a further extensive aggragation. The ability of a particular porphyrin to dimerize appears to parallel that of the corresponding iron(III) complexes (ferrihaems), although it is thought that ferrihaems do not exhibit further aggregation under these conditions.


Kinetic studies on glucoamylase of rabbit small intestine. The kinetic properties of a maltase-glucoamylase complex with a neutral pH optimum, purified to homogeneity from the brush borders of the rabbit small intestine, are described. It has a broad range of substrate specificity, hydrolysing di- and poly-saccharides with alpha-1,4 and alpha-1,6 linkages. The Km and Vmax, values of the enzyme for the various substrates were determined. Starch and maltose were its best substrates. The kinetics of hydrolysis of two synthetic linear maltosaccharides, namely maltotriose and maltopentaose, were studied. Mixed-substrate incubation studies revealed the presence of at least two interacting sites on the enzyme, and the data were further analysed by the use of a number of non-substrate inhibitors.


Bile acids of snakes of the subfamily Viperinae and the biosynthesis of C-23-hydroxylated bile acids in liver homogenate fractions from the adder, Vipera berus (Linn.). 1. Analysis of bile salts of four snakes of the subfamily Viperinae showed that their bile acids consisted mainly of C-23-hydroxylated bile acids. 2. Incubations of 14C-labelled sodium cholate (3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholan-24-oate) and deoxycholate (3 alpha, 12 alpha-dihydroxy-5 beta-cholan-24-oate) with whole and fractionated adder liver homogenates were carried out in the presence of molecular oxygen and NADPH or an NADPH-generating system. The formation of C-23-hydroxylated bile acids, namely bitocholic acid (3 alpha, 12 alpha, 23xi-trihydroxy-5 beta-cholan-24-oic acid) and 3 alpha, 7 alpha, 12 alpha, 23 xi-tetrahydroxy-cholanic acid (3 alpha, 7 alpha, 12 alpha, 23 xi-tetrahydroxy-5 beta-cholan-24-oic acid), was observed mainly in the microsomal fraction and partly in the mitochondrial fraction. 3. Biosynthetic pathways of C-23-hydroxylated bile acids are discussed.


Isolation and some molecular parameters of elastase-like normal proteinases from horse blood leucocytes. Cytoplasmic granules were isolated from horse blood polymorphonuclear leucocytes by the heparin method and extracted with 0.9% NaCl by repeated freezing. Soluble proteins were separated on a column of Sephadex G-75 followed by chromatography on a column of CM-Sephadex with a NaCl gradient. Gel filtration, density-gradient centrifugation, isoelectric focusing and 0.1% sodium dodecyl sulphate/polyacrylamide-gel electrophoresis at pH 7.0 and at pH 4.5 were used to determine molecular parameters of proteinases. Three enzymes hydrolysing both casein and N-benzyloxycarbonyl-L-alanine nitrophenyl ester were found in the granule extract: proteinase 1, mol.wt. 38000, pI5.3; proteinase 2A, mol.wt. 24500, pI8.8; and proteinase 2B, mol.wt. 20500, pI above 10. The latter two elastase-like proteinases were purified to apparent homogeneity.


Substrate specificity and modifications of the active centre of elastase-like neutral proteinases from horse blood leucocytes. Two proteinases (2A and 2B) purified from the granular fraction of horse blood leucocytes degrade casein (Km values 12.8 and 6mg/ml respectively) with maximum activity at pH 7.4 and in the presence of 2m-urea. Urea-denatured haemoglobin, fibrinogen, albumin and resorcin/fuchsin-stained elastin are digested at a slower rate. The enzymes hydrolyse synthetic substrates of elastase, N-benzyloxycarbonyl-L-alanine 4-nitrophenyl ester (Km 0.114 and 0.178 mM) and N-acetyl-tri-L-alanine methyl ester (Km 5.55 and 0.98 mM), but they do not hydrolyse synthetic substrates of trypsin, chymotrypsin and thrombin. The examined proteinases are completely inhibited by 2 mM-di-isopropyl phosphorfluoridate and show a sensitivity to butyl and octyl isocyanates similar to that of pancreatic elastase. The pH-dependence of their photoinactivation in the presence of Rose Bengal indicates the presence of histidine in the active centre. Proteinase 2A rather insensitive to iodination by IC1 as is pancreatic elastase, whereas proteinase 2B is totally inactivated after incorporation of five iodine atoms per enzyme molecule.


Purification and properties of arylsulphatase B of human liver. 1. A purification scheme for an arylsulphatase B from human liver is described. Specificity of purification was achieved by the use of the affinity chromatography on an agrose-4-hydroxy-2-nitrophenyl sulphate derivative. The scheme provides a rapid and convenient method for preparation of a highly purified enzyme. 2. The purified enzyme was examined by isoelectric focusing electrophoresis on polyacrylamide gel and by ultracentrifugation and was found to be catalytically homogenous, with an apparent molecular weight of 50000 and a specific activity of 93.3 units/mg of protein. 3. The kinetic properties of the purified preparation and the effect of various amino acid group-specific reagents on the catalysis of the enzyme are described. The involvement of histidine residues in the active site of the enzyme is suggested. 4. The purified enzyme lost activity rapidly on freezing. The implication of this observation is discussed in terms of a possible dissociation-reaggregation phenomenon induced by cold treatment.


Estimation of the dissociation constants of enzyme-substrate complexes from steady-state measurements. Interpretation of pH-independence of Km. If the Michaelis constant of an enzyme-catalysed reaction is independent of pH under conditions where the catalytic constant varies with pH, it is equal to the thermodynamic dissociation constant of the enzyme-substrate complex. This is true for realistic mechanisms in which binding and catalytic steps, are clearly distinguished, as well as for the simpler mechanisms that have been considered previously. It is also true for a mechanism in which a bell-shaped pH profile for the catalytic constant results from a change of rate-limiting step with pH. The relaxation time for ionization of a typical group in unbuffered solutions at 25 degrees C is of the order of 0.1 ms at the longest, and is much shorter in buffered solutions. Thus ionizations in almost all enzyme mechanisms can properly be treated as equilibria, provided that ionization is not accompanied by a slow, compulsory change in conformation.


Purification and some properties of acetyl-coenzyme A carboxylase from rabbit mammary gland. 1. Acetyl-Coa carboxylase from lactating-rabbit mammary gland was purified to homogeneity by the criterion of polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. 2. Use of phosphate buffer throughout the purification gave low recovery of enzyme. Consequently, Tris buffers were used in the extraction and in selected stages of the purification procedure. 3. The purified enzyme had a specific activity of 5.15 +/- 0.3 mumol of bicarbonate incorporated/min per mg of protein (mean +/- S.E.M. of five preparations). This represents a purification of 257 +/- 16-fold and a yield of 4.3 +/- 0.13%. 4. The kinetic parameters of the purified enzyme were similar to those reported for the enzyme from other tissue sources. 5. The enzyme was assayed by a spectrophotometric assay and by a [14C]bicarbonate-fixation assay. Short incubation were used in the radio-chemical assay to avoid substantial loss of [14C]bicarbonate.


Density-gradient sedimentation in silica sols. Anomalous shifts in the banding densities of polystyrene "latex" beads. When polystyrene "latex" beads are centrifuged to equilibrium in gradients of Nalcoag 1030, Nalcoag 1034A and Ludox 130M, which are commercial formulations of colloidal silica, the beads at densities that may be markedly lower or higher than the bulk density. Addition to the gradient of small amounts of certain polymers restores the banding densities towards the expected value. These findings closely mimic previously observed "density shifts" of biological particles. A possible model for this anomalous behavior is discussed.


Some observations on the NADP+-linked oxidation of methylglyoxal catalysed by 2-Oxoaldehyde dehydrogenase. In the oxidation of methylglyoxal by 2-oxoaldehyde dehydrogenase, the apparent Km value for NADP+ was about 2.5 times lower than the corresponding Km for NAD+; the apparent Km values for methylglyoxal and for the amine activator L-2-aminopropan-1-ol, with NADP+ as cofactor, were also different from those obtained with NAD+. In the presence of NADP+, the enzyme was not activated by P1, in contrast with the activation of the enzyme when NAD+ was used. The significance of the results is discussed.


Lipid biosynthesis in liver slices of the foetal guinea pig. Lipid synthesis as measured by the incorporation of acetate or 3H2O into slices of foetal liver, is much higher than in slices of adult liver and shows a peak at about two-thirds of gestation. At this time the synthesis from glucose was low and reached a peak 10 days later. The changes in the activity of ATP citrate lyase, which mirrored acetate incorporation, and the effect of glucose and pyruvate on acetate corporation into lipid suggests that some of the lipid synthesis occurs via intramitochondrial acetyl-CoA production from acetate. Despite this, lipid synthesis was not inhibited by (-)-hydroxycitrate. The low rate of synthesis from glucose at two-thirds of gestation is ascribed to the low activity of pyruvate carboxylase at this time and a role for a phosphoenolpyruvate carboxykinase in providing oxaloacetate for lipogenesis is proposed. The activity of fatty acid synthetase broadly agreed with the changes in lipid synthesis, whereas the activity of acetyl-CoA carboxylase was barely sufficient to account for the rates of lipid synthesis in vivo. Acetate and short-chain fatty acids are likely to be the major precursors for lipid synthesis in vivo.


Protein phosphorylation in respiring slices of guinea-pig cerebral cortex. Evidence for a role for noradrenaline and adenosine 3':5'-cyclic monophosphate in the increased phosphorylation observed on application of electrical pulses. 1. Exposure of slices of cerebral cortex from guinea pigs to electrical pulses for 10s or to noradrenaline, 5-hydroxytryptamine or histamine increases the rate of phosphorylation of unidentified proteins in the tissue; the increases in protein phosphorylation due to electrical pulses and noradrenaline were non-additive, whereas the increases due to pulses and 5-hydroxytryptamine or histamine were additive. 2. The stimulating effects of electrical pulses and noradrenaline on protein phosphorylation were antagonized by the beta-adrenergic blocking agents L-propranolol, dichloroisoprenaline, practolol and ICI 66082, but not by the alpha-adrenergic blocking agents, phentolamine and phenoxybenzamine. 3. The increase in protein phosphorylation associated with electrical pulses was antagonized by 10 mum-trifluoperazine and 0.5 mum-prostaglandin E1. 4. It is postulated that under the experimental conditions used the action of electrical pulses on protein phosphorylation is mediated by noradrenaline acting through a beta-adrenergic receptor mechanism probably involving adenylate cyclase.


Localization of inhibition by adenosine diphosphate of phosphoglycerate-dependent oxygen evolution in a reconstituted chloroplast system. ADP was shown to inhibit phosphoglycerate-dependent O2 evolution in a simplified reconstituted chloroplast system containing 3-phosphoglycerate kinase and triose phosphate dehydrogenase. Rates of O2 evolution in the simplified system are comparable with those obtained by using stromal protein rather than purified enzymes. ADP does not inhibit O2 evolution with glycerate 1,3-biphosphate as substrate nor does it inhibit triose phosphate dehydrogenase. The inhibitory effect of ADP is attributed to an increase in the rate of conversion of glycerate biphosphate into phosphoglycerate. The results are discussed in terms of control by ADP of phosphoglycerate-dependent oxygen evolution.


Regulation of pyruvate dehydrogenase and pyruvate dehydrogenase phosphate phosphatase activity in rat epididymal fat-pads. Effects of starvation, alloxan-diabetes and high-fat diet. 1. Pyruvate dehydrogenase phosphate phosphatase activity in rat epididymal fat-pads was measured by using pig heart pyruvate dehydrogenase [32P]phosphate. About 80% was found to be extramitochondrial and therefore probably not directly concerned with the regulation of pyruvate dehydrogenase activity. The extramitochondrial activity was sensitive to activation by Ca2+, but perhaps less sensitive than the mitochondrial activity.


Tyrosine aminotransferase activity in the rabbit placenta. Evidence for decreased inducibility by insulin and cortisol as a function of gestational age. Tyrosine aminotransferase is present at low activity in the rabbit placenta. The activity can be enhanced by injecting the pregnant mother with insulin or cortisol. This response decreases over the last half of gestation (14-28 days gestational age).


Changes in the activities of the enzymes of hepatic fatty acid oxidation during development of the rat. 1. Changes in the activities of several enzymes involved in mitochondrial fatty acid oxidation were measured in livers of developing rats between late foetal life and maturity. The enzymes studied are medium- and long-chain ATP-dependent acyl-CoA synthetases of the outer mitochondrial membrane and matrix, GTP-dependent acyl-CoA synthetase, carnitine acyltransferase, enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase, general 3-oxoacyl-CoA thiolase and acetoacetyl-CoA thiolase.


Sex differences in anaesthetic toxicity: fluroxene and trifluoroethanol in mice. A sex difference in postanaesthetic mortality after fluroxene anaesthesia was found in Swiss Webster mice. More males succumbed than females. This toxicity was biotransformation-dependent and could be reversed by pretreatment with "opposite" sex hormones. The toxicity of the fluroxene metabolite trifluoroethanol also was more marked in male mice, but was only partially influenced by microsomal enzyme inhibitors or stimulators, or by sex hormones.


Light-induced membrane potential and pH gradient in Halobacterium halobium envelope vesicles. Illumination of envelope vesicles prepared from Halobacterium halobium cells causes translocation of protons from inside to outside, due to the light-induced cycling of bacteriorhodopsin. This process results in a pH gradient across the membranes, an electrical potential, and the movements of K+ and Na+. The electrical potential was estimated by following the fluorescence of a cyanine dye, 3,3'-dipentyloxadicarbocyanine. Illumination of H. halobium vesicles resulted in a rapid, reversible decrease of the dye fluorescence, by as much as 35%. This effect was not seen in nonvesicular patches of purple membrane. Observation of maximal fluorescence decreases upon ilumination of vesicles required an optimal dye/membrane protein ratio. The pH optimum for the lightinduced fluorescence decrease was 6.0. The decrease was linear with actinic light intensity up to about 4 X 10(5) ergs cn-2 s-1. Valinomycin, gramicidin, and triphenylmethylphosphonium ion all abolished the fluorescence changes. However, the light-induced pH change was enhanced by these agents. Conversely, buffered vesicles showed no pH change but gave the same or larger fluorescence changes. Thus, we have identified the fluorescence decrease with a light-induced membrane potential, inside negative. By using valinomycin-K+-induced membrane potentials, we calibrated the fluorescence decrease with calculated Nernst diffusion potentials. We found a linear dependence between potential and fluorescence decrease of 3 mV/%, up to 90 mV. When the envelope vesicles were illuminated, the total proton-motive force generated was dependent on the presence of Na+ and K+ and their concentration gradients across the membrane. In general, K+ appeared to be more permeable than Na+ and, thus, permitted development of greater pH gradients and lower electrical potentials. By calculating the total proton-motive force from the sum of the pH and potential terms, we found that the vesicles can produce proton-motive forces near--200 mV.


A novel phosphodiesterase from cultured tobacco cells. A novel phosphodiesterase was purified from cultured tobacco cells to a state which appeared homogeneous on polyacrylamide gel electrophoresis. The enzyme hydrolyzed various phosphodiester and pyrophosphate bonds, including p-nitrophenyl thymidine 5'-phosphate, p-nitrophenyl thymidine 3'-phosphate, cyclic nucleotides, ATP, NAD+, inorganic pyrophosphate, dinucleotides, and poly(adenosine diphosphate ribose), which is a polymer synthesized from NAD+. However, it did not hydrolyze highly polymerized polynucleotides. The molecular weight of the native enzyme was estimated as 270 000 to 280 000 by gel filtration on Sephadex G-200 and Bio-Gel A-5m. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the enzyme was composed of subunits with molecular weights calculated to be 75 000. The enzyme did not require divalent cations for activity being fully active in the presence of ethylenediaminetetraacetic acid. The pH optimum for the enzyme was approximately 6 with p-ni-trophenyl thymidine 5'-phosphate or adenosine cyclic 3',5'monophosphate, and 5.3 with NAD+. Double reciprocal plots of the initial velocity against the concentration of p-nitrophenyl thymidine 5'-phosphate gave two apparent Km values of 0.17 and 1.3 mM, suggesting the presence of at least two active sites.


Carbon-13 nuclear magnetic resonance of heme carbonyls. Cytochrome c and carboxymethyl derivatives of cytochrome c. Carbonyl complexes of horse cytochrome c and various carboxymethylated derivatives have been examined using 13C NMR (carbon-13 nuclear magnetic resonance) spectroscopy. The multiplicity and chemical shift of the 13CO resonance were found to be functions of pH and the extent of alkylation. Correlations have been made among prominent features of the chemical shift titration curves and changes in the environment of the heme. A simple model compatible with the bulk of previous observations has been suggested to account for the several carbonyl resonance peaks and the complex behavior of the chemical shift with changes in pH.


Complete tyrosine assignments in the high-field 1H nuclear magnetic resonance spectrum of bovine pancreatic trypsin inhibitor selectively reduced and carboxamidomethylated at cystine 14-38. The low-field portions of the 250-MHz 1H nuclear magnetic resonance spectra of native and chemically modified basic pancreatic trypsin inhibitor have been studied as a function of pH over the range pH 5-13. In derivatives selectively reduced and carboxamidomethylated at cystine 14-38, resonances associated with 15 of the 16 protons of the aromatic rings of the four tyrosines of the inhibitor have been located and assigned to specific tyrosyl residues. Titrations of pH yielded pK's for tyrosines 10, 21, 23, and 35 in the modified inhibitor of 9.9, 10.6, 11.6, and 11.0, respectively. Resonances associated with the three nitrotyrosine 10 protons of the mononitrated derivative and the six nitrotyrosine 10 and 21 protons of the dinitrated derivative have been similarly located, assigned, and titrated, yielding pK's for nitrotyrosines 10 and 21 of 6.5 and 6.4, respectively. Previously reported results for derivatives with cystine 14-38 intact have been revised on the basis of new data. Comparison of these revised results with the new data for derivatives with modified cystine 14-38 reveals no changes in pK's for any tyrosine or nitrotyrosing ring and no changes in chemical shift for resonances of nitrotyrosine 21 or tyrosines 21 and 23. However, modification of cystine 14-38 causes significant changes in chemical shifts of resonances of the nearby nitrotyrosine 10 and tyrosine 10 and 35 rings. Tyrosine 35 remains relatively immobile, rotating less than 1600 times/s at 25 degrees C for pH's in the range 5-13.


The pH dependence of the conformation of angiotensin peptides by nuclear magnetic resonance: cis-trans isomerism of proline 7. The pH dependence of the proton NMR spectrum of [Asn1, Val5] angiotensin II in aqueous solution shows the existence of one major and one minor conformation above pH 6.5, the minor conformation representing 12 +/- 2% of the total peptide. A similar observation has been made for (Asn1, Val5) angiotensin I and Val-Tyr-Val-His-Pro-Phe. This effect is not due to the presence of angiotensin-like impurities in the peptide samples. We have shown two expected impurities, [beta-Asp1, Val5] angiotensin II and [Asn1, 3-Bzl-Ty4, Val5] - angiotensin II, to be absent, and a third impurity [Asn1, Val5, D-His6] angiostensin II, to be present at less than or equal to 2.1 mol%, too little to account for the observed amount (12 +/- 2%) of minor conformation. The carbon-13 spectrum of the hexapeptide at high pH shows that the major conformation has Pro7 in the trans form and the minor conformation has Pro7 in the cis form.


Stereochemical analysis of the elimination reaction catalyzed by D-amino-acid oxidase. The stereochemistry of the intramolecular proton transfer catalyzed by the flavoenzyme, D-amino-acid oxidase, during the elimination reaction of beta-chloro-alpha-amino acid substrates (Walsh et al. (1973), J. Biol. Chem. 248, 1964) has been established. Both D-erythro- and D-threo-2-amino-3-chloro(2-3H) butyrate have been shown to yield (3R)-2-keto (3-3H)-2- butyrate predominantly. Tritium kinetic isotope effects on the rate of the reaction (4.7 for the D-erythro, and 3.8 for the D-threo compound) and percentages of intramolecular triton transfer (7.2% for the D-erythro- and 2.6% for the D-threo compound) have been measured. Their implications on the mechanism of this unusual elimination reaction are discussed.


The subcellular distribution and partial characterization of cholinesterase activities of canine platelets. The multiple cholinesterase activities in canine platelets have been investigated. Platelets were homogenized by rapid decompression under nitrogen, glass tube/Teflon pestle, and glycerol lysis techniques. Rapid decompression under nitrogen technique was found to be the most efficient and gentle method for cell disruption. Homogenates were subfractionated using sodium diatrizoate density gradients. Marker enzyme assays and pulse labeling experiments with 5-hydroxyl[14C] tryptamine and [125I] thrombin on prepared subcellular fractions confirmed that the soluble, plasma membrane and the granule-1 fractions were all in reasonably pure form. Furthermore, labeling of the plasma membrane with [125I] thrombin is cited as the first successful attempt at attaining significantly bound marker for this structure. Cholinesterase activity distributions measured in these fractions indicated that about 30% of the activity was present in the plasma membrane, 50% in granule-1 and 5% in soluble fractions. Kinetic data of cholinesterase activities obtained from intact platelets, plasma membrane preparations and platelet release supernatants indicated that they are strikingly similar.


Effect of Ca2+, ruthenium red and ageing on pregnenolone production by mitochondrial fractions from normal and luteinizing hormone treated rat testes. The effect of Ca2+ in vitro on pregnenolone production rates under various incubation conditions by mitochondrial fractions isolated from testes of normal rats and of rats after in vivo treatment with luteinizing hormone has been investigated. Concentrations of Ca2+ in the range of 0.1-0.5 mM stimulated succinate supported pregnenolone production in mitochondrial fractions from both control and luteinizing hormone treated testes. When mitochondrial fractions were isolated in 0.25 M sucrose without additions, Ca2+ in vitro increased succinate supported pregnenolone production rates in mitochondrial fractions isolated from control testes to a greater extent than in mitochondrial fractions, from luteinizing hormone treated testes. Production rates in control mitochondrial fraction, incubated in the presence of initial Ca2+ concentrations of 0.7 mM and higher were almost similar to production rates in relevant luteinizing hormone treated mitochondria.


Subcellular localizations of guanylate cyclase and 3',5'-cyclic nucleotide phosphodiesterase in sea urchin sperm. The subcellular localizations of guanylate cyclase and 3',5'-cyclic nucleotide phosphodiesterase in sea urchin sperm were examined. Both the specific and total activities of these two enzymes were much higher in sperm flagella (tails) than in the heads. In addition to the observation that guanylate cyclase in the flagella was particulate-bound and solubilized by Triton X-100, more than 80% of the cyclase activity in the flagella was found in the plasma membrane fraction, whereas the activity of cyclic nucleotide phosphodiesterase was observed in both the axonemal and plasma membrane fractions. The observations indicated that the cyclase in the flagella appeared to be associated with the plasma membrane. Cyclic nucleotide phosphodiesterase in the plasma membrane fraction as well as the axonemal fraction hydrolyzed both cyclic GMP and cyclic AMP; however, the rates of hydrolysis for cyclic GMP were obviously higher than those for cyclic AMP. The enzymic properties of guanylate cyclase and cyclic nucleotide phosphodiesterase in sperm flagella were also briefly described.


Transport and utilization of the biosynthetic intermediate shikimic acid in Escherichia coli. Auxotrophic mutants of Escherichia coli W or K12 blocked before shikimic acid in the aromatic biosynthetic pathway grew poorly on shikimic acid as sole aromatic supplement. This poor growth response was correlated with a relatively poor ability to transport shikimic acid. If citrate was present in the growth medium (as it is in some commonly used basal media) the growth of some of the E. coli K12 mutants on shikimate was further reduced. Mutants were derived from pre-shikimate auxotrophs which grew rapidly on media containing shikimic acid. These derivatives all had an increased ability to transport shikimic acid. Thus, it is proposed that the growth on shikimate observed in the parent cells is restricted by their relatively poor uptake of shikimate from the medium and that this restriction may be removed by a mutation which enhances shikimate transport. Transduction analysis of the mutations which enhanced utilization and transport of shikimic acid by E. coli K12 strains indicated at least two classes. Class 1 was about 20% cotransduced with the histidine region of the E. coli K12 chromosome and appeared to be coincident with a known shikimate transport locus, shiA. Class 2 was not cotransduced with his. The locus (or loci) of this class is unknown. Kinetic measurements suggested that both classes had shikimate uptake systems derived from the wild-type system. Two class 1 mutants had increased levels of otherwise unaltered wild-type transport while one class 2 mutant had an altered Michaelis constant (Km) for shikimate transport.


Enzymatic synthesis of 2-O-alpha-D-mannopyranosyl-methyl-alpha-D-mannopyranoside by a cell-free particulate system of Mycobacterium smegmatis. A cell-free particulate enzyme preparation of Mycobacterium smegmatis ATCC 607 catalyzed the transfer of labeled mannose from GDP[14C] mannose to methyl-alpha-D-mannopyranoside (an exogenously added acceptor) to form a product that was characterized to be 2-O-alpha-D[14C] mannopyranosyl-methyl-alpha-D-mannopyranoside. This transmannosylase activity was specific for both the sugar nucleotide donor and methyl monosaccharide acceptor. The reaction was stimulated by the addition of various metal ions and had a pH optimum of 6.0. The apparent Km of this transmannosylase reaction for methyl-alpha-D-mannopyranoside was 35 mM. The possible relationship between this "artificial" mannosyl-transfer system and the "natural" system which leads to the formation of the oligomannosides and glycoproteins is discussed.


Biosynthesis of yeast mannan. Diversity of mannosyltransferases in the mannan-synthesizing enzyme system from yeast. 1. A microsomal enzyme preparation from the yeast Saccharomyces cerevisiae catalyzes the transfer of mannosyl units from GDPmannose to mannose and a number of mannose-containing oligosaccharides and glycosides whereby different glycosidic bonds are formed. 2. Of the compounds tested besides mannose, only those containing an alpha-linked mannosyl unit at the nonreducing position of their molecule were effective as acceptors. Monodeoxyanalogues of mannose as well as alpha-mannose phosphates did not serve as acceptors in the above reaction. 3. The structure of the product formed with mannose as acceptor was determined to be O-alpha-D-mannosyl-(1 leads to 2)-mannose; with alphaMan (1 leads to 6)mannose as the acceptor, the product was alphaMan(1 leads to 6)mannose and with alphaMan-(1 leads to 2)mannose the product was tentatively characterized as a mixture of alphaMan-(1 leads to 3)alphaMan(1 leads to 2)mannose and alphaMan(1 leads to 2)alphaMan(1 leads to 2)mannose. 4. The enzymes catalyzing the formation of different types of glycosidic bonds differed in their acceptor specificity, pH-activity curves and rates of heat denaturation. 5. Radioactive disaccharides were unable to enter the mannan protein molecule in the cell-free system while free radioactive mannose did incorporate into polysaccharide to a minor extent under the same conditions.


Regulation of 6-hydroxy-2,4,5-triaminopyrimidine synthesis by riboflavin and iron in riboflavin-deficient mutants of Pichia guilliermondii yeast. The effect of riboflavin and iron on 6-hydroxy-2,4,5-triaminopyrimidine synthesis rate was investigated in the cultures of the yeast Pichia guilliermondii (rib2 mutants) with the blocked second reaction to flavinogenesis. It was shown that riboflavin inhibited the 6-hydroxy-2,4,5-triaminopyrimidine synthesis rate in iron-rich and iron-deficient cells of mutants with low riboflavin requirements. Cycloheximide did not prevent the stimulation of 6-hydroxy-2,4,5-triaminopyrimidine synthesis caused by riboflavin starvation. 7-methyl-8-trifluoromethyl-10-(1'-D-ribityl)isoalloxazine strongly inhibited the 6-hydroxy-2,4,5-triaminopyrimidine synthesis, while 7-methyl-8-trifluoro-methyl-10-(beta-hydroxyethyl)izoalloxazine and galactoflavin exerted only a slight effect on this process. The 6-hydroxy-2,4,5-triaminopyrimidine synthesis rate in iron-deficient cells was significantly higher than in iron-rich cells. The 2,2'-dipyridyl treatment of iron-rich cells caused the stimulation of 6-hydroxy-2,4,5-triaminopyrimidine synthesis and cycloheximide abolished this effect. The results suggest that the activity of the first enzyme of flavinogenesis (guanylic cyclohydrolase) is under the control of feedback inhibition by flavins and the biosynthesis of this enzyme is regulated by iron.


Resistance of Pediococcus cerevisiae to amethopterin as a consequence of changes in enzymatic activity and cell permeability. I. Dihydrofolate reductase, thymidylate synthetase and formyltetrahydrofolate synthetase in amethopterin-resistant and -sensitive strains of Pediococcus cerevisiae. Pediococcus cerevisiae/AMr, resistant to amethopterin, possesses a higher dihydrofolate reductase (5, 6, 7, 8-tetrahydrofolate: NADP+ oxidoreductase, EC 1.5.1.3) activity than the parent, a folate-permeable and thus amethopterin-susceptible strain and than the wild-type. The properties of dihydrofolate reductase from the three strains have been compared. Temperature, pH optima, heat stability, as well amethopterin binding did not reveal significant differences between the enzymes from the susceptible and resistant strains. The enzyme from the wild-type was 10 times more sensitive to inhibition by amethopterin and more susceptible to heat denaturation. The apparent Km values for dihydrofolate in enzymes from the three strains were in the range of 4.8--7.2 muM and for NADPH 6.5--8.0 muM. The amethopterin-resistant strain exhibited cross-resistance to trimethoprim and was about 40-fold more resistant to the latter than the sensitive parent and the wild-type.  The resistance to trimethoprim appears to be a direct result of the increased dihydrofolate reductase activity. Inhibition of dihydrofolate reductase activity by this drug was similar in the three strains. 10--20 nmol caused 50% inhibition of 0.02 enzyme unit. Trimethoprim was about 10 000 times less effective inhibitor of dihydrofolate reductase than amethopterin. The cell extract of the AMr strain possessed a folate reductase activity three times higher than that of the sensitive strain. The activities of other folate-related enzymes like thymidylate synthetase and 10-formyltetrahydrofolate synthetase (formate: tetrahydrofolate ligase (ADP-forming), EC 6.3.4.3) were similar in the three strains studied.


Resistance to Pediococcus cerevisiae to amethopterin as a consequence of changes in enzymatic activity and cell permeability. II. Permeability changes to amethopterin and other folates in the drug-resistant mutant. the accumulation of amethopterin in a Pediococcus cerevisiae strain resistant to this analogue was about 30% of that in P. cerevisiae/PteGlu, the sensitive parent. The uptake in the resistant strain was strictly glucose dependent, whereas in the sensitive parent about 16% accumulation occurred in absence of glucose. The transport in both strains was inhibited by iodoacetate and KF. Amethopterin uptake exhibited saturation kinetics with an apparent Km of 5 muM in P. cerevisiae/AMr and 0.5 muM in P. cerevisiae/PteGlu. The apparent V was 0.2 nmol per min per mg cells (dry weight); the same for both strains. The optimum pH for the uptake of amethopterin by P. cerevisiae/AMr and P. cerevisiae/PteGlu was pH 6.0. Folate and methyltetrahydrofolate competitivity inhibited amethopterin uptake with apparent Ki values of 8 and 0.7 muM, respectively. The uptake of folate exhibited a slightly increased Km value as compared to that of the sensitive strain, whereas the uptake activity velocity was in the same range. Methyltetrahydrofolate accumulated up to about 60-fold higher intracellular concentration than that of the medium, which is a markedly lower accumulation from that in the sensitive strain. The uptake was glucose dependent and inhibited by iodoacetate and KF. The pH optimum for methyltetrahydrofolate uptake in the resistant strain was the same as that in the sensitive parent (pH 5.7--6). In contrast to the increase in the apparent Km value for amethopterin in the resistant strain, the affinity of the carrier for methyltetrahydrofolate was apparently unchanged, whereas the V value was about 16 times lower than that in the sensitive strain. The Ki for amethopterin when added to increasing concentrations of methyltetrahydrofolate was 5.2 muM, a value about the same as that of the Km.


The interaction between vitamin B-12 and micelles in aqueous solution. The apparent pK for benzimidazole displacement of a number of cobalamins is markedly affected by the presence of sodium lauryl sulfate micelles. However, micelles of cetyltrimethylammonium bromide or Triton X have little or no effect on the pK. By measuring the apparent pK as a function of sodium lauryl sulfate concentration, the association constants between the micelles and both base on and base off methylcobalamin were calculated. This calculation indicates that the base off form is strongly associated with the micelle while the base on form is not.


Functional heterogeneity and pH-dependent dissociation properties of human transferrin. Human diferric transferrin was partially labeled with 59Fe at low or neutral pH (chemically labeled) and by replacement of diferric iron previously donated to rabbit reticulocytes (biologically labeled). Reticulocyte 59Fe uptake experiments with chemically labeled preparations indicated that iron bound at near neutral pH was more readily incorporated by reticulocytes than iron bound at low pH. The pH-dependent iron dissociation studies of biologically labeled transferrin solutions indicated that Fe3+, bound at the site from which the metal was initially utilized by the cells, dissociated between pH 5.8 and 7.4. In contrast, lower pH (5.2--5.8) was required to effect dissociation of iron that has remained bound to the protein after incubation with reticulocytes. These findings suggest that each human transferrin iron-binding site has different acid-base iron-binding properties which could be related to the observed heterogenic rabbit reticulocyte iron-donating properties of human transferrin and identifies that the near neutral iron-binding site initially surrenders its iron to these cells.


pH dependence of the oxidation-reduction potential of cytochrome c2. The pH dependence of the spectra and of the oxidation-reduction potential of three cytochromes c2, from Rhodopseudomonas capsulata, Rhodopseudomonas sphaeroides and Rhodomicrobium vannielii, were studied. A single alkaline pK was observed for the spectral changes in all three ferricytochromes. In Rps. capsulata cytochrome c2 this spectroscopic pK corresponds to the pK observed in the dependence of oxidation-reduction potential on pH. For the other two cytochromes the oxidation-reduction potential showed a complex dependency on pH which can be fitted to theoretical curves involving three ionizations. The third ionization corresponds to the ionization observed in the spectroscopic studies but the first two occur without changes in the visible spectra. The possible structural bases for these ionizations are discussed.


Iron-sulfur proteins of the green photosynthetic bacterium Chlorobium. The iron-sulfur proteins of the green photosynthetic bacterium Chlorobium have been characterized by oxidation-reduction potentiometry in conjunction with low-temperature electron paramagnetic resonance spectroscopy. Chlorobium ferredoxin was the only iron-sulfur protein detected in the soluble fraction; no high-potential iron-sulfur protein was observed. In addition, high-potential iron-sulfur protein was not detected in the chromatophores. Four chromatophore-bound iron-sulfur proteins were detected. One is the "Rieske" type iron-sulfur protein with a g-value of 1.90 in the reduced state; the protein has a midpoint potential of + 160 mV (pH 7.0), and this potential is pH dependent. Three g=1.94 chromatophore-bound iron-sulfur proteins were observed, with midpoint potentials of -25, -175, and about -550 mV. A possible role for the latter iron-sulfur protein in the primary photochemical reaction in Chlorobium is considered.


Binding of fluorescent lanthanides to rat liver mitochondrial membranes and calcium ion-binding proteins. (1) Tb3+ binding to mitochondrial membranes can be monitored by enhanced ion fluorescence at 545 nm with excitation at 285 nm. At low protein concentrations (less than 30 mug/ml) no inner filter effects are observed. (2) This binding is localized at the external surface of the inner membrane and is unaffected by inhibitors of respiration or oxidative phosphorylation. (3) A soluble Ca2+ binding protein isolated according to Lehninger, A.L. ((1971) Biochem. Biophys. Res. Commun. 42, 312-317) also binds Tb3+ with enhanced ion fluorescence upon excitation at 285 nm. The excitation spectrum of the isolated protein and of the intact mitochondria are indicative of an aromatic amino acid at the cation binding site. (4) Further characterization of the Tb3+-protein interaction revealed that there is more than one binding site per protein molecule and that these sites are clustered (less than 20 A). Neuraminidase treatment or organic solvent extraction of the protein did not affect fluorescent Tb3+ binding. (5) pH dependency studies of Tb3+ binding to the isolated protein or intact mitochondria demonstrated the importance of an ionizable group of pK greater than 6. At pH less than 7.5 the amount of Tb3+ bound to the isolated protein decreased with increase in pH as monitored by Tb3+ fluorescence. With intact mitochondria the opposite occurred with a large increase in Tb3+ fluorescence at higher pH. This increase was not observed when the mitochondria were preincubated with antimycin  A and rotenone.


A nonspecific inhibitory effect of tRNA on the activity of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase from Saccharomyces cerevisiae. The inhibitory effect of tRNA on yeast 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase (EC 4.1.2.15) has been reinvestigated. From earlier studies the inhibition by tRNAPhe appeared to be quite specific. This study shows that tRNAPhe is indeed a potent inhibitor but so is unfractionated tRNA, as well as ribosomal RNA and heparin. Complete digestion to mononucleotides relieves the inhibition. Since the enzyme requires a metal ion (Co2+) we suggest that the RNA and heparin are inhibitory by virtue of their capacity to chelate the Co2+.


Effects of increased intracellular pH-buffering capacity on the light response of Limulus ventral photoreceptor. Aspects of a possible involvement of hydrogen ions in the electrophysiological responses to light of Limulus ventral photoreceptors were investigated. A 1 M solution of either a zwitter-ionic pH buffer or a weakly-buffering control substance was pressure injected through a micropipette into a ventral photoreceptor cell. To estimate the amount injected, 35SO4 was included in the solution. Membrane currents induced by light flashes were measured by a voltage-clamp technique. The buffer-filled micropipette passed current and a 3M KCl filled micropipette monitored membrane voltage. The sensitivity (peak light-induced current/stimulus energy) was measured, after dark adaptation, before and after injection. Injections of buffers, pH 6.3-7.2, to intracellular concentrations of at least 40-200 mM produced only a small mean decrease in sensitivity, approximately equal to that caused by injections of control substances. Excitation, therefore, apparently is not mediated by a change in intracellular pH. Buffers with pH values 5.4-8.4 were also injected. The time to peak of the response depended on pH, being shortened by up to 20% at pH values below 7.7 and lengthened at higher pH values. The time to peak of the response appeared to be shortened by an increase in intracellular pH-buffering capacity even when there was no change in intracellular pH.


Lipid-protein interactions in membrane models. Fluorescence polarization study of cytochrome b5-phospholipids complexes. According to previous authors, cytochrome b5, when extracted from bovine liver by a detergent method, is called cytochrome d-b5. On the other hand, the protein obtained after trypsin action, which eliminates an hydrophobic peptide of about 54 residues, is called cytochrome t-b5. Fluorescence polarization of the dansyl phosphatidylethanolamine probe inserted into phospholipid vesicles is very sensitive to the binding of proteins, and so is a useful method to study lipid-protein interactions. The chromophore mobility, R, decreases markedly when dipalmitoyl phosphatidylcholine vesicles are incubated with cytochrome d-b5, whereas R does not change for cytochrome c and cytochrome t-b5. This can be interpreted as a strengthening of bilayer, only due to the interaction of the hydrophobic peptide tail. Interaction of dipalmitoly phosphatidylcholine vesicles with cytochrome d-b5 occurs either below or above the melting temperature of the aliphatic chains (41 degrees C). Even for a high protein to lipid molar ratio (1 molecule of protein for 40 phospholipid molecules), the melting temperature is apparently unaffected. Phosphatidylserine and phosphatidylinositol do not interact at pH 7.7 with cytochrome d-b5, because electrostatic forces prevent formation of complexes. At low pH, the interaction with the protein occurs, but the binding is mainly of electrostatic nature.


Phosphatidic acid phosphatase and phospholipdase A activities in plasma membranes from fusing muscle cells. Plasma membrane from fusing embryonic muscle cells were assayed for phospholipase A activity to determine if this enzyme plays a role in cell fusion. The membranes were assayed under a variety of conditions with phosphatidylcholine as the substrate and no phospholipase A activity was found. The plasma membranes did contain a phosphatidic acid phosphatase which was optimally active in the presence of Triton X-100 and glycerol. The enzyme activity was constant from pH 5.2 to 7.0, and did not require divalent cations. Over 97% of the phosphatidic acid phosphatase activity was in the particulate fraction. The subcellular distribution of the phosphatidic acid phosphatase was the same as the distributions of the plasma membrane markers, (Na+ + k+)-ATPase and the acetylcholine receptor, which indicates that this phosphatase is located exclusively in the plasma membranes. There was no detectable difference in the phosphatidic acid phosphatase activities of plasma membranes from fusing and non-fusing cells.


Amino acid stimulation of ATP cleavage by two Ehrlich cell membrane preparations in the presence of ouabain. Two membrane fractions prepared from the Ehrlich ascites-tumor cell show non-identical stimulatory responses to certain amino acids in their Mg+2 -dependent activity to cleave ATP, despite the presence of ouabain and the absence of Na+ or K+. The first of these, previously described, shows little (Na+ + K+)-ATPase activity, and is characteristicallly stimulated by the presence of certain diamino acids with low pK2, and at pH values suggesting that the cationic forms of these amino acids are effective. The evidence indicates that these effects are not obtained through occupation of the kinetically discernible receptor site serving characteristically for the uphill transport of these amino acids into the Ehrlich cell. The second membrane preparation was purified with the goal of concentrating the (Na+ +K+)-ATPase activity. It also is stimulated by the model diamino acid, 4-amino-1-methylpiperidine-4-carboxylic acid, and several ordinary amino acids. The diamino acids were most effective at pH values where the neutral zwitterionic forms might be responsible. Among the optically active amino acids tested, the effects of ornithine and leucine were substantially stronger for the L than for the D isomers. The list of stimulatory amino acids again corresponds poorly to any single transport system, although the possibility was not excluded that stimulation might occur for both preparations by occupation of a membrane site which ordinarily is kinetically silent in the transport sequence. The high sensitivity to deoxycholate and to dicyclohexylcarbodiimide of the hydrolytic activity produced by the presence of L-ornithine and 4-amino-1-methyl-piperidine-4-carboxylic acid suggests that the stimulatory effect is not merely a general intensification of the background Mg+ -dependent hydrolytic activity.


[Partial purification and study of gamma-glutamyl transpeptidase from sheep cerebral capillaries]. Gamma-glutamyl transpeptidase was purified 53 times from Sheep brain cortex capillaries. On gel filtration it appears homogeneous with a M.W. = 350 000. The enzyme is likely a glycoprotein, the properties of which are close to hog kidney gamma-glutamyl transpeptidase ; gamma-glutamyl aminoacid formation is assayed electrophoretically. The results obtained using several aminoacids are in favour of the existence of different units, specific of each group of aminoacids ; together with the data from structural analogs, they support the hypothesis that gamma-glutamyl transpeptidase participates in aminoacid transport accross blood brain barrier.


[Biosynthesis of fatty acids in mouse brain mitochondria in the presence of malonyl-CoA or acetyl-CoA]. Incorporation of malonyl-CoA or acetyl-CoA is studied in mouse brain mitochondrial fatty acids. Rupture of mitochondria is necessary ; Triton X-100 gives the best result. Other detergents or sonication are of lesser efficiency. Cofactor requirements have been studied : NADH and NADPH have been tested ; ATP increases biosynthesis and CoA causes an inhibition. Two systems of biosynthesis are involved : -- One is a de novo system using malonyl-CoA. Malonyl-CoA alone is incorporated and synthesizes mainly C16, indicating the existence of a malonly-CoA decarboxylase although elongation of short chain fatty acids cannot be excluded. Addition of acetyl-CoA increases the biosynthesis and palmityl-CoA when added causes an inhibition. -- The other system, using acetyl-CoA, elongates exogenous palmityl-CoA ; endogenous acyl-CoAs are not elongated by acetyl-CoA. All these results are confirmed by radiogas chromatographic studies of the reactions products.


[Relationship between the magnitude of Km and pH for L-asparaginase]. The dependency of L-asparagine hydrolysis rate on L-asparagine concentration in the presence of L-asparaginases from E. coli and Erw. carotovora is studied in a broad pH range. Km values are calculated from the data obtained. It is found that Km insignificantly depends on pH value with the pH range of 5-9 for both asparaginases. Sharp Km maximum is observed at pH greater than 9 in both cases. The maximum position does not coinside with enzyme isoelectric points and with the region of the substrate transition from zwitterionic form into anionic one.


[Comparative study of endogenous RNA-polymerase activity of the cell nuclei and chloroplasts of pea leaves]. The endogenous RNApolymerase activity of isolated cell nuclei and chloroplasts from young pea plants (Pisum sativum) has been studied. The presence of all four nucleoside triphosphates and Mg2+ ions is necessary for the reaction. The missing of one of these nucleotides from the reaction mixture, especially ATP, sharply decreases the transcription. Chloroplasts synthesize RNA per DNA unit more intensively, than nuclei. In spring such predominance is especially pronounced. Maximal synthesis of RNA is observed at pH 8.3 both in nuclei and chloroplasts. Maximal transcription was observed at 25 degrees in chloroplasts and at 30--35 degrees in nuclei. Actinomycin D inhibited the process of transcription both in nuclei and some stimulation in chloroplasts were observed, when rifamicin B was added. It is suggested that there are differences in nuclear and chloroplast forms of RNA polymerase.


[Regulation of fodder yeast Candida tropicalis glutamine synthetase activity by the end products of glutamine metabolism]. Effect of different products of glutamine metabolism on the activity of glutamine synthetase in the presence of Mg2+, and Mn2+ and Co2+ as cofactors is studied. All the metabolites studied are found to inhibit the glutamine synthetase activity in the presence of any cation listed. The degree and the character of the inhibition by one or other metabolite depended in a considerable degree on the nature of the cation presented in the reaction mixture (Mg2+, Mn2+ or Co2+). The mechanism of the cumulative effect of retroinhibitors under the change of Mg2+ or Mn2+ in the reaction mixture was the same.


[Carbonic anhydrase of blue-green alga Spirulina platensis]. Carboanhydrase (carbonate-hydroliase EC 4.2.1.1.) is found in the extract of Spirulina platensis cells. A linear dependency of the enzyme activity on the protein concentration; pH optimum is found to be 8.0. Specific activity of carboanhydrase is 3 muM/min-mg of protein under the concentration of CO2 of 4-10(-3) M, appearing Michelis constant being 4.9-10(-3) M. The enzyme was stabilized with 10 mM of cisteine, its activity was inhibited by 50% with sulphanylamide (1-10(-5) M), acetazolamide (8--10(-7) M) and Cl- ions (5-10(-2) M). The activity of carboanhydrase, as well as the rate of NaH14CO3 fixation, depended on the pH value of cultural medium.


[Isolation of mitochondrial DNA, purified of nuclear DNA, from animal tissues (degree of methylation and level of pyrimidine nucleotide clustering--criteria of purity)]. A method of preparation of mitochondria free of nuclear DNA and its fragments by treatment of mitochondria with DEAE-cellulose has been developed. This method is based on binding nuclear nucleic acids and nucleoproteins to DEAE-cellulose particles in the media used for isolation of mitochondria. Treatment with DEAE-cellulose under the conditions described does not induce any visible degradation of mitochondria and mitochondrial DNA. The mitochondrial DNA preparations obtained from beef and rat liver are represented with closed circular molecules of contour length about 5.5 mu. The 5-methylcytosine content in beef and rat mitochondrial DNA (3.03 and 2.0 mole %, respectively) is twice as much as in corresponding nuclear DNA. Besides, mitochondrial DNA strongly differs from nuclear ones by a lower degree of pyrimidine clustering: the amount of mono- and dipyrimidine fragments (about 32 mole %) in mitochondrial DNA is 1.5 times as large and the content of long pyrimidine clusters (hexa- and others) is 2--4 times as low as those in nuclear DNA. The methylation level and the pyrimidine clustering degree may be used as criteria for the purity of mitochondrial DNA from nuclear DNA.


[Study of the structure of the histidine decarboxylase of Micrococcus sp. n. by the method of circular dichroism]. Ring dichroism spectra (RD) of histidine decarboxylase (HDC) from Micrococcus sp. n. at the regions of peptide bonds (200-240 nm) and aromatic amino acids (250-300 nm) absorption are studied. The treatment of RD spectra according to methods of Greenfield-Fasman, Saksena-Vetlaufer and Mayer permits to conclude that at the pH range within 4-8 the content of ordered structures of alpha-helix type comprises 20%, that of beta-structure type-40%, while the rest 40% are represented with polypeptide chain in a disordered globular state. When pH is varied from 1 to 12, the content of alpha-helices decreases from 17 to 5%. There are two distinct dichroic bands in the spectrum of aromatic chromophores absorption (at 270 and 290 nm), the former containing tirosine, tryptophane and phenylalanine residues and the latter being induced with triptophane residues. The study of HDC RD spectra at the regions of peptide bonds and aromatic acids absorption at different temperatures has shown that a part of triptophane, tyrosine and phenylalanine residues is in an ordered structure of the alpha-helix type. The HDC undergoes irreversible changes under heating to 70 degrees and in 8 M urea. 5 M guanidine chloride eliminates the ordered HDC structure, while sodium dodecylsulphate at concentrations up to 1% does not affect the enzyme structure.


[Isolation and several properties of Streptomyces griseus carboxypeptidase]. Enzymatic and physico-chemical properties of homogenous preparation of carboxypeptidase from Streptomyces griseus are studied. pH-Optimum is found to be 7.9 and 8.2 under the hydrolysis of cbs-Gly-Leu and hyppuryl-arg respectively, temperature optimum --60 degrees C. The enzyme splits more efficiently basic amino acids and leucine from N-terminal-protected dipeptides. Str. griseus carboxypeptidase is activated by reducting agents (NaCN, cisteine, ascorbic acid), it is inhibited by KMnO4 and it does not belong to "serine" type enzymes. SH-groups are essential for the enzyme activity. No significant effect of metal ions on the enzyme activity is observed. The inhibitory effect of EDTA developed only after the prolonged treatment. The enzyme has one N-terminal group (alanine), which evidences the presence of one polypeptide chain in the enzyme molecule.


[Soluble, nuclear and mitochondrial forms of dehydrogenases, pentose-phosphate pathway transferases and nucleases in chicken liver]. The sub-cellular topography of oxidative and non-oxidative enzymes of the pentose phosphate pathway of carbohydrates metabolism and enzymes of the nucleic exchange (acid and alkaline deoxyribonucleases and ribonucleases) in chicken liver is studied. Nuclear and mitochondrial forms of the enzymes are discovered. The activity of the enzymes studied of carbohydrates metabolism is shown to correlate with that of the enzymes of nucleic metabolism in cytosol, nucleic and mitochondrial liver fractions.


[Purification and properties of the riboflavin kinase of the yeast Pichia guilliermondii]. Riboflavin kinase (E.C.2.7.1.26) was isolated from the cells of the yeast Pichia guilliermondii. The enzyme was 680-fold purified uzing ammonium sulphate fractionation, chromatography on DEAE-Sephadex A-50 and CM-Sephadex C-50 and gel-filtration through Sephadex G-75. Purified enzyme preparation was free from phosphatases and FAD-synthetase. The pH optimum was 8,7, the temperature optimum-45 degrees C. The enzyme was activated by Zn2+, Mg2+ and Co2+ ions. Km for riboflavin was 1,0x10(-5) M, for ATP -- 6,7X10(-6) M. Riboflavin kinase catalyzed the phosphorylation of riboflavin analogues with the substitution of methyl groups at positions 7 and 8. UTP, GTP, ADP and CTP, besides ATP, were phosphate donors. AMP inhibited the enzyme activity. Molecular weight of the enzyme was 28000, as estimated by gel-filtration through Sephadex G-150. Purified riboflavin kinase was stable under storage.


[Catalytic properties of alpha-ketoglutarate decarboxylase from bovine brain]. Investigation of the effect of different buffer systems on the rate of alpha-ketoglutarate decarboxylase reaction have shown that the pH optimum is 6.8 in tris-maleic, tris-H3PO4 and KH2PO4-KOH buffers, and it is 7.5 in imidazole buffer. The highest reaction rate was observed when using phosphate containing buffers. The increase of phosphate concentration increased considerably the rate of alpha-ketoglutarate decarboxylase reaction. Mg2+ and Ca2+ were shown to affect slightly the reaction rate. Co2+ and Ag+ slightly inactivated the enzyme. Cu2+ turned to be a very efficient inhibitor of alpha-ketoglutarate decarboxylase reaction. Apparent Mikhaelis constants are determined to be 1.6-10(-3) M for alpha-ketoglutaric acid and 1.7-10(-2)M for 2,6-dichlorphenolindophenol.


[Immobilized phospholipase D].

[Immobilized phospholipase D]. Conditions of phospholipase D adsorption on silica gels have been studied. The immobilized phospholipase D is shown to differ from the soluble form in thermostability, pH optima and activation conditions. A question is discussed as to the connection of the use of activators and the adsorption immobilization. It is assumed that phospholipase D belongs to enzymes, functioning only in the immobilized state.


Evidence for stem cell function of resting bone marrow lymphocytes identified by the complete 3H-thymidine labelling method. Resting bone marrow lymphocytes, recongised as small lymphocytes by light microscopy, were labelled by the complete 3H-thymidine labelling technique, enriched by fractionation on a discontinuous albumin gradient and investigated for their stem cell properties by culture in diffusion chambers. Fraction 3, with the highest enrichment of labelled small lymphocytes and slight enrichment of other labelled cells (reticulum and endothelial cells), produced substantial growth, in contrast to fraction 4, with no enrichment of these cells. The number of labelled small lymphocytes per chamber in fraction 3 remained constant or tended to increase. This is assumed to be an indication of some degree of self-replication in the small lymphocyte population. Since, however, their labelling intensity decreased only slowly, it must be further concluded that part of the labelled small lymphocyte population probably remained resting. Some labelled transitional and blast cells appeared before the development of recognisable myelopoietic and erythropoietic precursors and of megakaryocytes, in agreement with the concept that small lymphocytes transfrom to transitional cells during the developmetn of normal haempopiesis.


Changes in the electric dipole vector of human serum albumin due to complexing with fatty acids. The magnitude of the electric dipole vector of human serum albumin, as measured by the dielectric increment of the isoionic solution, is found to be a sensitive, monotonic indicator of the number of moles (up to at least 5) of long chain fatty acid complexed. The sensitivity is about three times as great as it is in bovine albumin. New methods of analysis of the frequency dispersion of the dielectric constant were developed to ascertain if molecular shape changes also accompany the complexing with fatty acid. Direct two-component rotary diffusion constant analysis is found to be too strongly affected by cross modulation between small systematic errors and physically significant data components to be a reliable measure of structural modification. Multicomponent relaxation profiles are more useful as recognition patterns for structural comparisons, but the equations involved are ill-conditioned and solutions based on standard least-squares regression contain mathematical artifacts which mask the physically significant spectrum. By constraining the solution to non-negative coefficients, the magnitude of the artifacts is reduced to well below the magnitudes of the spectral components. Profiles calculated in this way show no evidence of significant dipole direction or molecular shape change as the albumin is complexed with 1 mol of fatty acid. In these experiments albumin was defatted by incubation with adipose tissue at physiological pH, which avoids passing the protein through the pH of the N-F transition usually required in defatting. Addition of fatty acid from soluion in small amounts of ethanol appears to form a complex indistinguishable from the "native" complex.


[Cloning stem cells in the bone marrow of irradiated mice]. Different amount of intact or irradiated bone marrow from syngenous donors was administered to mice irradiated with a lethal dose. There was revealed a linear dependence of the number of the 8-9-day colonies grown in the bone marrow of the femur on the amount of the administered cells, and an exponential dependence on the irradiation dose. Regularity of the stem cell cloning in the bone marrow was analogous to such in the spleen. Radiosensitivity of the colony-forming units (CFU) differed depending on the site (the spleen, the bone marrow) of their colony formation. The CFU settling in the marrow proved to be more radioresistant (D(0) equalled 160-200 P) in comparison with the CFU settling in the spleen (D(0) constituted 80-100 P). It is supposed that a different radiosensitivity of the CFU was caused by the presence of heterogenic population of the stem cells and also by specific peculiarities of the organ (the spleen, the bone marrow) in which the colonies formed.


[The effect of psychotropic substances (aminazin, majeptil, trisedil) on protein synthesis in different regions of the rat brain]. Experiments were conducted on rats; a study was made of methionineS35 incorporation into the sum total proteins isolated from various portions of the brain after a single administration of chlorpromazine, majeptil and tricedil. A generalized depression of protein synthesis in all the structures, except the medulla oblongata, followed chlorpromazine administration in one and three hours. A stimulating effect is characteristic of majeptil in the majority of the brain portions. The action of tricedil was accompanied by reduction of methionine-S35 incorporation into the proteins of the majority of the brain structures and by an increase in its incorporation into the olfactory lobes. As supposed, changes in the protein synthesis in individual structures of the brain served as an important link in the action mechanism of psychotropic preparations on the organism.


[Effect of noradrenaline on the electrical and contractile properties of smooth muscle cells in the pulmonary artery]. Experiments were performed on the smooth muscle cells of rabbit a. pulmonalis using the microelectrode technique. No spontaneous electrical or mechanical activity was recorded in normal Krebs solution. The current-voltage relation in these smooth muscle cells showed marked rectification. No changes in the isometric tension were observed due to the anodal or cathodal stimulating currents. Strong depolarization of the muscle cells produced only local potentials on the cathelectrotone which never developed into a spike. Noradrenaline (10(-8) g/ml) caused depolarization of the 5-7 mV in the muscle cell membrane and a considerable contraction of the muscle strip as well. Under such conditions the contractile apparatus of the muscle cells became sensible to the resting potential level. Anodal stimulation was accompanied by relaxation of the muscle strip, whereas cathodal stimulation--by its contraction. The alpha-adrenoblocking agent (phentolamine) blocked the effect of noradrenaline evidencing the fact that noradrenaline exerted its excitatory action on the smooth muscle cells of the a. pulmonalis through the alpha-adrenoreceptors.


[Hydroxylation in isolated liver cells]. Ethylenediamine tetraacetate and mechanical treatment were applied to obtain the isolated hepatocytes. Oxidative phosphorylation was found to be preserved in the isolated cells. Hepatocytes were capable of hydroxylation of dimethylaniline (DMA), ethylmorphine and aminopyrine. VMAX for the hydroxylation of DMA calculated as per 1 nmol of the cytochrome P--450 was higher in the cells than in the microsomes. NADH formed during the oxidation of glutamate and malate can be used for hydroxylation.


[Effect of carbidine on the content and storage of adrenergic neurotransmitter in the synaptic vesicles]. The influence of carbidine, an original psychotropic drug, on the adrenergic neurotransmitter content and storage in the sympathetic nerves was studied with the use of cytochemical electron microscopy. The influence of carbidine on the uptake of the exogenous noradrenaline (NA) in the synaptic vesicles was also studied. Carbidine was found to be capable to decreasing the NA storage in the synaptic vesicles and failed to block the accumulation of the exogenous NA in the synaptic vesicles.


[Activity of NAD- and NADP-dependent malate dehydrogenase isoenzymes in the myocardium of rabbits with alloxan diabetes]. Isoenzymes NAD-and NaDP MDH were detected in the cardiac muscle of rabbits by disc electrophoresis in polyacrylamide gel. Alloxan diabetes proved to be accompanied by a significant reduction in the activity of mitochondrial NADP MDH (in the reaction of malic decarboxylation) and its increase in cytozol. The activity of NAD-MDH (in the reaction of oxyacetate reduction) was also decreased in various isoenzymes in the myocardium (particularly in the mitochondria) in diabetes. Insulin restored the correlation of the activities of the isoenzymes NAD- and NADP-MDH in the cytostructures of the myocardium disturbed in diabetes.


[Cyproheptadine as an inhibitor of the effects of bradykinin]. A study was made of the influence of ciproheptadin, serotonin and histamine inhibitor, on the effects of exo- and endogenous bradykinin. Ciproheptadin inhibited the effect of bradykinin on a section of a guinea pig intestine in vitro, eliminated the hypotensive effect of bradykinin in rats in vivo, and also blocked the constriction of the vessels of the ear in rabbit and a fall of the blood kininogen level caused by the administration of pyrogenal.


[Mg, Ca-activated ATP-ase of Pacinian corpuscles]. Adenosine triphosphatase (ATPase) activated by Mg2+ or Ca2+ ions was detected in single mechanoreceptors (Pacini's corpuscles) of cat; addition of Ca2+ (10(-5)M) to Mg-ATP-ase increased the activity by the factor of 1.6. The activity optimum of Mg- or Co-ATPase was in the alkaline pH zone. A high substrate specificity of Mg, Ca-ATPase was shown. Parachlorinemercury-benzoate (5muM) considerably reduced the activity of Mg, Ca-ATPase, whereas oubain (10(-5)M) failed to affect it significantly. It is supposed that Mg, Ca-ATPase of Pacini's corpuscles was close to actomyosine -like proteins.


[Activity of adrenal cytoplasmic dehydrogenase following prolonged ACTH administration]. The activity of cytoplasmic dehydrogenases of the adrenal cells was examined during the prolonged injections of ACTH to Wistar rats. The indices of specific steroid synthesis function of the glands remained relatively high in the course of the whole experiment. Changes in the rats of corticosterone synthesis and dehydrogenase activities were phasic in character; they included an initial synchronic activation the first  two days with its subsequent decrease (7 days of ACTH injections). The prevalence of activities of NADP-dependent dehydrogenases in combination with reactivation of steroidogenesis was marked during the concluding phase (13 days) of the experiment. A possibility of the adaptive role of selective activation of NADP-dependent enzymes in the maintenance of a high level of hormone biosynthesis under conditions of prolonged ACTH stimulation is discussed.


[Effect of hypercapnia on tyrosine and tryptophan metabolism]. Experiments were conducted on albino rats; it was revealed that an increase in CO2 content in the inspired air (3.8%) caused disturbances in tyrosine and tryptophane metabolism. The activity of tyrosine-aminotranspherase and of tryptophane-oxygenase proved to increase in the liver; blood serum displayed a reduced concentration of free tyrosine and free total tryptophane, but the level of free tryptophane obtained by dialysis proved to rise. A possible significance of these deviations in endogenous blastomogenesis is discussed.


[Mechanism of the analgesic effect of narcotic analgetics]. A study was made of the effect of morphine, promedol, phentanyl, pentazacine and psychostimulant d,l-amphetamine on the threshold of pain sensitivity and self-stimulation of the hypothalamus and the septum in rats. Electrical stimulation of the systems of positive reinforcement of the hypothalamus and the septum, and also analgetics increased the threshold of pain sensitivity, whereas d,l-amphetamine failed to influence it. D,l-amphetamine and morphine facilitated, promedol failed to influence, phentanyl decreased and pentazacine completely depressed the hypothalamic self-stimulation. The septal self-stimulation remained unaltered under the effect of morphine, promedol, phentanyl, but was decreased under the effect of pentazacine and increased against the background of d,l-amphetamine. A conclusion was drawn that the analgetic action and that activating the positive emotion were independent effects of the psychotropic agents.


[Effect of neuroleptics on tyrosine hydroxylase from rat hypothalamus synaptosomes]. A study was made of the effect of a number of neuroleptics of various chemical structure of tyrosine-hydroxilase isolated from the synaptosomes of rat hypothalamus. A direct spectrophotometric method of determination of the activity of the enzyme was used; it was based on measurement of the absorbence at 335 nm (at the isobestic point for oxidized forms of a synthetic cofactor 6,7-dimethyl-5,6,7,8-tetrahydropterine). At the tyrosine concentration of 0.15 muM haloperidol, haloanizon, and fluorophenazin were found to increase, and triperidol, droperidol and carbidin -- to decrease the initial rate of thyrosinehydroxilase reaction. All the neuroleptics under study proved to be capable of eliminating the substrate inhibition of the enzyme occurring with a rise of tyrosine concentration to 0.3 mM. The KM value for tyrosine failed to alter with the action of neuroleptics. The effect of neuroleptics was assumed to be of allosteric nature.


[Elimination of allogeneic inhibition of hematopoietic stem cells by treatment of the recipient mice with cyclophosphane]. The experiments demonstrated that pretreatment of lethally irradiated recipient (CBA X C57BL/6) F1hybrid mice with cyclophosphamide (200 mg/kg of body weight) on day before the bone marrow transplantation (4 hours after the irradiation) suppressed the allogeneic inhibition of hematopoietic stem cells to 24% (while the inhibition in the untreated animals was 92.5%). It is suggested that cyclophosphamide acted on the recipient's radioresistant lymphoid cells effecting the allogeneic inhibition of stem cells.


[Effect of polyinosinic-polycytidylic acid on the colony-forming ability of hematopoietic stem cells in conditions of allogeneic inhibition]. It was found that the colony-forming capacity of parental bone marrow transplant (C57BL/6) was partially restored in the (CBA X C57BL/6) hybrid recipient irradiated with 800 rad when poly I -- poly C preparation was injected. The effect of poly I -- poly C injection on the colony formation was equivalent to addition of the thymus cells syngeneic with the marrow. In either case the number of splenic colonies was more than double that in the control. On the other hand, it was found that in a completely syngeneic system the number of splenic colonies was not influenced by the thymus cells and poly I -- poly C preparation. Poly I -- poly C doses ranging from 50 to 100 mug and thymus cell doses ranging from 4-10(6) to 8-10(6) did not increase the efficiency of the colony formation with a stable bone marrow dose transplant.


[Proliferation and differentiation of hematopoietic stem cells in hypokinesia]. Using the method of exogenous cloning in vivo of the hemopoietic stem cells of the bone marrow and spleen in the femur and the spleen of mice it was shown that during hypokinesia the kinetics of the stem cells differed in both organs (the spleen and the bone marrow). Differentiation of transplanted stem cells from different sources was unchanged in the spleen, but stem cells of the bone marrow seeding in the femur changed the character of their differentiation in the direction of increase of the erythopoietic function, whereas stem cells of the spleen failed to alter the direction of differentiation.


Torsion of the testis and allied conditions. In 15 years at Bristol there have been 293 cases of torsion of the testis, 55 cases of torsion of a testicular appendage and 5 cases of testicular ischaemia due to other causes. The risk of a male developing torsion of the testis or its appendix by the age of 25 is about 1 in 160. Both conditions occurred primarily in adolescents, but among prepubertal boys torsion of an appendage was as common as torsion of a normally descended testis. There was a slight left-sided preponderance in testicular torsion, more marked in torsion of the appendages; the incidence of bilateral torsion was 2-0 and 1-8 per cent respectively. The clinical features and differential diagnosis of the two conditions are compared. Torsion of a testicular appendage is the most commonly misdiagnosed scrotal lesion, the preoperative diagnosis being correct in only 11 per cent of cases compared with 90 per cent for torsion of the testis. Twenty-one cases of recurrent torsion underwent prophylactic bilateral orchidopexy. There were 20 cases of torsion of undescended testes, with a salvage rate of only 20 per cent. The overall testicular survival rate was 55-3 per cent. Viability depends upon the possibility of spontaneous reduction, the preoperative delay after the onset of symptoms, the degree of torsion of the cord and the length of follow-up in doubtful cases. Urgent scrotal exploration is advised in every case of acute testicular pain unless there is overwhelming evidence of epididymoorchitis. Exploration of the opposite side is mandatory in torsion of the testis and advisable in torsion of an appendage.


Raynaud's phenomenon as side effect of beta-blockers in hypertension. A series of 102 hypertensive patients were assessed for the frequency of symptoms of Raynaud's phenomenon and absent peripheral pulses. Out of 21 patients receiving methyldopa alone only one had cold hands and feet whereas among patients on beta-blockers the incidence was 50%. The frequency of both symptoms and absent pulses was highest in patients taking propranolol compared with those taking atenolol or oxprenolol. Patients without a foot pulse were much more likely to have cold hands. A change from propranolol to oxprenolol in some symptomatic patients resulted in improvement. In two patients the skin temperature fell after an 80-mg dose of propranolol. The mechanism by which beta-blockers induce Raynaud's phenomenon is still not clear.


Histologic and enzymatic studies of the mesolimbic and mesostriatal serotonergic pathways. Selective lesions of the dorsal (B7), median (B8), or lateral (B9) raphe nuclei were made stereotaxically in male rats 4 weeks before sacrifice. The extent of damage to each raphe nucleus was quantified histologically by means of a simplified formaldehyde histochemical method for visualization of serotonin in cryostat sections. A detailed mapping of the distribution of the yellow-fluorescent raphe perikarya provided the basis for quantification. Tryptophan hydroxylase activity was measured in 6 forebrain regions from each animal, and the results were correlated with the per cent damage to each raphe nucleus. Tyrosine hydroxylase was also assayed in 5 of these regions; it was not significantly affected by any of the raphe lesions. Dorsal raphe lesions reduced tryptophan hydroxylase activity in the striatum, thalamus, cortex, and hypothalamus, but not in the septal nuclei or hippocampus. Damage to B8 resulted in decrements in this serotonergic enzyme in the septal nuclei, hippocampus, cortex, and hypothalamus, but not in the striatum or thalamus. Lesions of the scattered B9 cells had no significant effect on enzyme activity in any region examined. These data suggest that the dorsal and median raphe nuclei provide two distinct though perhaps overlapping serotonergic systems innervating different parts of the forebrain: a mesostriatal pathway originating in B7 and a mesolimbic system derived from B8. Behavioral studies on the animals, which are presented in a companion paper, indicated that damage to the median nucleus is responsible for many of the behavioral effects previously reported after combined lesions of both major raphe nuclei.


Evidence that the rapid binding of newly accumulated noradrenaline within synaptosomes involves synaptic vesicles. When rat brain synaptosomes were incubated with [3H]noradrenaline for 1 min and then exposed to osmotic shock, only about 20% of the newly accumulated [3H]noradrenaline was released. It would appear that most, but possibly not all of the newly accumulated [3H]noradrenaline is rapidly bound to some particulate cytoplasmic constituent within the synaptosome. [3H]Dopamine and [3H]5-hydroxytryptamine were also rapidly bound within synaptosomes but [3H]glycine and [3H]gamma-aminobutyric acid were not. Reserpinization (5 mg/kg, i.p., 24 h before preparation) only slightly reduced the initial rate of [3H]noradrenaline uptake by synaptosomes. However, when reserpinized synaptosomes were osmotically shocked, most of the newly accumulated radioactivity was released; this radioactivity was identified chromatographically as [3H]noradrenaline. On the basis of the findings with reserpinized preparations, it seems likely that (1) the rapid intrasynaptosomal binding involves synaptic vesicles and (2) the neuronal membrane transport system itself may be capable of driving the uptake of noradrenaline by nerve-terminals. The rapid vesicular binding observed may not be essential for the accumulation of the amine by presynaptic terminals during brief exposures.


Time-course variations in tyrosine hydroxylase activity in the rat locus coeruleus after electrolytic destruction of the nuclei raphe dorsalis or raphe centralis. Time-course variations in tyrosine hydroxylase activity were measured in the locus coeruleus of the albino rat after electrolytic coagulation of either the nucleus raphe dorsalis or the nucleus raphe centralis. Highly significant increases were measured at 4 days after lesioning of the raphe dorsalis (30.33%) and the raphe centralis (81.55%) compared with control values, whereas the activity in groups A9 and A10 was unchanged at this time-point. In conjunction with other experimental evidences, an hypothesis is proposed that the catecholaminergic neurons located in the locus coeruleus are directly and/or indirectly controlled by the serotonin-containing neurons located in the anterior raphe system nuclei.


Resting and stimulated values of model parameters governing transmitter release at a synapse in Aplysia californica. Transmitter release (R) at a synpase in Aplysia californica can be analyzed in terms of a model with the following parameters: A, the available pool of transmitter; F, the fraction of available pool released by a presynaptic action potential; M, the rate of transmitter mobilization into the available pool; D, the rate constant of demobilization of transmitter from the available pool. In the present paper we show that: (1) beginning with an analysis of the recovery from depression of the second of a pair of disolated EPSPs separated by a series of intervals of about 10-60 sec, and assuming that the recovery is due to refilling of a depleted A, it is possible to estimate resting equilibrium values of these parameters; (2) changes in these parameters when a new equilbrium state is reached after prolonged stimulation (e.g., 300 stimuli at 1/sec) can then be quantitatively determined; (3) the increased rate of transmitter release observed during and after repetitive stimulation is the consequence of increases in F and M with changes in A passively following; and (4) there are significant correlations among certain resting parameters and between the values of certain resting parameters and these parameters upon stimulation. Preparations with a large resting F tend to have a relatively small resting A. Preparations with a large resting F or M tend to increase these less with stimulation than preparations with smaller resting values of these parameters. Preparations with large stimulus-dependent increases in F tend to have large stimulus-dependent increases in M.


Synaptic depression at a synapse in Aplysia californica: analysis in terms of a material flow model of neurotransmitter. When a pair of stimuli separated by an appropriate interval is given to the right visceropleural connective of Aplysia californica the amplitude of the second EPSP elicited in cell R15 is usually smaller than the amplitude of the first EPSP. In the present paper we show that this phenomenon, synaptic depression, can be analyzed in terms of the material flow model of neurotransmitter economics developed in our preceding publications. We specifically show how changes in the 4 model parameters; A, the available pool of transmitter; F, the fraction of the available pool released by a presynaptic action potential; M, the rate of transmitter mobilization into the available pool; and D, the rate constant of demobilization of transmitter from the available pool, all effect synaptic depression. In addition, we show how transient changes in F and M, that are observed immediately and for seconds after a stimulus, influence the time course of synaptic depression. Using this analysis we then tested our previous inferences about changes in the model parameters produced either by pharmacological manipulations or repetitive stimulation, by comparing the observed effects of these manipulations on synaptic depression with the theoretical predictions. The theoretical and experimental findings agreed, thereby strengthening both our previous conclusions of the mode of action of these manipulations and the model itself.


Biochemical differentiation of mechanically dissociated mammalian brain in aggregating cell culture. Mouse and rat brain cells were dissociated by a simple mechanical sieving technique and studied in culture for the formation of aggregates and the activities of choline acetyltransferase, acetylcholinesterase, glutamic acid decarboxylase, tyrosine 3-monooxygenase, aromatic L-amino acid decarboxylase, catechol methyltransferase, and monoamine oxidase. Cells from fetal and neonatal tissue formed aggregates but not cells from tissue older than two days after birth. The pattern of development of enzyme activities in these aggregates varied with the age of starting tissue. The highest levels of specific activity for the neuron-specific enzymes were found after 3-4 weeks in culture for aggregates of cells derived from relatively undeveloped brains.


Stimulation of tyrosine hydroxylase activity by cyclic AMP in synaptosomes and in soluble striatal enzyme preparations. Dibutyryl cyclic AMP (dB-cAMP) elicits a concentration-dependent stimulation of tyrosine hydroxylase activity in the striatal and mesolimbic synaptosomes. The per cent of stimulation is significantly higher in the mesolimbic synaptosomes than in the striatal synaptosomes. dB-cAMP and depolarizing agents (ouabain or veratridine) have an additive effect on synaptosomal tyrosine hydroxylase activity, indicating that they stimulate tyrosine hydroxylase activity by different mechanisms. cAMP does not stimulate soluble striatal tyrosine hydroxylase activity unless it is added in combination with ATP and Mg2+, compounds required for the activity of cAMP-dependent protein kinase. The cAMP elicited per cent stimulation of soluble tyrosine hydroxylase activity is dependent upon the concentration of added protein kinase and upon the pH of the reaction. dB-cAMP has the same effect on the kinetic state of tyrosine hydroxylase in synaptosomes as cAMP on the soluble tyrosine hydroxylase. The nucleotide does not alter the apparent Km for tyrosine, reduces the Km for the pteridine cofactor and increases the Ki for dopamine. Thus, cAMP increases the affinity of tyrosine hydroxylase for the pteridine cofactor and concomitantly decreases the affinity for the end-product inhibition.


Choline acetyltransferase, glutamate decarboxylase and tyrosine hydroxylase in the cochlea and cochlear nucleus of the guinea pig. Activities of choline acetyltransferase (ChAC), glutamate decarboxylase (GAD) and tyrosine hydroxylase (TH), enzymes catalyzing the synthesis of acetylcholine (ACh), gamma-aminobutyric acid (GABA) and catecholamines, respectively, were measured in the cochlea and cochlear nucleus of the guinea pig. ChAc activity in the organ of Corti, third turn, was 1270 pmole ACh formed/min/mg protein (ChAc, 1270) and was higher than in turn 4 (ChAc, 543). ChAc activity was higher when the preparation included the inner hair cell region than when not. GAD activity in samples of turn 3 and 4 combined was low, 0.17 nmole GABA formed/min/mg protein (GAD, 0.17). All 3 enzymes were low in auditory nerve: ChAc, 1.7, GAD, 0.10 and TH, 1.0 pmole DOPA formed/min/mg protein. In the cochlear nucleus, the values were: ChAc, 129, GAD, 1.70 and TH, 2.7. The findings on the distribution of ChAc activity in the organ of Corti fit the hypothesis that the olivocochlear nerve fibers are cholinergic. Because of low GAD in the cochlea, GABA is unlikely to be transmitter in the organ of Corti. Similarly, it is unlikely that ACh, GABA or a catecholamine is a transmitter between the auditory nerve and the cochlear nucleus.


An evaluation of L-glutamate as the transmitter released from optic nerve terminals of the pigeon. The possibility was investigated that L-glutamic acid is the excitatory transmitter released from the optic nerve terminals of the pigeon optic tectum. (1) Superficial layers of the tectum contained high levels of endogenous glutamate and accumulated L-[3H]glutamate by a high affinity uptake process. (2) Subcellular and autoradiographic studies indicated that 10-30% of the exogenously accumulated L-[3H]glutamate was localized within synaptosomes, and that 11-15% of the synaptosomes had been labelled. (3) The glutamate-accumulating synaptosomes sedimented to the same isopycnic density as pinched-off optic nerve terminals. (4) GABA-and noradrenaline-accumulating synaptosomes were also associated with this subcellular population. (5) Retinal ablation did not change endogenous glutamate concentrations or the high affinity uptake of glutamate. The results are discussed in relation to a possible role for L-glutamate as the 'optic nerve transmitter' and in the context of previous evidence implicating glutamate as an excitatory transmitter.


[Hemodynamic modifications induced by hypoxia in the dog]. In non hypercapnic hypoxia (inhalation of a 4,5% O2 mixture during 10 minutes) blood pressure, heart rate, cardiac output, femoral and carotid blood flow inhance so that, in less extent, vertebral blood flow. Such a reaction is consequent with an hypoxic stimulation of adrenal glands.


[Respiratory stimulant effect of S 2620 in the dog anesthetized with pentobarbital]. In the dog under pentobarbitone anesthesia, the intravenous injection of 1 mg/kg S 2620 is followed by a significant increase in respiratory rate, PaO2 and pHa and by a large decrease in PaCO2. Cervical vagotomy and chemoreceptor denervation reduced and even abolished these effects. They can be induced again by a second intravenous injection or by direct instillation in the fourth ventricle. These results suggest a central action of the drug.