bluuebunny/crossref_metadata_2025 huggingface dataset:
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.
Localization of the Binding Sites of Prokaryotic and Eukaryotic RNA Polymerases on Simian Virus 40 DNA
Authors: SARAGOSTI Sentob , CROISSANT Odile , YANIV Moshe
Published: July 2010

The binding sites of calf thymus RNA polymerase (B) II, wheat germ RNA polymerase B and of the Escherichia coli RNA polymerase were mapped on the simian virus 40 genome by observation of enzyme‐linear DNA complexes by electron microscopy. Three to four major sites and several minor sites are observed for each enzyme; common binding sites for the three enzymes are found in positions 0.17, 0.53 and 0.90 of the viral physical map. Initiation complexes with these enzymes can be stabilized with specific ribodinucleotides and a single ribonucleoside triphosphate. Whereas ApA and ATP greatly enhances the binding of the E. coli enzyme at position 0.17, they stabilize the binding of the eukaryotic enzyme at many sites, some of them located in close proximity of the origin of replication.
Circular Dichroism Studies of 6‐N‐Methylated Adenylyladenosine and Adenylyluridine and Their Parent Compounds
Authors: OLSTHOORN Cornelis S. M. , HAASNOOT Cornelis A. G. , ALTONA Cornelis
Published: July 2010

The temperature dependence of the circular dichroism spectra of two adenylyladenosine and three adenylyluridine dinucleotides in aqueous solution have been studied. The effect of methylation of the N‐6 position of the adenylyl moieties on the thermodynamic parameters of the stacking equilibrium was investigated applying a two‐state model for the thermodynamic analysis of the experimental data. It is shown that the methylated compounds allow a more accurate analysis of the data and that methylation stabilizes the stacking interaction by a relative decrease of the entropy of unstacking.
DNA Replication in Physarum polycephalum
Authors: HARDMAN Norman , GILLESPIE David A. F.
Published: July 2010

DNA has been isolated from Physarum polycephalum nuclei obtained from macroplasmodia at different stages in the mitotic cycle, and examined using the electron microscope. Putative replicating structures were identified, the majority of which contained clusters of 2–37 ‘microbubbles’, each microbubble corresponding to a segment of DNA 100–5000 nucleotides long. The microbubble‐containing structures are unstable in the formamide hyperphase used to prepare specimens for electron microscopy, possibly due to dissociation of newly replicated nascent DNA fragments from the parental DNA template during manipulation. The microbubble clusters present in early S‐phase DNA extend over segments averaging 16400 nucleotide residues, and are separated by non‐replicated regions of DNA varying in length from 10000 to 50000 nucleotides. It is suggested that each micro‐bubble cluster may represent a ‘replicon’, and that many ‘replicons’ in Physarum DNA may contain several sites for the initiation of DNA synthesis that are active during S‐phase.
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.
Purification and Characterization of a Mutant tRNA Nucleotidyltransferase
Authors: McGANN Robert G. , DEUTSCHER Murray P.
Published: July 2010

tRNA nucleotidyltransferase has been extensively purified from a mutant strain of Escherichia coli which displays greatly decreased AMP incorporation, but normal CMP incorporation. The defect in AMP incorporation is retained throughout the purification of the mutant protein. The mutant protein behaves identically to the wild‐type protein with regard to elution position on various chromatographic columns, and both have similar molecular weights of about 50000. The defect in the mutant protein is accentuated by the use of yeast tRNA rather than E. coli tRNA‐C–C as substrate, by decreased pH, by increased ionic strength and by decreased divalent cation concentration. Substitution of Mn2+ for Mg2+ greatly increases the relative activity of the mutant enzyme. In all these cases, CMP incorporation by the mutant enzyme remains the same as the wild‐type enzyme. The Km values of the mutant enzyme for its tRNA and triphosphate substrates are unchanged, and the mutant protein is as stable as the wild type with respect to temperature inactivation. These results strongly suggest that the mutation is in the structural gene for tRNA nucleotidyltransferase, and that the mutation probably does not affect the overall structure of the mutant protein, but only a localized region near the AMP‐incorporating site.
Purification of Mouse Immunoglobulin Heavy‐Chain Messenger RNAs from Total Myeloma Tumor RNA
Authors: AUFFRAY Charles , ROUGEON François
Published: July 2010

A procedure is described for the large‐scale purification of light (L) and heavy (H) chain mRNAs from plasmacytomas produced in mice. Intact RNA is selectively precipitated in high yield from frozen tumors homogenized in 3 M LiCl and 6 M urea. L and H‐chain mRNAs were purified by oligo(dT)‐cellulose chromatography and either sucrose gradient centrifugation in conditions preventing aggregation or by means of high‐resolution preparative gel electrophoresis under non‐denaturing conditions. γ2a and α H‐chain mRNAs sedimented as major components at 15.5 S and 16.5 S respectively, when L‐chain mRNAs sedimented as 12‐S species. H‐chain mRNAs isolated by continuous elution during preparative gel electrophoresis were completely separated from both L‐chain mRNA and residual 18‐S rRNA, and migrated as single components of 1900 ± 50 nucleotides on analytical denaturing gels. The partially purified H‐chain mRNAs were translated into major components of molecular weights of 56000 (γ2a) and 60000 (α) in an mRNA‐dependent rabbit reticulocyte lysate, whereas L‐chain mRNAs yielded polypeptides of molecular weights of 25000 (λ) and 27000 (к). Up to 95% of the translation products directed by the purified mRNAs were immunoprecipitated using specific antisera. The purity of L and H‐chain mRNAs was assessed by hybridization of corresponding cDNAs with excess recombinant plasmid DNA. The results indicated a minimum purity of 47% (γ2a), 62% (α), for H‐chain mRNAs and 60% (к), for L‐chain mRNAs.
Post‐transcriptional Polyadenylation Is Probably an Essential Step in Selection of Balbiani Ring Transcripts for a Cytoplasmic Role
Authors: EGYHÁZI Endre
Published: July 2010

Some aspects of poly(A) addition on to the RNA of the two major Balbiani rings 1 and 2 and of the fate of polyadenylated and nonpolyadenylated 75‐S RNA transcripts in salivary gland cells of Chironomus tentans have been investigated. RNA derived from the Balbiani ring portion of chromosome IV, essentially representing a spectrum of nascent growing transcripts, lacks poly(A) segments, while about 16% of the finished 75‐S RNA molecules released into the nuclear sap is associated with poly(A) stretches. More than 90% of the recently arrived cytoplasmic 75‐S RNA molecules contain poly(A) chains that consist of about 103 AMP residues. Poly(A)‐containing 75‐S RNA rapidly disappears from the nucleus and no significant amount of nonpolyadenylated Balbiani ring RNA molecules can be chased into the cytoplasm. A large proportion of Balbiani ring transcripts remains without poly(A) segments in the nuclear sap even after prolonged incubation periods and probably never leaves the nucleus. It seems probable therefore that the molecular mechanism operating in the selection of 75‐S RNA chains for cytoplasmic use includes post‐transcriptional poly(A) addition as an obligatory event in the biogenesis of Balbiani ring RNA. Another defined mRNA fraction of non‐Balbiani‐ring origin, designated 35‐S RNA, which emerges in the nuclear sap after 45 min of labelling with tritiated RNA precursors, appears to be polyadenylated up to 100% and becomes to a large extent converted to cytoplasmic 35‐S RNA. A small but significant proportion, about 13%, of hnRNA produced by non‐Balbiani‐ring genes on the chromosomes I–III is polyadenylated. The corresponding figure for hnRNA in the nuclear sap is about 25%, and for mRNA in the cytoplasm 56%.
Inhibitory Effect of Activated Protein C on Activation of Prothrombin by Platelet‐Bound Factor Xa
Authors: DAHLBÄCK Björn , STENFLO Johan
Published: July 2010

Factor Xa binds to a receptor available on the platelet surface after the release reaction. The receptor consists of phospholipid and factor V. Factor Xa bound to the receptor catalyses the activation of prothrombin effectively. The effects of bovine protein C, a vitamin‐K‐dependent zymogen of a serine protease, on prothrombin activation by platelet‐bound bovine factor Xa has been studied. Protein C was found to be activated (protein Ca) by thrombin formed in the prothrombin‐platelet‐factor Xa incubation. Protein Ca in contrast to the zymogen, protein C, or protein Ca inactivated with diisopropylphosphofluoridate inhibited prothrombin activation by factor Xa in the presence of platelets. Protein Ca was found to destroy the receptor by proteolysis whereas direct binding of protein Ca to the receptor could not be demonstrated. The inhibition by protein Ca could be monitored as a parallel decrease in factor Xa binding and prothrombin activation. The receptor was protected by factor Xa from proteolysis by protein Ca. Protein Ca was also found to inhibit the interaction between prothrombin and the factor Xa platelet receptor. These results indicate that protein C after activation may have a role as a regulator of prothrombin activation in vivo.
A Conformational Study of Human Spectrin
Authors: CALVERT Rosaleen , UNGEWICKELL Ernst , GRATZER Walter
Published: July 2010

Urea denaturation profiles of spectrin dimer, measured by circular dichroism in the regions of the peptide and aromatic Cotton effects, reflect the existence of several independently unfolding domains, as well as the presence of flexible, non‐globular structure. As shown by sedimentation velocity and cross‐linking experiments, dissociation of the two subunits largely precedes unfolding. The flexible, segmentally mobile structure reveals itself further in the appearance of sharp signals in the high‐resolution proton magnetic resonance spectrum. These spectra reveal that some 20% of the chain is in the segmentally mobile form, regardless of ionic strength, and that its compositon is highly hydrophobic, with few polar side chains. This suggests the possibility that this part of the molecule may penetrate into the lipid bilayer. Conformational stability of the spectrin dimer, as measured by circular dichroism, is substantially unaffected by the state of phosphorylation and by the ionic strength, even though the latter is known to affect the size or shape of the molecule.
Novel Intermediates in the Synthesis of Maltose‐Binding Protein in Escherichia coli
Authors: RANDALL Linda L. , JOSEFSSON Lars‐Göran , HARDY Simon J. S.
Published: July 2010

Nascent intermediates in the synthesis of maltose‐binding protein, a periplasmic protein in Escherichia coli, were demonstrated both in vivo and in vitro. They are likely to result from a drastic reduction in the rate of elongation at specific sites on the mRNA leading to detectable accumulation of distinct species of incomplete polypeptides. In order to reach its final destination in the periplasmic space, maltose‐binding protein is transferred across the cytoplasmic membrane as it is elongated. It is possible that variations in the rate of elongation are involved in this export process.
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.
A Study of the Mechanism of Ferritin Formation
Authors: PÂQUES Eric P. , PÂQUES Antoinette , CRICHTON Robert R.
Published: July 2010

The rate of ferritin formation in the buffers 4‐morpholinepropanesulphonic acid (Mops), 4‐morpholineethanesulphonic acid (Mes) and imidazole at pH values from 5.0 to 6.5 is quite similar. However, the rate of iron deposition is much greater in Mops and Mes at pH values above 6.5 than in imidazole. Increasing the concentration of imidazole inhibits ferritin formation and also leads to a transformation in the shape of the kinetic curves observed. This inhibition is also observed at constant ionic strength but is not found for non‐complexing buffers such as Mops. An inhibition of ferritin formation in imidazole and in Mops buffers is also observed with increasing ionic strength. We conclude that the unprotonated form of imidazole inhibits iron deposition, possibly by binding to the active site of the apoferritin molecule. The temperature dependence of iron deposition was examined. An optimum temperature of 50°C was found but the Arrhenius plots were non‐linear. On the basis of these and previous results, a kinetic model is developed which accounts well for ferritin formation at pH values below 6.5 and above 7.0 in non‐complexing buffers. The model does not account for the kinetics observed at pH values close to neutrality.
Simultaneous Decrease in Deamination of 5′‐Adenylic Acid and 5′‐Deoxy‐5′‐S‐isobutylthioadenosine in Chick‐Embryo Fibroblasts Infected by Rous Sarcoma Virus
Authors: LAWRENCE Françoise , RICHOU Mireille , ROBERT‐GERO Malka
Published: July 2010

The rate of deamination of 5′‐deoxy‐5′‐S‐isobutylthioadenosine [(iBuS)5′Ado] in chick embryo fibroblasts was substantially reduced after their infection and morphological transformation by Rous sarcoma virus. Concomitant with the reduction in rate of (iBuS)5′ Ado deamination there was a decrease in adenosine deaminase and 5′‐adenylic acid deaminase activities. The drop of these activities was related to infection and not to the expression of the src gene.(iBuS)5′Ado was deaminated by at least three enzymes or isoenzymes whose apparent molecular weights have been estimated to be 295000, 121000 and 37000 respectively. Two of these enzymes have been characterized as 5′‐adenylic acid deaminase and the heavy form of adenosine deaminase, respectively.
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.
Characteristics of the Carbohydrate Moiety and the Intermolecular Associations of the High‐Molecular‐Weight Forms of α2u‐Globulin
Authors: HAARS Laura J. , PITOT Henry C.
Published: July 2010

We have investigated some of the biochemical characteristics of the glycosylated forms of α2u‐globulin, the major protein of adult male rat urine. Labeled α2u‐globulin that was secreted into the culture medium of primary monolayer cultures of hepatocytes was immunoprecipitated from the medium with antibody specific for urinary α2u‐globulin and the complex used for further study. α2u‐Globulin existed in the medium as a high‐molecular‐weight aggregate that could be dissociated with reducing agents and detergents into two glycosylated molecules of Mr 75000 and 50000 and one nonglycosylated molecule of Mr 20000. The glycosylated forms of α2u‐globulin were shown by kinetic studies not to be precursors of the molecule of Mr 20000. When the glycoproteins were synthesized in the presence of radioactive sugar and amino acid precursors, the ratio of sugar to amino acid suggested that the highest‐molecular‐weight glycoprotein had 1.5–2 times the carbohydrate content of the faster‐migrating glycoprotein. The oligosaccharide moiety, isolated from an exhaustive pronase digest of the combined glycoprotein molecules, was of Mr 5000 as determined by gel nitration methods. Sequential glycosidase digestion with neuraminidase, β‐galactosidase, and β‐N‐acetylglucosaminidase produced a core oligosaccharide of Mr 1200. Anion‐exchange chromatography demonstrated that the charge microheterogeneity of the α2u‐globulin glycopeptides could be abolished by treatment with neuraminidase. Our results indicate that the oligosaccharide moiety of α2u‐globulin is similar to those oligosaccharides found on other serum proteins, and suggest that formation of the species of α2u‐globulin of Mr 75000 and 50000 requires either the attachment of many oligosaccharide chains to a single polypeptide of Mr 20000 or the covalent linkage of poly‐peptides containing one or more oligosaccharide chains to form dimers and trimers.
Amino‐Acid Sequence of the L‐1 Light Chain of Chicken Cardiac‐Muscle Myosin
Authors: MAITA Tetsuo , UMEGANE Toshiyo , KATO Yukio , MATSUDA Genji
Published: July 2010

The light chain fraction was separated from myosin extracted from chicken cardiac muscle. Two light chain components, L‐1 and L‐2 in the fraction were isolated by chromatography on a column of DEAE‐cellulose (DE‐52) in the presence of 4 M urea. After performic acid oxidation, the L‐1 chain was digested with trypsin and the resulting peptides were isolated. The amino acid sequences of the peptides were established. The ordering of these tryptic peptides in the L‐1 chain was deduced from the amino acid compositions and the partial sequences of peptic peptides from S‐carboxymethylated L‐1 chain. Comparing the whole sequence of the L‐1 chain thus established with that of alkali light chain of rabbit skeletal muscle myosin, 67 amino acid substitutions and two insertions were recognized.
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%.
Optical Study of the Light‐Induced Protonation Changes Associated with the Metarhodopsin II Intermediate in Rod‐Outer‐Segment Membranes
Authors: BENNETT Nelly
Published: July 2010

The kinetics of the protonation changes and of the formation of the metarhodopsin II intermediate are measured in the presence of the pH indicator dye bromocresol purple after two consecutive flashes on the same suspension of right‐side‐out vesicles from cattle rod‐outer‐segment membranes. A rapid proton binding followed by partial proton release is observed. The formation of metarhodopsin II is found to be biphasic. The rapid phase of metarhodopsin II formation is slightly faster than the proton binding, and the kinetics of the slow phase are similar to that of the proton release. The protein groups which are protonated and deprotonated are shown to be situated on the outside of the vesicles.

These results provide evidence that a structural change of the protein involving pK changes occurs without spectral modification before the formation of metarhodopsin III (half‐time of the order of seconds).
Effect of Taurodeoxycholate, Colipase and Temperature on the Interfacial Inactivation of Porcine Pancreatic Lipase
Authors: GRANON Simone , SÉMÉRIVA Michel
Published: July 2010

Beside their inhibitory effect upon lipase adsorption, bile salts at low concentration (around 0.2 mM) have repeatedly been shown to enhance lipolysis slightly. From the data reported in this paper, this activation has been attributed to a stabilization of the adsorbed lipase brought about by low concentrations of bile salts. This hypothesis relies on the following observations. (a) For a given temperature, the activation by bile salts depends on the substrate. It is maximum for trihexanoin (trihexanoylglycerol) and does not exist for tripropionin (tripropionylglycerol). (b) In the absence of bile salts, the optimal activities are obtained for different temperatures depending on the substrate. (c) For a given substrate, the activation by a low concentration of bile salts depends on the temperature. It increases when the temperature is raised (UP to 35–40° C) and completely disappears at a sufficiently low temperature (around 10° C). (d) This temperature efect does not seem to be due to a modification of the physical parameters of the interface as measured by the interfacial tension. (e) Colipase, like bile salts, increases the lipase activity on short‐chain triglycerides but only at high temperature when lipase denaturation occurs. It has no influence upon the activity when the temperature is sufficiently low (around 10° C).
Light‐Induced Appearance of Polysomal Poly(A)‐Rich Messenger RNA during Greening of Barley Plants
Authors: HEINZE Hartmut , HERZFELD Frank , KIPER Manuel
Published: July 2010

Changes in polysotnal poly(A)‐rich tnRNA during greening of etiolated barley plants were studied by the technique of cDNA‐mRNA hybridization. Hybridization data of the homologous reactions reveal that in etiolated as well as in greened shoots a complexity of 5 × 107 nucleotides or about 33000 different average‐sized mRNAs are present. These are organized in different abundancy classes with 94% of the total complexicity present in each of the slowest reacting class representing rare messengers.

Heterologous hybridizations indicate that 92% of all polysotnal poly(A)‐rich mRNAs in etiolated shoots are complementary to those of greened and 82%, of ‘green’ poly(A)‐rich mRNAs are complementary to white ones.

It is shown that the abundant mRNA classes are essentially responsible for these diferences. The prevalent classes making up 15% (‘white’) and 31% (‘green’) of the poly(A)‐rich mRNA mass but comprising only a complexity of 1.8 × 104 and 2.1 × 104 nucleotides are identical to 50% with each other. Hybridization of isolated prevalent ‘green’ cDNA with whole ‘white’ poly(A)‐rich mRNA indicates that the additionally appearing 50% prevalent green messengers must be regarded as greenspecific, only present in polysomal poly(A)‐rich mRNA after illumination.

This conclusion is underlined by the hybridization of the ‘green’ cDNA with total polysomal RNA of etiolated shoots. Evidently appearance of these prevalent messengers in functional polysomes is not caused by a shift from poly(A)‐free mRNA to poly(A)‐rich mRNA.

The results clearly demonstrate that light induces greening by turning on genes or influencing post‐transcriptional processing to produce mature green‐specific poly(A)‐rich tnRNA.
Cytochrome b‐565 in Saccharomyces cerevisiae: Use of Mutants in the Cob‐Box Region of the Mitochondrial DNA to Study the Functional Role of this Spectral Species of Cytochrome b
Authors: MEUNIER‐LEMESLE Danielle , CHEVILLOTTE‐BRIVET Paule , PAJOT Patrick
Published: July 2010

In order to study the functional role of the spectral species of cytochrome b‐565 observed in mitochondria, genetically manipulated strains of Saccharomyces cerevisiae have been used. Strains have been found which are devoid of cytochrome b‐565 under certain conditions and which nevertheless are able to grow on a respirable substrate. Two different methods have been used to determine the cytochrome b‐565 content: anaerobic titrations and antimycin‐A‐induced reduction of cytochrome b‐565. Both yield the same results.
Cytochrome b‐565 in Saccharomyces cerevisiae: Use of Mutants in the Cob‐Box Region of the Mitochondrial DNA to Study the Functional Role of this Spectral Species of Cytochrome b
Authors: CHEVILLOTTE‐BRIVET Paule , MEUNIER‐LEMESLE Danielle
Published: July 2010

A wild‐type strain of Saccharomyces cerevisiae and temperature‐dependent revertants of isonuclear box mutants partially or completely devoid of cytochrome b‐565, were used to study the role of this cytochrome in oxidative phosphorylation.

At the mitochondrial level, the phosphorylating data and the succinate oxidase activity at three different temperatures (16° C, 28° C and 36° C) were measured in these strains showing various cytochrome b‐565 contents. It is concluded that this cytochrome b‐565 is not in the main pathway of the electron transfer chain.

At the optimum growth temperature (28° C), the measurements of the P/O ratio for the wild phenotype strains, with ethanol as substrate, led to the conclusions that two phosphorylation sites of the respiratory chain are functional in these strains.

The growth yields for wild phenotype strains and revertants grown in vivo in complex media, with ethanol or galactose as the energy source (at 16° C, 28° C and 36° C), were compared with the cytochrome b‐565 content.

The growth yields showed small variations if compared to the reference strain, when the cytochrome b‐565 content was greatly diminished or absent.

Thus the ATP production at site II is independent of the cytochrome b‐565 content. Cytochrome b‐565 does not play an essential role in oxidative phosphorylation.
The Protochlorophyllide Holochrome of Barley (Hordeum vulgare L.)
Authors: APEL Klaus , SANTEL Hans‐Joachim , REDLINGER Tom E. , FALK Heinz
Published: July 2010

The NADPH:protochlorophyllide oxidoreductase of barley has been solubilized from etioplast membranes and purified to apparent homogeneity. The highest specific activity measured for the purified enzyme was 1.6 nmol chlorophyllide formed (mg protein−1) per flash. Electrophoretic analysis of the purified enzyme on sodium dodecylsulfate/polyacrylamide gels revealed only, one polypeptide of Mr 36000. During glycerol gradient centrifugation the enzyme migrates as a low‐molecular‐weight component. It is proposed that each enzyme molecule contains only one polypeptide chain. Assuming a molecular weight of 36000 for the enzyme, it was calculated that two or three protochlorophyllide molecules are bound to each enzyme molecule.
Circular Dichroism Studies of Glutathione Reductase
Authors: CARLBERG Inger , SJÖDIN Torgny , MANNERVIK Bengt
Published: July 2010

The circular dichroic spectrum of glutathione reductase was studied. The far‐ultraviolet region of the spectrum indicated a low content of helix and β structures, as calculated on tire basis of commonly used spectral parameters of secondary structure. The fractions of amino acid residues present in helix and β structures, respectively, constitute approximately 30% each of the total number in crystals of the enzyme from human erythrocytes [G. E. Schulz, R. H. Schirmer, W. Sachsenheimer, and E. F. Pai (1978) Nature (Lond.) 273, 120–124]. This discrepancy between the estimates may originate from the presence of supersecondary structures in the enzyme, which invalidate the use of parameters defined for circular dichroic spectra of simple secondary structures. Nevertheless, the similarities between the spectra of glutathione reductase from calf liver, porcine erythrocytes, and human erythrocytes strongly suggest that the secondary and supersecondary structures of the three enzymes are very similar. Furthermore, the far‐ultraviolet circular dichroic spectrum of the apoenzyme is not significantly different from that of the holoenzyme, indicating lack of major structural differences between the two forms of the protein. The circular dichroism of the near‐ultraviolet region indicates changes in the interaction of FAD with the protein matrix as well as local conformational changes involving aromatic side‐chains of amino acid residues during redox changes of the holoenzyme. Differences between near‐ultraviolet spectra of apoenzyme and holoenzyme likewise indicate reversible local conformational changes upon binding and release of the prosthetic group FAD. The reconstituted holoenzyme had a spectrum indistinguishable from the native enzyme. The 300–600‐nm region of the spectrum could be resolved into six Gaussian functions corresponding to the vibronic bands identified in other flavin‐dependent dehydrogenases. The rotational strength of the Cotton effects in the 300–600‐nm region increased and the positions of the maxima in the circular dichroism spectrum shifted upon reduction of the enzyme The increase in the 400–510‐nm region indicates a stronger asymmetric interaction between the isoalloxazine ring of FAD and the protein than that observed in the oxidized form of the enzyme. The enhancement may be effected by the dithiol, which is formed upon reduction of the enzyme.