bluuebunny/crossref_metadata_2025_split huggingface dataset:
Membrane Adenosine Triphosphatase of Micrococcus lysodeikticus
Authors: Muñoz E. , Salton M. R. J. , Ng M. H. , Schor M. T. 
Published: July 2010

A protein component which accounts for 10% of the total protein of isolated cell membranes of Micrococcus lysodeikticus has been partially characterized. The protein can be released into solution from the membrane complex and in this “soluble” state it possesses Ca++‐dependent adenosine triphosphatase activity. This enzyme has been purified about 50‐fold, by a procedure based on its association‐dissociation properties with the membrane. The progressive purification and degree of homogeneity have been demonstrated by examining the various stages in the soluble ATPase preparation by disc electrophoresis and by gel diffusion against membrane antiserum. On treatment with 8 M urea or 1% sodium dodecyl sulfate followed by disc electrophoresis, clear evidence of dissociation was obtained, indicating the existence of subunits. The purified protein has a sedimentation coefficient of 14–15 S as determined by sucrose gradient centrifugation or analytical ultracentrifugation. A sedimentation coefficient of 3.5 S has been estimated for the main product of the protein dissociated by sodium dodecyl sulfate again an indication of the dissociation of the protein into subunits, probably identical on the basis of molecular weight. The “soluble” enzyme was activated by Ca++, optimum activation being observed at ATP/Ca++ ratio of 1/1 and pH 7.5 under which conditions 50–60 μmoles of ATP were hydrolyzed per mg protein per 10 min at 37°. The ATPase was quite stable at 4°. The Ca++‐dependent ATPase activity was inhibited by ADP, sodium azide, and Mg++.When the membrane‐ATPase complex remains stable, no hydrolytic activity for ATP is shown unless trypsin activation. Our system shows similarities with the “allotopic” properties described for some mitochondrial enzymes. From this and previous work in our laboratory a close relationship between the “stalked particles” seen in M. lysodeikticus cell membranes by electron microscopy and ATPase appears to be evident.
Studies of Glutamate Dehydrogenase
Authors: Sund H. , Pilz I. , Herbst M. 
Published: July 2010

The size and shape of beef liver glutamate dehydrogenase were investigated by X‐ray small‐angle measurements in the range of protein concentration between 1 and 33 mg/ml. It was shown that the cross‐section of the glutamate dehydrogenase particles of varying sizes were independent of the molecular weight of the particles. The radius of gyration of the cross‐section (Rq) was found to be 30.3 Å and the mass per unit length (M/1Å) to be 2,340. From the comparison of the experimental scattering curves with theoretical cross‐section curves for elliptical cylinders it follows that the glutamate dehydrogenase molecule has a very isotropic cross‐section. The experimental data lie between the axial ratio 1:1 (circular cross‐section) and 0.8:1. For the circular cross‐section a diameter of 86 Å and a cross‐section area of 5,800 Å2 is calculated. For an axial ratio of 0.8:1 the long and the short axes are calculated to be 95 Å and 76 Å, respectively, and the cross‐section area is found to be 5,700 Å2. Again the cross‐section is independent of protein concentrations and therefore independent of the state of association of glutamate dehydrogenase. This result confirms our earlier conclusion from diffusion, sedimentation and viscosity measurements, that the dissociation of the prolate glutamate dehydrogenase molecule into enzymatically active subunits involves a transverse cleavage.A linear relationship exists between the length and the molecular weight of the glutamate dehydrogenase particles. For a molecular weight of about two millions a length of 800–900 Å was found, for molecular weights of one and one‐half millions the length of the molecules were calculated to 400 Å and 200 Å, respectively. These data are confirmed by comparison of the experimental data with theoretical scattering curves of circular cylinders. The results support the conclusion that the associated enzyme molecule is formed in a stepwise association‐dissociation equilibrium.The scattering curves of glutamate dehydrogenase show a pronounced side maximum at an angle corresponding to a Bragg's value of 56 Å. The comparison with theoretical cross‐section curves of hollow cylinders suggests that glutamate dehydrogenase is loosely built and possesses voids. This suggestion is supported by the difference of the volume of glutamate dehydrogenase per 1 Å length calculated from the mass per 1 Å length (4,200 Å3, even if one assumes an average swelling of proteins of 0.33 g H2O per 1 g protein) and from the cross‐section area (5,700 to 5,800 Å3).
Déficience en cytochrome oxydase chez les souches de Saccharomyces cerevisiae exigeant la thréonine pour leur croissance
Authors: de Robichon‐Szulmajster H. , Surdin Y. , Slonimski P. P. 
Published: July 2010

In the previous paper, a metabolic relationship between a cytochrome oxidase deficiency and threonine biosynthesis in Saccharomyces cerevisiae was proposed.It has been found that high concentrations of threonine or its immediate precursors l‐aspartate‐β‐semi‐aldehyde or homoserine, are able to restore cytochrome oxidase synthesis in threonine auxotrophs, grown in the presence of glucose as a carbon source. On the other hand, these strains are able to synthesize cytochrome oxidase when the carbon source is ethanol or glycerol even without an excess of threonine.The results show that this phenomenon is concerned with the regulation of cytochrome oxidase synthesis. Different hypotheses are presented to explain the possible role of threonine in this regulation. One of them stresses the possible role of a differential auxotrophy for the cytoplasmic and the mitochondrial protein synthesis systems.
Modification of Regulatory Properties of Phosphofructokinase by Acetylation
Authors: Chapman A. , Sanner T. , Pihl A. 
Published: July 2010

The effect of acetylation and of sulfhydryl blocking agents on phosphofructokinase from rabbit muscle has been studied.Treatment of phosphofructokinase with N‐acetylimidazole led to rapid loss of its allosteric function, as measured by the ability of AMP to stimulate the ATP‐inhibited enzyme. The catalytic activity was far less sensitive to acetylation. The loss of allosteric function on acetylation was due to the fact that the modified enzyme was insensitive to ATP inhibition. The presence of ATP during acetylation prevented the loss of allosteric activity. The results indicate that tyrosine residues may play an important role in the inhibitory binding sites of ATP.Incubation of phosphofructokinase with a 50‐fold excess of para‐chloromercuribenzoate led to complete inactivation of the enzyme, an effect which could be reversed by the addition of excess thiol. Sulfhydryl blocking had no effect on the ability of AMP to stimulate the ATP‐inhibited enzyme. The results confirm that sulfhydryl groups are closely associated with the catalytic site of phosphofructokinase, and demonstrate that they are not involved in the inhibition of the enzyme by ATP and its reversal by AMP.
The Substrate‐Dependent Steric Course of the Ethanolamine Ammonia‐Lyase Reaction
Authors: DIZIOL Peter , HAAS Helga , RÉTEY János , GRAVES Steven W. , BABIOR Bernard M. 
Published: July 2010

The adenosylcobalamin‐dependent ethanolamine ammonia‐lyase from Clostridium sp. deaminates (2S)‐2‐aminopropanol to propionaldehyde about three times faster than it does (2R)‐2‐aminopropanol.
(2S)‐2‐Amino(1‐2H2)propanol and (1S, 2S)‐2‐amino(1‐2H1)propanol are deaminated by the enzyme with a large kinetic deuterium isotope effect (kH/k2H∼ 6), whereas (1R, 2S)‐2‐amino(1‐2H1)‐propanol behaves like the unlabelled substrate.
(2R)‐2‐Amino(1‐2H2)propanol reacts about 1.7 times more slowly than unlabelled (2R)‐2‐aminopropanol or (1R, 2R)‐2‐amino(1‐2H1)propanol.
From the above results it follows that the enantiomeric 2‐amino(1‐2H2)propanols react at the same rate, irrespective of their absolute configuration and the lyase reaction involves the 1‐Hsi atom in both enantiomers.
(2S)‐2‐Amino[U‐14C, 2‐3H, 3‐3H]propanol was prepared by a combination of enzymic and chemical reactions and the distribution of tritium between C‐2 and C‐3 was determined by its degradation to acetic acid. This doubly labelled substrate was converted on ethanolamine ammonia‐lyase into (2R)‐[U‐14C, 2‐3H, 3‐3H]propionaldehyde.
On the other hand, the (2S) configuration was proved for all three samples of [2‐3H]propionaldehyde recovered from incubations of [5′‐3H]adenosylcobalamin with ethanolamine ammonia‐lyase irrespective of whether the (2S) or (2R) enantiomer of 2‐aminopropanol or propionaldehyde itself (in the presence of ammonium ions) were used as substrates.
Incubations with enzyme plus [5′‐3H]adenosylcobalamin also yielded tritiated 2‐amino‐propanols. These were in turn incubated with horse liver alcohol dehydrogenase and diaphorase in deuterium oxide, which lead to exchange of the 1‐HRe atom with deuterium. Retention of the tritium in the samples provided evidence for the (1S) configuration. By co‐crystallization as the 4‐methyl‐oxazolidone derivative the (2S) configuration of the tritiated 2‐aminopropanol samples was also proved.
These results show that ethanolamine ammonia‐lyase deaminates (2S)‐2‐aminopropanol with retention, whereas (2R)‐2‐aminopropanol reacts with inversion of configuration. Tritium from [5′‐3H]adenosylcobalamin is transferred in all cases to the 2‐pro‐S position of propionaldehyde and to the 1‐pro‐S position of 2‐aminopropanol. In the case of (2R)‐2‐aminopropanol, tritium transfer is accompanied by inversion at the asymmetric centre. All these results, together with the previously reported racemization during the conversion of 2‐aminoethanol, can be explained by a simple mechanistic scheme.
Ca2+, K+ Redistributions and α‐Adrenergic Activation of Glycogenolysis in Perfused Rat Livers
Authors: ALTHAUS‐SALZMANN Marianne , CARAFOLI Ernesto , JAKOB Andrea 
Published: July 2010

The α‐adrenergic activation of glycogenolysis was investigated in isolated rat livers perfused in a non‐recirculating system. Net uptake and/or release of Ca2+, K+ and H+ by the liver (measured by ion‐selective electrodes) were correlated with the glycogenolytic effects of phenylephrine. Uptake and retention of 45Ca by the mitochondria of perfused livers were studied to obtain information on the role played by exchangeable mitochondrial calcium in α‐adrenergic activation of glycogenolysis.
Between 1 and 5 min after starting the addition of phenylephrine a net release of Ca2+ was observed, this was paralleled by an uptake of K+. Production rates of glucose and lactate from endogenous glycogen started to increase at the same time. During the following minutes K+ was released. 2mM EGTA and a high concentration of Mg2+ strongly diminished the ionic and metabolic responses to phenylephrine, 0.2 mM EGTA was less effective.
High concentrations of K+ prevented the metabolic response to phenylephrine but had no effect on the release of Ca2+ into the extracellular medium. Tetracaine activated glycogenolysis and suppressed all the effects of the α‐adrenergic agonist.
Experiments with 45Ca provided no evidence for an α‐adrenergic release of Ca2+ from the exchangeable mitochondrial pool. Incorporation of 45Ca into the mitochondria of perfused livers was enhanced by phenylephrine.
We propose that the a‐adrenergic release of Ca2+ from a pool located close to the surface of the cell is capable of triggering the glycogenolytic response.
Heme Deficiency and Phosphorylation of Ribosome‐Associated Proteins
Authors: FLOYD Georgia A. , TRAUGH Jolinda A. 
Published: July 2010

Phosphorylation of proteins in the protein‐synthesizing complex from rabbit reticulocyte lysates was examined under conditions of optimal protein synthesis and during inhibition of protein synthesis by hemin deprivation. A creatine phosphate–ATP–GTP–Pi exchange system was used to maintain [γ‐32P]ATP and [γ‐32P]GTP at high, constant specific activity. Phosphate incorporation into proteins in the 0.5 M KCl wash fraction and in salt‐washed ribosomes was examined before and after inhibition of protein synthesis in hemin‐deprived lysates and compared to phosphorylated proteins observed in hemin‐supplemented lysates. Two of the phosphoproteins in the 0.5 M KCl wash fraction showed a twofold to fourfold increase in phosphate incorporation prior to inhibition of protein synthesis under conditions of hemin deprivation. One of these phosphoproteins was identified as the small subunit of initiation factor 2 (eIF‐2), the initiation factor involved in the GTP‐dependent binding of Met‐tRNAf to 40‐S ribosomal subunits. A second protein, with a molecular weight of 55000, did not correspond to any of the highly purified initiation factors from reticulocytes, but was a major constituent of the salt‐wash fraction. No changes in phosphate incorporation into the major phosphorylated 40‐S ribosomal protein, S6, were observed; however, several minor components, which were not completely removed by the high‐salt wash, showed alterations in phosphorylation.Protein synthesis and phosphorylation were also examined in intact reticulocytes. When iron and the iron‐transporting protein, transferrin, were present, protein synthesis was linear for at least 2 h. When the cells were incubated in the nutritional media from which iron and transferrin had been omitted, inhibition occurred between 15 min and 30 min. Cells incubated in the absence of iron and transferrin showed increased phosphorylation of two proteins in the high‐salt‐wash fraction. These had molecular weights of 38000 and 55000 and corresponded to the two proteins showing increased phosphorylation in the lysate system in the absence of hemin.
Substrate‐Induced Dissociation of Glycerol‐3‐phosphate Dehydrogenase and Its Complex Formation with Fructose‐bisphosphate Aldolase
Authors: BATKE József , ASBÓTH Gergely , LAKATOS Susan , SCHMITT Bernard , COHEN René 
Published: July 2010

A threefold decrease in specific activity of glycerol‐3‐phosphate dehydrogenase was found on going from 800 nM to 10 nM enzyme concentration. According to ultracentrifugal analyses the dimeric glycerol‐3‐phosphate dehydrogenase (molecular weight 78000) dissociates into monomers in the equilibrium mixture of its substrates and products. The concentration‐dependent decrease in the specific activity is interpreted as a consequence of subunit dissociation and the estimated dissociation constants are 0.7 μM and 3.5 μM at 38°C and 20°C respectively.According to active‐enzyme‐band centrifugation experiments and kinetic analysis aldolase forms a complex with glycerol‐3‐phosphate dehydrogenase and this complex formation influences the specific activity of the dehydrogenase.The interaction between glycerol‐3‐phosphate dehydrogenase and aldolase can provide a regulatory mechanism at the branching point of glycolytic and lipid metabolic pathways.
Direct Oxidation of NADPH by Submitochondrial Particles from Saccharomyces cerevisiae
Authors: DJAVADI Fereydoun H. S. , MORADI Mahnaz , DJAVADI‐OHANIANCE Lisa 
Published: July 2010

It has been accepted that in Saccharomyces cerevisiae Submitochondrial particles do not oxidize the NADPH and that the NADPH: cytochrome c reductase is not a mitochondrial enzyme but rather a microsomal one.The present study provides clear evidence that in S. cerevisiae a direct oxidation of NADPH occurs through the mitochondrial electron transport system.The following results were obtained: Submitochondrial particles from S. cerevisiae are capable of oxidizing NADPH with a relatively high rate. The oxidation of NADPH is sensitive to antimycin A and NaN3 but insensitive to rotenone as is known for NADH oxidation. Also NADPH: cytochrome c reductase activity is inhibited by antimycin A. NADPH‐induced reduction of cytochromes b, c+c1, and aa3 is as fast as NADH‐induced reduction. Cytochromes are reduced to the same extent with either NADH or NADPH.The changes of the ratio of NADH/NADPH oxidation rate and the ratio of NADH‐K3Fe(CN)6/NADPH‐K3Fe(CN)6 reductase activities at various phases of growth suggest that two distinct pyridine nucleotide dehydrogenases could be responsible for NADH and NADPH oxidation. This problem remains to be elucidated.
Glycogen Synthase Kinase‐3 from Rabbit Skeletal Muscle
Authors: EMBI Noor , RYLATT Dennis B. , COHEN Philip 
Published: July 2010

A glycogen synthase kinase was partially purified from rabbit skeletal muscle by precipitation with ammonium sulphate, chromatography on DEAE‐cellulose and chromatography on hydroxyapatite.
The enzyme was highly specific for glycogen synthase. In the standard assay, the relative rates of phosphorylation were: glycogen synthase (100), phosvitin (2.5), phosphorylase kinase (< 1), casein (0.3), protein phosphatase inhibitor‐1 (< 0.1), phosphorylase(< 0.01), mixed histones (< 0.01). The enzyme was separated from virtually all phosvitin kinase and casein kinase activity by the chromatography on DEAE‐cellulose.
The Km values for ATP and GTP were 0.02 mM and 0.5 mM respectively, and a similar maximum reaction velocity was obtained with each nucleoside triphosphate. The activity of the enzyme was unaffected by cyclic AMP, cyclic GMP, calcium ions, calcium ions plus calmodulin, and the specific protein inhibitor of cyclic‐AMP‐dependent protein kinase.
The properties of the enzyme demonstrated that it was distinct from both cyclic‐AMP‐dependent protein kinase and phosphorylase kinase, the two well characterized glycogen synthase kinases in skeletal muscle. This enzyme was therefore termed glycogen synthase kinase‐3.
The phosphorylation of glycogen synthase by glycogen synthase kinase‐3 reached a pleateau near 1.5 molecule phosphate incorporated per subunit under optimal conditions. The activity of glycogen synthase measured in the absence of glucose 6‐phosphate was decreased fivefold and the apparent Ka for glucose 6‐phosphate was increased 15‐fold, when 1.2 molecule phosphate per sub‐unit had been introduced into the enzyme. Phosphorylation to a similar extent with either cyclic‐AMP‐dependent protein kinase or phosphorylase kinase produced smaller changes in activity.
Glycogen synthase was phosphorylated by cyclic‐AMP‐dependent protein kinase, phosphorylase kinase and glycogen synthase kinase‐3, using conditions where the phosphorylation by any one protein kinase reached a plateau near one molecule of phosphate incorporated per subunit. The different protein kinases were used separately and in combination to generate seven different phosphorylated species of glycogen synthase. The phosphorylation of glycogen synthase approached two molecules per subunit when any two protein kinases were combined, and three molecules per subunit when all three protein kinases were combined, and the inactivation produced by the different protein kinases was essentially additive. The results imply that each protein kinase preferentially phosphorylates a different site(s) on glycogen synthase, and this is confirmed by the amino acid sequence analysis described in the following paper in this journal.
The Translational Repression of Globin mRNA in Free Cytoplasmic Ribonucleoprotein Complexes
Authors: CIVELLI Olivier , VINCENT Alain , MAUNDRELL Kinsey , BURI Jacques‐F. , SCHERRER Klaus 
Published: July 2010

The 20‐S globin mRNA‐protein complex isolated from the post‐polyribosomal supernatant of duck reticulocyte lysates was tested for its translational capacities in two different cell‐free protein‐synthesizing systems, one derived from wheat germ, the other derived from nuclease‐treated rabbit reticulocyte lysates. In both translational systems, the cytoplasmic free globin mRNA‐protein complex did not support protein synthesis whereas its fully deproteinized mRNA efficiently directed the synthesis of both the α and β globin polypeptide chains. Sedimentation of the 20‐S mRNA‐protein complex through exponential sucrose gradients containing 0.5 M KCl fractionated this complex into four different particles, from which two globin‐mRNA‐containing core particles were isolated. Neither of these globin‐mRNA‐containing core particles was translated in either the wheat germ or rabbit reticulocyte lysate system, whereas their extracted mRNA efficiently directed globin synthesis in vitro.These observations led to the conclusion that factors repressing translational activity are tightly bound to the free cytoplasmic globin mRNA in its ribonucleoprotein form; our data are also consistent with the notion that the associated translational repressing activity is, at least partly, mediated by protein. The results correlate in a functional manner, the different protein compositions observed in the active and inactive forms of globin mRNA‐protein complexes isolated respectively from the polyribosomal and the post‐polyribosomal cytoplasmic fractions of duck reticulocytes.
An Approach to the Structure of Thyroglobulin
Authors: MARRIQ Claudine , ARNAUD Christiane , ROLLAND Marcel , LISSITZKY Serge 
Published: July 2010

The mixture of CNBr peptides obtained by treatment of porcine thyroglobulin with cyanogen bromide was separated into three‐fractions by Sephadex G‐200 gel filtration in 1 M propionic acid. When one of these fractions was reduced and S‐alkylated, a hormone‐containing CNBr peptide was purified by filtration on Biogel A‐1.5 m and ion‐exchange chromatography on DEAF‐Sephadex. Similarly, two other hormone‐containing CNBr peptides were separated from another reduced and S‐alkylated fraction by Sephacryl S‐200 gel filtration and DEAE‐Sephadex chromatography.The three purified CNBr peptides have the same Mr (15000), differ in amino acid composition and contain 60–70% of the hormones present in the initial thyroglobulin. Fragmentation of these peptides into smaller hormone peptides was carried out by trypsin digestion followed by gel filtration. This resulted in the purification of eight discrete hormone‐containing tryptic peptides whose homogeneity was established by the study of their N‐terminal and C‐terminal sequences. At least four sites for the synthesis of thyroxine and two sites for the synthesis of triiodothyronine have been identified in the three CNBr peptides. A possible additional site was recovered in the form of the dipeptides seryl‐triiodothyronine and probably seryl‐thyroxine.To gain information oil the number of hormone‐forming sites in thyroglobulin iodinated in vivo and on the distribution of these sites in the different hormone‐containing peptides isolated, estimation of thyroxine and triiodothyronine content of batches of thyroglobulin of iodine content comprised between 0.73% and 1.6% was carried out, together with the estimation of the hormone content of all the CNBr peptides separated from two preparations of thyroglobulin of different iodine contents (0.7%and 1.23%). It was shown that the number of thyroxine and triiodothyronine residues increased linearly with increasing thyroglobulin iodine content in the range 0.73–1.6% and reached six residues of thyroxine and two residues of triiodothyrqnine/mol protein for a thyroglobulin iodine content of 1.6% with no indication of saturation. This result is at variance with previous findings on porcine thyroglobulin iodinated in vivo which suggested that the thyroxine content reached a plateau of three to four residues/molecule for an iodine content comprised between 0.7% and 1.1%.