bluuebunny/crossref_metadata_2025_split huggingface dataset:
Further Study of Nucleotide‐Binding Site on Chloroplast Coupling Factor 1
Authors: GIRAULT Guy , GALMICHE Jean‐Michel 
Published: March 2005

The adenine nucleotide binding sites on spinach chloroplast coupling factor 1 have been investigated by measuring the fluorescence changes of the substrate or of the enzyme as a function of the amount of nucleotide bound to the protein.The decrease of the fluorescence of ethenoadenine nucleotide and the quenching of the intrinsic fluorescence of the protein has been studied when these nucleotides bind to the factor. The enhancement of the fluorescence of a probe, 4‐nitrobenzofurazan (Nbf), is analyzed when adenine nucleotides bind to the protein. These three spectral changes do not parallel the total amount of nucleotide bound to the coupling factor.We propose that the first molecule of nucleotide which binds to the factor exhibits, after binding, specific properties and induces structural changes of the protein. Thus the first molecule of ethenoadenine nucleotide bound to the factor has a very low fluorescence yield and brings about a quenching of the intrinsic fluorescence of the enzyme. Furthermore the first molecule of adenine nucleotide which binds to the factor induces a conformation change which brings the Nbf amino groups from an hydrophilic to an hydrophobic surrounding and consequently causes an enhancement of the fluorescence of the N‐Nbf group.The action of the different nucleotides on the fluorescence intensity of the N‐Nbf group of the factor has been studied.The properties of the solubilized factor are discussed in terms of intersubunit interactions modified by ligand binding on only one site of one subunit. Properties of the factor in vitro are compared with those of the factor embedded in the chloroplast membrane.
Sensitivity of Hemoglobin Thiol Groups within Red Blood Cells of Rat during Oxidation of Glutathione
Authors: KOSOWER Nechama S. , KOPPEL Rela L. , KOSOWER Edward M. 
Published: March 2005

The intracellular thiol‐oxidising diazenes, diazenedicarboxylic acid bis‐N,N‐dimethylamide and diazenedicarboxylic acid bis‐N′‐ethylpiperazinide, have been used in the study of red cells. A difference in the consequences of diazene oxidant treatment between the human red cell and rat red cell has been found in respect to the quantity of oxidant needed for glutathione (GSH) oxidation, to the fate of GSH, and to the reactivity of hemoglobin. In the first place, significantly more oxidant is needed for GSH oxidation in the rat red cell than in the human cell. Secondly, in the human cell, all of the GSH is converted to glutathione disulfide (GSSG), from which GSH is regenerated. In the rat cell, GSH disappears without being converted to GSSG, and GSH is not regenerated. Thirdly, a decrease in rat hemoglobin thiol groups, but no change in human hemoglobin, is found.Sterically unhindered thiol groups in the rat hemoglobin are thought to react with the usual adduct intermediate in GSH oxidation by diazene (formed from RCON=NCOR + GSH →RCON(SG)NHCOR) to produce mixed disulfides, from which GSH is not easily regenerated. The results support the idea that reduction of mixed disulfides of GSH and protein is not carried out directly by GSSG reductase but necessitates thiol transferase and GSH.The thiol‐oxidising diazenes may be of use in mapping of exposed, reactive thiol groups in proteins.
Preparation of Polypeptide Subunits of Cytochrome Oxidase from Neurospora crassa
Authors: WERNER Sigurd 
Published: March 2005

Cytochrome oxidase was purified from Neurospora crassa by ammonium sulfate fractionation in the presence of bile salts. The enzyme preparations contained 10–13 nmol of heme a per mg of protein; no other hemoproteins could be detected. Dodecylsulfate gel electrophoresis resolved the enzyme complex into seven major bands, representing seven polypeptide subunits.A procedure is described that allows the isolation of these enzyme subunits on a large scale starting from a single batch of oxidase preparation. It involves dissociation of the enzyme complex by dodecylsulfate and subsequent separation of the obtained polypeptides by chromatography in the presence of various dodecylsulfate concentrations. Purification of subunits 3, 4, 5, 6 and 7 was achieved by column chromatography using molecular sieves (Sephadex G‐100, Bio Gel P‐60) and hydroxylapatite. For the purification of subunits 1 and 2 an electrophoretic separation on a preparative polyacrylamide gel was required.The advantages and disadvantages of the separation procedure of the enzyme polypeptides are discussed. As a special point of interest, the conservation of antigenic determinants of the polypeptide chains during the dodecylsulfate treatment is considered.
Reaction of Yeast Fatty Acid Synthetase with Iodoacetamide
Authors: KRESZE Georg‐Burkhard , STEBER Liesel , OESTERHELT Dieter , LYNEN Feodor 
Published: March 2005

When yeast fatty acid synthetase is treated with iodoacetamide at 0°C, three carbamoylmethyl residues are incorporated into the protein under concomitant complete loss of the enzymatic activity [Oesterhelt, D., Bauer, H., Kresze, G.‐B., Steber, L. and Lynen, F. (1977) Eur. J. Biochem. 79, 173–180]. Here we report that, in this reaction, only cysteinyl residues are alkylated. During protein hydrolysis of the modified enzyme, S‐carboxymethyl cysteine is formed which, when treated with performic acid in the presence of a strong acid and chloride ions, is oxidized only partially to yield open‐chain S‐carboxymethyl cysteine sulfone but, for the greater part, is transformed into S‐carboxymethyl cysteine lactam sulfone (3‐carboxy‐5‐oxo‐tetrahydro‐1,4‐thiazine‐1,1‐dioxide), a novel derivative of S‐carboxymethyl cysteine which cannot be detected with ninhydrin. If, on the other hand, the carbamoylmethylated enzyme is treated first with performic acid and hydrolyzed afterwards, a large part of the carbamoylmethyl residues is lost due to decarboxylation of the α‐sulfonyl carboxylic acid S‐carboxymethyl cysteine sulfone.When carboxamidomethylated fatty acid synthetase was digested with trypsin at 0°C, two different carbamoylmethyl peptides were found which could be separated by gel filtration. The larger peptide TA was cleaved, during prolongated incubation with trypsin at 30°C, to yield the smaller peptide TB which was purified and its amino acid sequence determined to be Thr‐Pro‐Val‐Gly‐Ala‐Cys(carbamoylmethyl). By comparison of tryptic and peptic peptides from acetylated and carboxamidomethylated synthetase, respectively, it is demonstrated that the same cysteinyl residues are alkylated by iodoacetamide which are the acetyl‐binding sites of the condensing enzyme component, i.e. the peripheral SH‐groups.
Comparison of the Properties of Plasma Membrane‐Bound and Mitochondria‐Bound ATPases in the Yeast Schizosaccharomyces pombe
Authors: DELHEZ Jacques , DUFOUR Jean‐Pierre , THINES Denise , GOFFEAU André 
Published: March 2005

Two distinct membrane‐bound ATPases of the glucose‐grown yeast Schizosaccharomyces pombe are separated by differential and isopycnic gradient centrifugation. The mitochondrial ATPase, with optimal activity at pH 9.0, equilibrates with cytochrome c oxidase at densities 1.17 and 1.19 g × cm−3 while the non‐mitochondrial ATPase, with optimal activity at pH 6.0 is associated with mannans in 1.21‐g × cm−3 particles. Cytochemical staining for carbohydrates confirms the plasma membrane origin of vesicles found in a subcellular fraction enriched about 10 times in pH‐6.0 ATPase activity.
The following analogies are found between the two purified membrane‐bound ATPases. They hydrolyse preferentially ATP and require magnesium. They exhibit a Km of 3.3 mM for ATP‐Mg at pH 6.0. The two activities are inhibited by ADP, Dio‐9, dicyclohexylcarbodiimide, triethyltin, miconazole, sodium fluoride and p‐hydroxymercuribenzoate.
Nevertheless, the plasma membrane‐bound ATPase is distinct from the mitochondria‐bound ATPase in the following properties. The plasma membrane‐bound ATPase is not sensitive to the antibody dressed against the purified mitochondrial enzyme. Excess of Mg2+ inhibits the mitochondria‐bound ATPase but not the plasma membrane one. The relative specificities for nucleoside triphosphates and cations of the two enzymes are different. In contrast to the mitochondrial enzyme, the plasma membrane‐bound ATPase is not stimulated by carbonate and maleate anions. The plasma membrane‐bound ATPase is not inhibited by the mitochondrial inhibitors oligomycin and venturicidin.
Biosynthesis of Triacylglycerols Containing Short‐Chain Fatty Acids in Lactating Cow Mammary Gland
Authors: MARSHALL Michael O. , KNUDSEN Jens 
Published: March 2005

Microsomal 1,2‐diacylglycerol acyltransferase from lactating cow mammary gland incorporated equal molar amounts of microsomal‐bound 1,2‐dipalmitoyl [2‐3H]glycerol and [1‐14C]‐butyrate, [1‐14C]hexanoate or [1‐14C]palmitate from their CoA esters into triacylglycerol. The enzyme could also utilize exogenous 1,2‐diacylglycerols in the presence of ethanol.
The pH optimum of the enzyme was 6.1 and 6.4 with butyryl‐CoA and hexanoyl‐CoA respectively. Values of V were approximately the same (2.7 and 2.4 nmol · min−1· mg−1, respectively), but values of Km were different (34 and 10 μM, respectively) with these two substrates. Mg2+ was not required as cofactor.
The presence of a Mg2+‐dependent phosphatidate phosphatase in the microsomal fraction was demonstrated.
It is proposed that triacylglycerols containing butyric and hexanoic acid are biosynthesized in cow mammary gland by the glycerolphosphate pathway, in which long‐chain 1,2‐diacylglycerols derived from phosphatidic acid are acylated at the sn‐3 position by short‐chain acyl‐CoA esters.
The Role of Iron‐Sulfur Center 2 in Electron Transport and Energy Conservation in the NADH‐Ubiquinone Segment of the Respiratory Chain in Paracoccus denitrificans
Authors: MEIJER Emmo M. , WEVER Ron , STOUTHAMER Adriaan H. 
Published: March 2005

The electron paramagnetic resonance spectra at 15 K of reduced membrane particles of Paracoccus denitrificans exhibit resonance signals with g values, line shapes and temperature profile which are similar to the signals of the iron‐sulfur centers observed in the NADH‐ubiquinone segment of mitochondrial respiratory chains. These iron‐sulfur centers are reducible with NADH, NADPH as well as chemically with dithionite.
Sulphate‐limited growth of Paracoccus denitrificans results in the loss of an electron paramagnetic resonance signal (gz∼ 2.05, gy∼gx∼ 1.92) which has properties similar to those of iron‐sulfur center 2 of the NADH dehydrogenase of mitochondrial origin. The loss of this signal is accompanied by a decrease in the NADH oxidase and NADH ferricyanide oxidoreductase activities to respectively 30 and 40% of the values found for succinate‐limited growth conditions. In addition respiration in membrane particles from sulphate‐limited cells loses its sensitivity to rotenone.
Since sulphate‐limited growth of Paracoccus denitrificans induces loss of site I phosphorylation [Arch. Microbiol. (1977) 112, 25–34] these observations suggest a close correlation between site I phosphorylation, rotenone‐sensitivity and the presence of an electron paramagnetic resonance signal with gz∼ 2.05 and gy∼gx∼ 1.92.
Aminopeptidase N from Escherichia coli: Ionizable Active‐Center Groups and Substrate Specificity
Authors: CHAPPELET‐TORDO Danielle , LAZDUNSKI Claude , MURGIER Maryse , LAZDUNSKI Andrée 
Published: March 2005

Effects of pH on the Michaelis constant and maximal velocity of hydrolysis of l‐alanine‐4‐nitroanilide by aminopeptidase N have been studied in the range between pH 6.5 and pH 9.5. V does not vary with pH in this range, therefore any ionizable group shown to be involved in the action of aminopeptidase N will likely be affecting binding and not the catalytic step. The pKmvs pH profile suggests that two ionizable groups (pKa= 7.2 and pKa= 8.2) affect binding of this substrate. The pKivs pH profile observed with the competitive inhibitor hexylamine does not show any inflection point in the alkaline region (pH > 7.2), which suggests that the pKa= 8.2 at 37°C corresponds to the pKa of the substrate ammonium group.In the neutral pH range the inflection points occur at pH 7.2 both in plots of pKmvs pH and pKivs pH. Thus the corresponding ionizable group should belong to the enzyme. Since the strategic–SH group of aminopeptidase N is protected from the action of N‐ethylmaleimide by hexylamine and other competitive inhibitors, we propose that the value of pKa= 7.2 corresponds to the ionization of this group. Aminopeptidase N is competitively inhibited by l‐amino acids having hydrophobic side‐chains such as leucine, isoleucine, phenylalanine, tyrosine and tryptophan. Blocking of the α‐amino group prevents binding, and blocking of the α‐carboxylic group reduces the inhibitory capacity of the amino acid. Aliphatic amines are also competitive inhibitors; the longer the aliphatic chain, the smaller the Ki, which varies by a factor 20 from butylamine to octylamine. Even though aminopeptidase N is not a metalloenzyme, divalent cations competitively inhibit the hydrolysis of l‐alanine‐4‐nitroanilide. The efficiency of binding is in the order: Cd2+ > Zn2+ > Cu2+ > Co2+ > Mn2+ > Mg2+ > Ca2+ with Cd2+ ions having a 103 higher binding constant than Ca2+ ions.The Ki values and initial rates of hydrolysis of 15 peptides by aminopeptidase N are reported. The peptides tested included: alanyl‐X, X‐alanine, di, tri and penta‐alanine, and dipeptides devoid of alanine, where X represents any one of several amino acids. The C‐terminal amino acid of alanyl‐X dipeptides contributes mainly to the binding constant, which depends on the hydrophobicity of their side‐chain. This affinity varies by a factor of 28 from glycine to phenylalanine whereas V varies only by a factor of 2. Effects of changes in the NH2‐terminal amino acid in X‐alanine dipeptides and in dipeptides devoid of alanine on the rate of hydrolysis and Ki, was also studied. V values for the enzyme of dipeptides devoid of alanine decreased with Ki of the peptides. For di, tri, and pentaalanine, the increase of the peptide size increases the affinity but not the rate of hydrolysis.
The Denaturation‐Renaturation of Chicken‐Muscle Triosephosphate Isomerase in Guanidinium Chloride
Authors: McVITTIE John D. , ESNOUF M. Peter , PEACOCKE Arthur R. 
Published: March 2005

The process of denaturation of the chicken muscle dimeric enzyme triosephosphate isomerase on addition of guanidinium chloride has been studied at pH 7.6, the pH at which the recovery of activity is optimal (100%) on removal of denaturant. Determinations of the sedimentation coefficient, intrinsic viscosity, molecular weight (by sedimentation equilibrium studies) and the absorption coefficient at 280 nm in various concentrations of guanidinium chloride concurred in showing a single, sharp transition at about 0.7 M guanidinium chloride at a protein concentration 1 – 5 mg/ml from the native enzyme to the dissociated, unfolded chains of the monomer. Relative fluorescent intensity measurements revealed a single transition at about 0.4 M guanidinium chloride at enzyme concentrations of about 0.05 mg/ml.
The process of denaturation in different guanidinium chloride concentrations was first order with respect to enzyme and about sixth order with respect to denaturant.
The rate of attainment of equilibrium during the renaturation obeyed second‐order/first‐order reversible kinetics. It was concluded that the rate‐determining step in renaturation at pH 7.6 must be the association of two subunits.
Hormone‐Induced Changes in Pyruvate Kinase
Authors: VAN BERKEL Theo J. C. , KRUIJT Johan K. , KOSTER Johan F. 
Published: March 2005

The kinetic properties of pyruvate kinase from isolated hepatocytes of fed rat liver incubated with and without glucagon (1 μM) are compared. Glucagon incubation of hepatocytes decreases the apparent affinities of the enzyme for the substrate phosphoenolpyruvate and the allosteric activator fructose 1,6‐bisphosphate while the apparent Ki values for alanine and ATP are lowered. The Km for the second substrate ADP is not influenced by glucagon incubation. The kinetic curves show intermediate plateaus indicative of the presence of two enzyme forms, an active form (called La) and a less active form (called Lb). It is concluded that the effect of glucagon on the kinetic parameters of pyruvate kinase is caused by a shift of the equilibrium between both enzyme forms to the less active Lb side.
Comparison of the kinetic parameters of pyruvate kinase of a total homogenate from a fed and a 48‐h‐starved rat indicates that starvation causes a decrease of the apparent affinities of the enzyme for phosphoenolpyruvate and fructose 1,6‐bisphosphate while the apparent Ki values for alanine and ATP are lowered. The Km for ADP is not influenced by starvation. This indicates that a change in kinetic properties of pyruvate kinase also occurs in the intact animal stressing its physiological importance. The change in kinetic parameters introduced by glucagon incubation of hepatocytes and starvation of the intact animal are similar and identical to the changes observed on in vitro phosphorylation of the enzyme purified from rat liver [Ekman et al. (1976) Biochim. Biophys. Acta, 429, 374–382] or from pig liver [Ljungström et al. (1976) Eur. J. Biochem. 68, 497–506]. This indicates that the La and Lb form of pyruvate kinase might represent the dephosphorylated and phosphorylated forms of the enzyme respectively.
The glucagon inhibited enzyme is partially reactivated in vitro by incubation at 37°C. This reactivation is promoted by the presence of 10mM Mg2+. The reactivation in vitro can be blocked completely by 20 mM KF. These properties of reactivation are most consistent with a protein‐phosphatase‐mediated process. The reactivation is inhibited by a high Ca2+ concentration (2.6 mM).
Incubation in vitro of a liver homogenate of a fed and starved rat with 10 mM Mg2++ 0.5 mM ATP in the presence of F− shows that starvation increases the pyruvate kinase inactivation reaction presumably due to protein kinase activity.
It is concluded that hormonal regulation of pyruvate kinase activity is exerted both by changing the degree of (de)phosphorylation of the enzyme and by changing the concentration of the allosteric activator fructose 1,6‐bisphosphate which depends, at least in part, on the hormonal control of the phosphofructokinase–fructose‐1,6‐bisphosphatase cycle.
Microheterogeneity in Lipid A Demonstrated by a New Intermediate in the Biosynthesis of 3‐Deoxy‐d‐manno‐octulosonic‐acid–Lipid A
Authors: LEHMANN Volker , RUPPRECHT Eckhard 
Published: March 2005

A previous report [Lehmann, Rupprecht & Osborn (1977) Eur. J. Biochem. 76, 41–49] described the characterisation of a new class of conditional lethal mutants which are temperature sensitive both in synthesis of 3‐deoxy‐d‐manno‐octulosonic acid 8‐phosphate and in growth. It was found that these mutants synthesize at restrictive temperatures lipid A intermediates consisting of glucosamine disaccharides carrying four 3‐hydroxymyristic acids and two unsubstituted phosphate groups (acidic precursor) [Lehmann (1977) Eur. J. Biochem. 75, 257–266]. The present paper describes the discovery of a new lipid A intermediate (neutral precursor) which is accumulated in the same mutant under slightly modified cultivation conditions. It differs from the acidic precursor by the presence of 4‐aminoarabinose, phosphorylethanolamine and polyamines and exhibits, like the complete lipid A structure of lipopolysaccharides, a considerable degree of intrinsic heterogeneity [Mühlradt, Wray & Lehmann (1977) Eur. J. Biochem. 81, 193–203].Application of different degradation pathways combined with 31P nuclear magnetic resonance spectroscopy showed that in the neutral precursor 4‐aminoarabinose is linked to the ester‐bound 4‐phosphate while phosphorylethanolamine is linked to the 1‐phosphate. The presence of polyamines such as putrescine, spermidine, cadaverine and lysine suggest that some species of the neutral precursor fraction are crosslinked by ionic interaction. Evidence will be presented that the neutral precursor, like the acidic one, is an intermediate in the 3‐deoxy‐d‐manno‐octulosonic acid–lipid A synthesis. Furthermore the findings altogether support a notion in which the acidic precursor could serve as acceptor for the transfer of 4‐aminoarabinose, phosphorylethanolamine and polyamines to the glucosamine backbone structure.
The Inhibition of Ribosomal Translocation by Viomycin
Authors: MODOLFLL Juan , VÁZQUEZ David 
Published: March 2005

The basic peptide antibiotic viomycin rapidly inhibits polypeptide chain elongation when added to purified endogenous Escherichia coli polysomes actively engaged in polypeptide synthesis. After inhibition is established, the polysomal nascent peptide chains react only slowly with puromycin, while the chains of uninhibited polysomes or tetracycline‐blocked polysomes react very rapidly. Since viomycin does not inhibit the peptidyl transferase reaction, these results indicate that the antibiotic confines peptidyl‐tRNA to the ribosomal acceptor site and, consequently, that it blocks polypeptide chain elongation by inhibiting ribosomal translocation.Viomycin at concentrations strongly inhibitory for translocation (10 μM) does not impair the elongation‐factor (EF)‐G‐plus‐ribosome‐dependent GTP hydrolysis, the formation of GDP · EF‐G · ribosome · fusidic‐acid complex and guanyl‐5′‐yl‐methylene‐diphosphonate · EF‐G · ribosome complex, or the non‐enzymic binding of AcPhe‐tRNA to the ribosomal donor site. In contrast, viomycin strongly inhibits the non‐enzymic translocation and the release of AcPhe‐tRNA from the ribosomal acceptor site promoted by depletion of NH4+ ions. It is suggested that viomycin inhibits the movement of peptidyl‐tRNA associated with translocation by interfering with its exit from the ribosomal acceptor site. Viomycin at high concentrations (10–100 μM) partially inhibits the EF‐G‐plus‐ribosome‐dependent GTP hydrolysis and the turnover of the GDP · EF‐G · ribosome · fusidic‐acid complex. These effects, however, are probably unrelated to the inhibition of ribosomal translocation.The binding of viomycin to AcPhe‐tRNA · ribosome · poly(U) complexes is readily reversible, since the antibiotic can be removed by filtration through molecular sieves.