bluuebunny/crossref_metadata_2025 huggingface dataset:
Mechanism of the Alcohol Dehydrogenases from Yeast and Horse Liver
Authors: Biellmann Jean‐François , Jung Michel J.
Published: March 2005

Studies of the alcohol‐acetaldehyde interchange, in the presence of analogues of NAD+ and brought about by yeast and horse liver alcohol dehydrogenases, have not provided any evidence in favour of the direct participation of the enzyme in the hydrogen transfer step. A new preparation of 1,4,5,6‐tetrahydro‐nicotinamide adenine dinucleotide is described. This analogue has been found to be a good competitive inhibitor for both enzymes, thus demonstrating the importance of fixation of the enzyme by a hydrogen bond to the group present in C‐3 of the nicotinamide nucleus.
Propriétés des ribosomes et du RNA synthétisés par Escherichia coli cultivé en présence d'éthionine
Authors: Beaud Georges , Hayes Donal H.
Published: March 2005

Escherichia coli EA1 (met−, bio−, RCstr) grown in the absence of methionine but in the presence of ethionine synthesises ribosomes containing ethionine in their proteins, and whose RNA is sub‐methylated. When the sedimentation behaviour of such ribosomes is examined in the presence of low concentrations (100 μM) of Mg++ they are found to consist mainly of 50 S and 30 S particles which resemble normal ribosomes, but to contain some particles which sediment between 30 S and 50 S. Furthermore, the ratio of 30 S to 50 S particles in the ethionine ribosomes is higher than that observed for normal ribosomes. These modifications, in the synthesis of ribosomes seem to result more from the slowing of bacterial growth in the presence of ethionine than from a specific inhibition of the formation of 50 S particles. 50 S and 30 S subunits formed in the presence of ethionine are defective in the capacity to associate at high Mg++ concentration (10 mM) to form 70 S particles. Reconstruction experiments suggest that this defect is inherent in the structure of ribosomes synthesised in the presence of ethionine. When bacteria, cultured in the presence of ethionine, are sub‐cultured in the presence of methionine, or in the presence of methionine and chloroamphenicol their defective ethionine ribosomes regain normal association properties without loss of incorporated ethionine. These experiments led us to study the level of methylability of the RNA of these ribosomes.

Using an in vitro system, with an S 100 as a source of methylases, we have shown that the RNA, isolated from ethionine ribosomes, accepts methyl groups from S‐adenosyl‐[Me‐14C]‐methionine while normal ribosomal RNA or RNA isolated from bacteria grown successively in the presence of ethionine and methionine does not. Moreover, we have shown that ethionine ribosomes can be methylated in vitro to a level corresponding to 5–6 methyl groups per 70 S particle. When in vitro methylated ethionine ribosomes are analysed in sucrose gradients the methyl groups are found to be associated both with particles which sediment approximately at 30 S and 50 S and with particles which sediment between 30 S and 50 S. These results suggest that the defect in the association properties of ethionine ribosomes results from the submethylation of their RNA moieties. Preparations of ethionine ribosomes have been found to contain methylases since methylation of these particles occurs when external methylases (S 100) are omitted.
Studies on the Protein Moiety of Endotoxin from Gram‐Negative Bacteria
Authors: Wober Wolfgang , AlaupoviĆ Petar
Published: March 2005

The so‐called simple proteins isolated from the endotoxin of Serratia marcescens 08 and whole cells Escherichia coli 0 141 :K85(B) by aqueous phenol treatment were characterized by the determination of hydrodynamic properties, electrophoretic behavior, immunochemical specificity and chemical analysis. The chemical composition of both proteins revealed the presence of small amount of lipid A constituents such as glucosamine, phosphorus and fatty acids; the β‐hydroxymyristic acid regarded as the characteristic marker for lipid A was present in all protein preparations. Since the treatment of intact endotoxin or bacterial cells with aqueous phenol results in the formation of two fragments (a lipopolysaccharide and a lipid A‐protein), both of which are characterized by the presence of lipid A constituents, this dissociation seems to be caused by cleavage of a phenol‐sensitive linkage within the molecule of lipid A rather than at its point of attachment to the protein moiety. Thus, the “simple” protein preparations consist of the protein moiety and a small segment of lipid A. Contrary to previous views, such protein preparations are not simple proteins if this designation implies absence of constituents other than amino acids. However, for a historical reason we suggest that the term simple protein be retained for protein fragments isolated by aqueous phenol treatment of intact endotoxins.

The simple protein from Serratia marcescens 08 was treated successively with trypsin and pronase. The resulting trypsin and pronase cores contained increasing amounts of lipid A constituents firmly bound to the residual protein moiety. Since lipid A was separated as an entity from the protein moiety only after acid hydrolysis of the pronase core, it is proposed that in intact endotoxin lipid A is covalently linked to the protein moiety. The absence of any detectable glucosamine and fatty acids in the mixture of peptides and amino acids released by trypsin and pronase, indicated a single point attachment between the lipid A and protein moieties.

The protein moieties from Serratia marcescens and Escherichia coli were immunogenic and possessed a common antigenic determinant. Both trypsin and pronase cores retained the immunogenicity and revealed the presence of a common antigen with the parent simple protein. This second set of antigenic determinant(s) different from the specific O‐antigens seems to be located in the protein moiety close to lipid A.
Specific Excitation Energy Transfer from Antibodies to Dansyl‐Labeled Antigen
Authors: Pecht Israel , Maron Elchanan , Arnon Ruth , Sela Michael
Published: March 2005

The “loop” peptide region of hen egg‐white lysozyme was labeled with the dansyl group. The binding of the labeled peptide to the anti‐“loop” antibodies led to a specific excitation energy transfer from the antibody to the dansyl group on the hapten. This sensitized fluorescence was used for quantitative evaluation of the binding and homogeneity parameters of the “loop”‐anti‐“loop” interaction. The association constant observed was 3.2 × 106M−1 and the homogeneity index was 0.98 ± 0.05, in excellent agreement with values obtained by equilibrium dialysis.
The Synthesis of the Alternating Copolymer Poly [r(A‐s4U)] by RNA Polymerase of Escherichia coli
Authors: Cramer Friedrich , Gottschalk Ellen M. , Matzura Hans , Scheit Karl‐Heinz , Sternbach Hans
Published: March 2005

The transcription of poly [d(A‐T)] by RNA polymerase from Escherichia coli in the presence of ATP and 4‐thiouridine triphosphate (s4UTP) lead to the synthesis of poly[r(A‐s4U)]. The investigation of the polymerisation kinetics furnished the kinetic parameter Km (ATP‐s4UTP) = 0.33 mM and Vmax (ATP‐s4UTP) = 204 nmoles AMP × mg enzyme−1× min−1.

The structure of poly[r(A‐s4U)] was proved by nearest neighbour analysis and calculation of the AMP/s4UMP ratio from the absorption spectrum of poly[r(A‐s4U)]. As shown by sedimentation velocity analysis poly[r(A‐s4U)] exhibited a mean s20, w value of 12 S. Enzymatic hydrolysis and thermal denaturation of poly[r(A‐s4U)] revealed the existence of a large hyperchromicity in the absorption spectrum. The absorption‐temperature profile of poly [r(A‐s4U)] measured at 0.08 M Na+ showe a sharp cooperative transition with a Tm of 52°C.
Delayed Fluorescence and the High‐Energy State of Chloroplasts
Authors: Wraight Colin A. , Crofts Antony R.
Published: March 2005

The relation between the “high energy state” of photophosphorylation and delayed fluorescence has been studied in spinach chloroplasts.

In the presence of an electron acceptor or cofactors, the kinetics of the rise in intensity of light emitted 1 millisecond after illumination showed two distinct phases,‐a rapid phase complete in less than 0.1 s, and a sigmoidal slow phase with a halfrise time of about 0.3 s.

The maximal intensity and time course of emission were found to vary with electron flow and the degree of coupling. With nigericin or with NH4CI, the slow phase of the intensity rise was abolished but the rapid phase was unaffected. With valinomycin the rapid phase was substantially inhibited, but the extent of the slow phase was increased to give the same maximal intensity as observed in the absence of ionophore. With carbony cyanide p‐trifluoromethoxy‐phenylhydrazone (FCCP), or a combination of valinomycin and nigericin, both phases were substantially inhibited. This spectrum of sensitivities has been interpreted as showing that the slow phase depends on the pH component, and a part of the rapid phase on the electrical component, of the electro‐chemical H+‐gradient. Addition of 3‐(3,4‐dichlorophenyl)‐1,1‐dimethyl‐urea always inhibited emission.

A mechanism relating delayed fluorescence to the “high energy state” is suggested. The photochemical act of System II is envisaged as occurring between acceptor and donor sites on opposite sides of the thylakoid membrane and in equilibrium with pools the mid point potentials of which are dependent on the pH of the phase. It is shown that in such a system the energy conserved in the oxido reductive poise and in the electrochemical H+ gradient are additive and that both contribute to a decreased activation energy for delayed fluorescence. By dissipating the H+ gradient, the activation energy requirement is increased so that the intensity of emission falls.

It is concluded that other mechanisms for emission also operate, in particular one dependent on electron flow as such.
Absorption and Enterohepatic Circulation of Neutral Steroids in the Rat
Authors: Eriksson Håkan
Published: March 2005

The absorption of labelled pregnenolone, corticosterone, pregnenolone sulphate and deoxycorticosterone sulphate after intragastric, intraduodenal and intracaecal injection was studied in female bile fistula rats. Efficient absorption was observed in all cases. Evidence was obtained for the absorption of both free steroids and sulphates from the small intestine, free steroids being absorbed more rapidly. Partial hydrolysis of the sulphates, probably bacterial, occurred in the intestine. Radioactivity in the portal blood was distributed between the free, glucuronide, monosulphate and diconjugate fractions. Glucuronides were present in the bile but not in faeces and urine.

The enterohepatic circulation of labelled pregnenolone and corticosterone was studied in a series of rats consisting of five animals connected by cannulae between the bile duct of one rat and the duodenum of the next rat. The average time for an enterohepatic circulation was calculated to be 3—4 h. The ratio between radioactivities eliminated in faeces and urine increased progressively and markedly with each enterohepatic cycle, as did the ratio between conjugated and free steroids. It is concluded that the steroids undergo extensive metabolism in each enterohepatic cycle and that labelled steroids injected into rats are unlikely to be fully mixed with the endogenous pools before being excreted.
Studies on the Structure, Biosynthesis and Bacterial Metabolism of 15‐Hydroxylated Steroids in the Female Rat
Authors: Eriksson Håkan , Gustafsson Jan‐Åke , SjÖVALL Jan
Published: March 2005

Gas‐liquid chromatography, gas chromatography‐mass spectrometry and radio‐gas chromatog‐raphy have been used to obtain a tentative identification of eight 15‐hydroxylated C21O4 steroids in faeces and urine from conventional female rats: 3α(and 3β),15α‐dihydroxy‐5α,14β‐pregnane‐11,20‐dione, 3α(and 3β),11β,15α‐trihydroxy‐5α,14β‐pregnan‐20‐one, 3α(and 3β),15α, 20β‐trihy‐droxy‐5α,14β‐pregnan‐11‐one and 5α,14β‐pregnan‐3α(and 3β),11β,15α,20β‐tetrol. The assign‐ment of stereochemistry is based on indirect evidence. The steroids were present in the free and monosulphate fractions and became labelled after administration of [4‐14C]pregnenolone and [4‐14C]corticosterone to the rats. Incubations in vitro with caecal contents from conventional rats showed that they were microbial metabolites of the 15α,21‐hydroxylated C21O5 steroids previously shown to be present in faeces and urine from germfree rats.
Fatty Acid Degradation in Escherichia coli
Authors: Klein Klaus , Steinberg Regina , Fiethen Brigitte , Overath Peter
Published: March 2005

The properties of a transport system for the uptake of fatty acids into Escherichia coli cells are described. The system is induced under the same conditions as the enzymes involved in fatty acid degradation. In accordance with the chain‐length specificity of the acyl‐CoA synthetase, only fatty acids with more than eight carbon atoms are taken up. The reduced rate of uptake of fatty acids in mutants with various lesions in fatty acid degradation indicates that transport and metabolism are tightly coupled processes. The absence of an efflux reaction and the inability of a mutant lacking the acyl‐CoA synthetase to take up fatty acids support the hypothesis that this enzyme is involved in a vectorial acylation during the permeation process.

Mutants lacking one or all of the enzymes enoyl‐CoA hydrase, β‐hydroxyacyl‐CoA dehydro‐genase and thiolase, when assayed with substrates having four carbon atoms, can partially degrade long‐chain length and medium‐chain length fatty acids. This suggests that there are at least two types of enzyme activity in the cell which differ in their specificities for acyl‐CoA substrates of various chain‐lengths.

A new old locus situated between the genes proA and proB on the E. coli linkage group is described. Lesions in this gene make the cell unable to degrade fatty acids of any carbon number, and must therefore lack an element common to the degradation of all fatty acids. Fatty acids taken up by these mutants are exclusively incorporated into membrane phospholipids.
Effect of Phytohemagglutinin on Lymphocyte Membrane Transport
Authors: Peters J. Hinrich , Hausen Peter
Published: March 2005

Uptake of labelled uridine into lymphocytes is restricted. Several lines of evidence, including experiments with dipyridamole, an inhibitor for membrane transport processes, and kinetic experiments indicate that transport of uridine through the cell membrane is the rate‐limiting step in uridine uptake. Addition of phytohemagglutinin results in an immediate increase of uridine uptake. Saturation experiments have shown that this change is due to an increase in V, whereas the apparent Km value remains constant. The hypothesis is advanced, that phytohemagglutinin induces an increase in the number of functional carrier sites of the membrane transport system.
Induction of Enzymes Involved in the Catabolism of Deoxyribonucleosides and Ribonucleosides in Escherichia coli K 12
Authors: Hammer‐Jespersen Karin , Munch‐Petersen Agnete , Nygaard Per , Schwartz Marianne
Published: March 2005

When cells of Escherichia coli are grown in the presence of deoxyribonucleosides with glucose or glycerol as carbon sources the following enzymes are induced: deoxyriboaldolase, thymidine phosphorylase, deoxyribomutase, and purine nucleoside phosphorylase. Experiments with mutants deficient in one of the enzymes support the conclusion that formation of deoxyribose‐5‐phosphate is required for this induction.

Two of these enzymes, deoxyribomutase and purine nucleoside phosphorylase, are also induced in the presence of purine ribonucleosides. Evidence is presented that in this case phosphorolysis of the added purine nucleosides is not required for induction and, furthermore, that adenosine gives rise to induction only after prior deamination to inosine.

Addition of adenosine to glycerol‐grown cells of strains deficient in purine nucleoside phosphorylase, gives rise to an unspecific induction of nucleoside catabolizing enzymes in general.
Subcellular Distribution of Sulfite Cytochrome c Reductase in Rat Liver Tissue
Authors: Wattiaux‐De Coninck Simone , Wattiaux Robert
Published: March 2005

The intracellular distribution of sulfite cytochrome c reductase was investigated in homogenates of rat liver and compared with the distribution of cytochrome oxidase, acid phosphatase, catalase and glucose‐6‐phosphatase which were used respectively as reference enzymes for mitochondria, lysosomes, peroxisomes and endoplasmic reticulum. 60% of the enzyme is recovered in the mitochondrial fraction, the remainder being found principally in the soluble fraction.

The total mitochondrial fraction was analyzed by differential centrifugation in a stabilizing sucrose gradient and by isopycnic centrifugation in a sucrose gradient. The distribution curves of sulfite cytochrome c reductase are similar to those of two mitochondrial enzymes, cytochrome oxidase and adenylate kinase, and quite different from those of reference enzymes of other particles.

Sulfite cytochrome c reductase exhibits a structure‐linked latency. The enzyme can be unmasked and solubilized by hypotonic treatment and by digitonin. In the activation experiments, its behaviour was similar to that of adenylate kinase and different from that of glutamate dehydrogenase.

These findings are discussed in the light of the results of other authors. It is concluded that most of the rat liver sulfite cytochrome c reductase is associated with the mitochondria and is probably situated in the intermembrane space of these granules.
Isolation and Stoichiometry of Beef Pancreas Tryptophanyl‐tRNA Synthetase Complexes with Tryptophan and Tryptophanyladenylate
Authors: Dorizzi Mireille , Labouesse Bernard , Labouesse Julie
Published: March 2005

Highly purified tryptophanyl‐tRNA synthetase from beef pancreas forms an enzymetryptophanyladenylate complex which has been isolated from reaction mixtures by gel filtration. The stoichiometry of the complex is 2 moles of tryptophanyladenylate per mole of native enzyme of 108000 molecular weight. The complex reacts with inorganic pyrophosphate to form 1 mole of ATP per mole of bound tryptophanyladenylate and with yeast‐tRNA to form tryptophanyl‐tRNA. An enzyme‐tryptophan complex can also be isolated by gel filtration. Its stability is lower than that of the enzyme‐tryptophanyladenylate complex. When the protein has been specially treated to remove bound tryptophan, no enzyme‐ATP complex can be isolated from a mixture of enzyme and ATP. The bound tryptophan can react stoichiometrically with ATP to give tryptophanyladenylate which remains bound to the enzyme.
C‐Phycocyanin Monomer Molecular Weight
Authors: Kao Oranda , Berns Donald S. , Maccoll Robert
Published: March 2005

The monomer molecular weight of C‐phycocyanin from three different algal sources was investigated. The techniques used for determining molecular weight included high speed sedimentation equilibrium, osmotic pressure measurements in 6 M guanidine hydrochloride, and cyanogen bromide peptide analysis. All measurements were consistent with a monomer molecular weight of 30000 ± 1000 for C‐phycocyanin isolated from all algal sources.
The Influence of pH and Methylene Blue on the Pathways of Glucose Utilization and Lactate Formation in Erythrocytes of Man
Authors: Albrecht Volker , Roigas Henn , Schultze Maria , Jacobasch Gisela , Rapoport Samuel
Published: March 2005

The influence of pH on the contributions of the Embden‐Meyerhof and of the oxidative pentose‐phosphate pathways in the utilization of glucose by human erythrocytes in the presence and absence of methylene blue were determined from yields of 14CO2 and [14C]lactate.

In the absence of methylene blue both the NADP+ concentration and the phosphofructokinase are controlling factors. At pH 7.0 the oxidative pentose‐phosphate pathway accounts for nearly one‐half of the metabolic flux. Its contribution nearly vanishes at pH 8.2.

In the presence of methylene blue the limitation by the NADP+ concentration disappears. At pH values of 7.4 and below the phosphofructokinase is completely inhibited and is circumvented for the formation of lactate. The limitation of the glucose influx resides in the hexokinase. At pH 8.2 both the Embden‐Meyerhof and the oxidative pentose‐phosphate pathway participate in the metabolic flux, since the inhibition of the phosphofructokinase is released.

With methylene blue metabolic pools are formed. They are derived exclusively from the oxidative pentose‐phosphate pathway up to pH 7.5–7.6 and entirely from the Embden‐Meyerhof pathway at pH 8.2.
Energy Transfer Inhibition and Ion Movements in Isolated Chloroplasts
Authors: Karlish Steven J. D. , Avron Mordhay
Published: March 2005

Valinomycin and low concentrations of gramicidin act as energy transfer inhibitors in chloroplasts.

Light‐induced H+ uptake and the H+/e− ratio were stimulated by valinomycin, especially above pH 7.5, while the basal electron transport (–ADP) was inhibited. The maximal H+/e− ratio observed in the presence of valinomycin was 4 (at pH 6.5).

The relationship between light‐induced K+ efflux and proton uptake, both in the presence or absence of valinomycin, is not a stoichiometric one.

Explanations for the observed phenomena, based on the possibility of H+ or K+ binding to fixed negative charges within the thylakoids are discussed.
Methionine Metabolism and Cephalosporin C Synthesis in Cephalosporium acremoniumd‐Amino Acid Oxidase
Authors: Benz F. , Liersch M. , Nüuesch J. , Treichler H. J.
Published: March 2005

The d‐amino acid oxidase was produced during the growth cycle of Cephalosporium acremo‐nium in submerged culture.

The enzyme is strictly sterospecific for d‐amino acids as substrates. It was isolated from the mycelial crude extract and purified 18‐fold. A relatively small number of d‐monoamino acids were deaminated. With d‐methionine as substrate the enzyme had a pH optimum of 8.5 and it was competitively inhibited by the d‐stereoisomers of norleucine, norvaline, leucine, isoleucine, phenylalanine and lysine.

The properties of the C. Acremonium enzyme are compared with those of other fungi and mammalian d‐amino acid oxidases. Its possible influence on the biosynthesis of the antibiotic Cephalosporin C is discussed.
Effect of Reduction of the Intestinal Microflora on the Excretion of Neutral Steroids in Human Faeces and Urine
Authors: Jänne Olli A. , Laatikainen Timo J. , Vihko Reijo K.
Published: March 2005

Neutral steroid glucuronides and mono‐ and disulphates in faeces and urine of normal adult males were analysed before and during phthalylsulphathiazole treatment in order to clarify whether faecal and urinary excretion of C19 and C21 steroids is modified by reduction of the intestinal microflora. For the same purpose, the unconjugated neutral steroids in faeces were analysed, too. During the sulphonamide treatment, the profile of faecal neutral steroid sulphates became very similar to that in bile. The amount of sulphate‐conjugated neutral steroids, especially disulphates, was increased and the pattern of the faecal steroids indicated a decrease in the oxidoreducing and epimerising activities of the intestinal contents. No neutral steroid glucuronides were detected in faeces either before or during the sulphonamide treatment. The urinary excretion of neutral steroid conjugates was very little affected by the administration of the drug. However, most of the 16‐hydroxylated C19 steroid monosulphates were excreted in urine in larger amounts during the treatment than before it.

The results obtained indicate that in normal adult subjects changes in the composition of the intestinal microflora greatly modify faecal steroid excretion, but seem to be of very little importance for the urinary output of a large number of neutral steroid conjugates.
Resolution of Nitrogenase of Mycobacterium flavum 301 into Two Components and Cross Reaction with Nitrogenase Components from Other Bacteria
Authors: Biggins David R. , Kelly Michael , Postgate John R.
Published: March 2005

Nitrogenase from the aerobe Mycobacterium flavum 301 was resolved into two protein fractions resembling those obtained from other nitrogen‐fixing bacteria as regards reduction of N2, C2,H2, cyanide and methyl isocyanide. Fraction 1 contained iron and molybdenum (about 9:1); fraction 2 contained iron only, was rapidly inactivated by oxygen and was cold‐labile. Neither fraction was detected in ammonia‐grown organisms.

The optimum ratio of fraction 1 to fraction 2 for acetylene reduction was about 3:1; that for cyanide or methyl isocyanide was greater than 6.5:1.

Fractions from M. flavum 301 reduced acetylene in combination with nitrogenase fractions from Bacillus polymyxa, Klebsiella pneumoniae and Rhodospirillum rubrum, weakly with Azotobacter chroococcum, not at all with Clostridium pasteurianum.

Fraction 1 from R. rubrum, reduced acetylene with fraction 2 from M. flavum 301, K. pneumonie, B. polymyxa and A. chroococcum, but not C. pasteurianum.
Synthesis of Glucose 1,6‐Bisphosphate by the Action of Crystalline Rabbit Muscle Phosphofructokinase
Authors: Eyer Peter , Hofer Hans Werner , Krystek Edeltraud , Pette Dirk
Published: March 2005

Crystalline rabbit muscle phosphofructokinase catalyzes the transfer of the γ‐phosphate of ATP to glucose‐1‐phosphate in the presence of Mg2+. The reaction product was characterized as glucose 1,6‐bisphosphate by evaluation of hydrolysis data and by means of enzymatic and chromatographic analyses. The enzyme responsible for this “glucose‐1‐phosphate kinase” reaction is most probably identical with phosphofructokinase. The activities of glucose‐1‐phosphate kinase and phosphofructokinase show identical behaviour during purification, anion exchange chromatography and sucrose density gradient centrifugation; furthermore the stability of glucose‐1‐phosphate kinase is highly correlated to that of phosphofructokinase. It is concluded from experiments in vitro that the glucose‐1‐phosphate kinase reaction is the main route for glucose‐1,6‐bisphosphate synthesis in rabbit skeletal muscle. This reactions seems to be more significant than other pathways discussed in the literature, e.g. transphosphorylation from fructose‐1,6‐bisphosphate to glucose‐6‐phosphate (or glucose‐1‐phosphate) as catalyzed by phosphoglucomutase.
Isolation of the Human Retinol Binding Protein by Affinity Chromatography
Authors: Vahlquist Anders , Nilsson Sten F. , Peterson Per A.
Published: March 2005

The human retinol binding protein, which in plasma is bound to thyroxine binding prealbumin, has been isolated in free form from urine of patients with tubular proteinuria and from normal serum. The isolation procedure for urinary retionl binding protein involved gel chromatography and affinity chromatography on a column of prealbumin coupled to Sepharose, whereas the isolation of serum retinol binding protein required three fractionation steps; DEAE‐Sephadex chromatography, and affinity chromatography.

The retinol binding protein from urine and serum was obtained in a yield of 31% and 42% respectively. The relatively low yield of the urinary protein was explained by the fact that two forms of retinol binding protein were present in the urine, one of which did not bind to prealbumin. The purity of the retinol binding protein isolated from urine and serum was established by the following criteria: immunoelectrophoresis, polyacrylamide gel electrophoresis, amino acid analyses, and sedimentation equilibrium ultracentrifugations.

The use of affinity chromatography offers a simple and rapid procedure for the isolation of moderate quantities of highly purified retinol binding protein from urine and serum.
The Subunit Structure of the Escherichia coli K‐12 Pyruvate Dehydrogenase Complex
Authors: Vogel Otto , Beikirch Helga , Müller Harald , Henning Ulf
Published: March 2005

The dihydrolipoamide transacetylase component of the Escherichia coli K‐12 pyruvate dehydrogenase complex has been shown to consist of identical or very nearly identical polypeptide chains with a molecular weight of 80 000 (± 5%).

Amino acid and tryptic peptide analyses gave a minimum chemical molecular weight of about 80 000. Carboxypeptidase degradation revealed one C‐terminal sequence …Arg‐Arg‐(Val,Leu)‐Met‐CO2H per 80 000 molecular weight protein. An N‐terminal amino acid was not found. Dodecylsulfate polyacrylamide gel electrophoresis of the transacetylase subunit (either native, performic acid oxidized, or carboxymethylated) showed one protein band moving as a species with molecular weight of 80 000.

These data are at variance with the results of Reed and associates who had found that the transacetylase component of the E. coli Crookes pyruvate dehydrogenase complex consists of identical 36000 molecular weight polypeptide chains. It has been found that in strain W945 T1 of E. coli K‐12, the 80 000 molecular weight transacetylase subunit is cleaved during the purification procedure, yielding a major fragment with a molecular weight of about 38000. It is possible that this cleavage explains the inconsistency between the transacetylase subunit molecular weights.

It has been confirmed that the dihydrolipoamide dehydrogenase consists of subunits with molecular weight 56000.

A new, relatively rapid purification procedure for the pyruvate dehydrogenase complex is described; it allows the preparation of 200–300 mg enzyme complex, more than 90% pure, within one week.
The pH6 Acetolactate‐Forming Enzyme from Aerobacter aerogenes Subunit Structure
Authors: Huseby Nils‐Erik , Christensen Terje B. , Olsen Bjørn Reino , Størmer Fredrik C.
Published: March 2005

The native and subunit molecular weights of the pH6 acetolactate‐forming enzyme from Aerobacter aerogenes have been investigated by sedimentation equilibrium analysis and gel electrophoresis. The values obtained are 220 000 for the native enzyme and 58 000 for its subunits. The native enzyme is dissociated into four equal‐sized subunits by exposure to guanidine hydrochloride, urea, or sodium dodecylsulfate.

Electron micrographs of enzyme molecules, negatively stained, show that they appear as spherical particles with a diameter of about 6 nm forming dimeric structures of two particles close together. The molecular weight as calculated from the diameter of the particles, are consistent with the values obtained by sedimentation equilibrium and gel electrophoresis. The native enzyme molecule is visualized as a morphological dimer with each particle containing two polypeptide chains of similar size.
Calcium Binding to Sarcoplasmic Membranes
Authors: Fiehn Walter , Migala Andrea
Published: March 2005

ATP‐Supported calcium accumulation of sarcoplasmic vesicles is abolished when the vesicles are disrupted or made leaky. The native as well as the leaky sarcoplasmic membranes are characterized by a high intrinsic affinity for calcium which does not depend on ATP. Neither the number of the calcium binding sites nor their affinity for calcium is affected when the membranes are deprived of the phospholipids which are essential for the activity of the transport ATPase.
Structure primaire de la caséine β bovine
Authors: Ribadeau‐dumas Bruno , Brignon Ghislaine , Grosclaude François , Mercier Jean‐Claude
Published: March 2005

All the expected tryptic and CNBr peptides from bovine β‐casein A2 were previously isolated and analyzed [1]. The complete positioning of these peptides was reported later on [2].

The present report deals with the sequencing of 65 amino acid residues at the carboxyl‐terminus of β‐casein A2: ‐His‐Gln‐Pro‐His‐Gln‐Pro‐Leu‐Pro‐Pro‐Thr‐Val‐Met‐Phe‐Pro‐Pro‐Gln‐Ser‐Val‐Leu‐ Ser‐Leu‐Ser‐Gln‐Ser‐Lys‐Val‐Leu‐Pro‐Val‐Pro‐Glu‐Lys‐Ala‐Val‐Pro‐Tyr‐Pro‐Gln‐Agr‐Asp‐Met‐Pro‐Ile‐Gln‐Ala‐Phe‐Leu‐Leu‐Tyr‐Gln‐Gln‐Pro‐Val‐Leu‐Gly‐Pro‐Val‐Arg‐Gly‐Pro‐Phe‐Pro‐Ile‐Ile‐Val · OH.

This result was obtained by using well known methods such as enzymic or chemical cleavages of the peptide chain, fractionation of peptides by Sephadex or Dowex column chromatography, paper electrophoresis or chromatography, and sequential Edman degradation.

The carboxyl‐terminal tryptic peptide is very likely identical with a peptide isolated by Matoba et al. [3] from a tryptic digest of whole casein, and characterized by its bitter taste.
Structure primaire de la caséine β bovine
Authors: Ribadeau dumas Bruno , Brignon Ghislaine , Grosclaude François , Mercier Jean‐Claude
Published: March 2005

In previous work from this laboratory, all the expected tryptic and CNBr peptides of the bovine β‐casein A2 were isolated, analyzed and positioned in the peptide chain [1,2]. The sequence of 65 residues from the carboxyl‐terminus of this casein was also given [3].

In this communication we report the sequence of 32 residues from the amino‐terminus: H · Arg‐Glu‐Leu‐Glu‐Glu‐Leu‐Asn‐Val‐Pro‐Gly‐Glu‐Ile‐Val‐Glu‐SerP‐Leu‐SerP‐Serp‐Serp‐Glu‐Glu‐Ser‐Ile‐Thr‐Arg‐Ile‐Asn‐Lys‐Lys‐Ile‐Glu‐Lys.

This result was obtained by using, as starting material, β‐A2 tryptic peptides T1, T9 and T11 [1], whose alignment, H.T1‐T9‐T11, was previously established [2].

The amino‐acid sequence of the phosphopeptide T1 was determined mainly from fragments resulting of hydrolysis by thermolysin.

This very acidic peptide contains 4 of the 5 phosphorus atoms of β‐casein A2, 5 seryl and 1 threonyl residues. The 5 seryl residues were obtained in a single peptide resulting of hydrolysis by thermolysin. This peptide (T1Th1) contains 4 phosphorus atoms that can be readily removed by an ortho‐phosphoric monoester phosphohydrolase. Thus it can be inferred that T1 contains 4 phosphoseryl and 1 free seryl residues. The position of the different seryl residues was deduced from analysis of T1, T1Th1 and of dephospho‐T1Th1. The only threonyl residue of T1 is not phosphorylated.

Since five seryl residues were found in peptide T1, instead of the four previously mentioned, the total number of residues in β‐casein A2 amounts to 209 instead of 208 [1–3].

The sequence given above for T1 is very similar to the one recently reported by Manson [4].
Conformational Changes in a Synthetic Antigen Induced by Specific Antibodies
Authors: Schechter Bilha , Conway‐jacobs Abigail , Sela Michael
Published: March 2005

The high molecular weight copolymer (mol. wt. = 75 000) containing the repeating sequence–(l‐Tyr‐l‐Ala‐l‐Glu)–and denoted as polypeptide (Tyr‐Ala‐Glu)n has previously been shown to possess an α‐helical structure under physiological conditions. Studies were performed to check whether monovalent fragments of antibodies derived from anti‐(Tyr‐Ala‐Glu)n antibodies could convert the partially helical oligopeptide (Tyr‐Ala‐Glu)13 into a more helical conformation.

Circular dichroic spectra of mixtures of the monovalent antibody fragments with (Tyr‐Ala‐Glu)13 were measured at two different concentrations of both components. Analogous experiments were done with the same fragments and the polypeptide (Tyr‐Ala‐Glu)n. When anti‐(Tyr‐Ala‐Glu)n antibody fragments were mixed with (Tyr‐Ala‐Glu)13. When the same fragments were mixed with the polypeptide (Tyr‐Ala‐Glu)n, a difference spectrum was observed at the ellipticity band of the polypeptide centered at 275 nm. It was concluded that anti‐(Tyr‐Ala‐Glu)n monovalent antibody fragments could not only convert the (Tyr‐Ala‐Glu)13 into a structure more similar to the polypeptide (Tyr‐Ala‐Glu)n, but could also stabilize the helical content of this polypeptide.
Biosynthesis of Melittin, a Toxic Peptide from Bee Venom
Authors: Kreil G. , Bachmayer H.
Published: March 2005

The biosynthesis of melittin, the main toxin of bee venom, was studied in vivo by feeding radioactive amino acids to honey bees. Extracts from venom glands were analyzed for the presence of labelled melittin and other components. Radioactivity was first incorporated into another peptide which is considered to be a precursor of melittin. This conclusion is based on the observed labelling kinetics demonstrating the transient formation of this peptide. Furthermore, the structural similarity between melittin and this component could be established. Evidence is presented showing that it differs from melittin at the amino end. The precusor peptide could not be detected in the ejected venom.
Kinetics of the Inhibition of Aspartate Aminotransferase Isozymes by dl‐Glyceraldehyde 3‐Phosphate
Authors: Kopelovich Levy , Sweetman Lawrence , Nisselbaum Jerome S.
Published: March 2005

The inhibitor dissociation constants and types of inhibition of the isozymes of aspartate aminotransferase from rat liver by the time‐dependent inhibitor dl‐glyceraldehyde 3‐phosphate were determined from residual enzyme activity after equilibration of each isozyme with the inhibitor and one substrate in the preliminary incubation mixtures.

The results showed that the inhibition of the cationic isozyme was completely competitive with respect to α‐ketoglutarate and completely noncompetitive with respect to l‐aspartate. The pyridoxamine form of the cationic isozyme was approximately 10 times more sensitive to inhibition by dl‐glyceraldehyde 3‐phosphate (Ki= 0.084 mM) than the pyridoxal form (Ki= 0.98 mM). Inhibition of the anionic isozyme was mixed partially competitive‐partially noncompetitive with respect to α‐ketoglutarate and partially noncompetitive with respect to l‐aspartate. The pyridoxamine form of the anionic isozyme was approximately 5 times more sensitive to inhibition by dlDL‐glyceraldehyde 3‐phosphate (Ki= 0.39 mM) than the pyridoxal form (Ki= 1.9 mM). Both forms of the cationic isozyme were more sensitive to inhibition than the corresponding forms of the anionic isozyme.

The degree to which inhibition of the anionic isozyme was partial was assessed by determining the interaction constants α and β. These were α= 6.9 and β= 0.32, and indicated that the anionic isozyme, when fully combined with dl‐glyceraldehyde 3‐phosphate, retained 32% of its catalytic capacity. The cationic isozyme was completely inhibited with α=∞ and β= 0.

Comparison of the Ki values for the pyridoxamine and pyridoxal forms of the isozymes of aspartate aminotransferase with the concentrations of d‐glyceraldehyde 3‐phosphate reported for perfused liver and for incubated erythrocytes suggests that inhibition may occur in vivo.
Correlation Between H+ and Anion Movement in Mitochondria and the Key Role of the Phosphate Carrier
Authors: Mcgivan John D. , Klingenberg Martin
Published: March 2005

The mechanism according to which anions such as di‐ and tricarboxylates are accumulated in mitochondria are investigated with respect to H+ movement and the role of the Pi carrier.

In the absence of respiration, malate is taken up mainly by net accumulation with H+/malate = 1.6. When corrected for exchange with Pi, H+/malate = 2 is found. Under aerobic conditions malate is taken up mainlay in exchange for Pi. The ratio H+/malate is largely independent of the pH despite strong pH dependence of the malate uptake.

The citrate/malate exchange is neutralized by the movement of 1 H+ in the direction of both citrate uptake or efflux.

The net uptake of malate and citrate is abolished by SH reagents demonstrating a key role of the Pi carrier in the net accumulation of di‐ and tricarboxylates.

The uptake of malate and citrate is increased by respiration. This increase is also dependent on the function of the Pi carrier.

For the movement of Pi a ratio H+/Pi= 1.5 at pH 6.9 is obtained, in fair agreement with a theoretical H+/Pi at pH =pK of the dissociation of Pi‐ to Pi2‐.

Anion distribution not coupled to the Pi carrier, such as the distribution of glutamate, is not linked to H+ movement and to energy‐linked accumulation.

The role of the H+ gradient and of energy coupling via the Pi carrier in maintaining different redox potentials across the inner mitochondrial membrane is discussed.
Crystallographic Evidence for the Tetrameric Subunit Structure of l‐Asparaginase from Escherichia coli
Authors: Epp Otto , Steigemann Wolfgang , Formanek Helmut , Huber Robert
Published: March 2005

The analysis of a monoclinic crystal modification of l‐asparaginase from Escherichia coli supports the chemical and physio‐chemical evidence for a tetrameric subunit structure.

The pseudo‐symmetries occurring in the crystal structure provide evidence that the molecules lie on positions with approximate point symmetry 222.
Subunits of Bakers' Yeast Cytochrome b2 (l‐Lactate Cytochrome c Oxidoreductase)
Authors: Lederer Florence , Simon Anne‐Marie
Published: March 2005

After reduction and carboxymethylation, crystallized cytochrome b2 has been separated by gel filtration into two main fractions and a minor one. The latter is probably an aggregate. The apparent molecular weight of the major fractions has been determined by acrylamide gel electrophoresis in the presence of 0.1% sodium dodecyl sulfate, and found to be of the order of 36000 and 21000. Their amino acid composition is very different. Their sum gives a composition in good agreement with the composition corresponding to the minimum molecular weight of unfractionated cytochrome b2. On the basis of the evidence presented in the paper and of the known molecular weight of crystallized b2, it is suggested that the protein is an oligomer of the type (αβ)4.
Subunits of Bakers' Yeast Cytochrome b2 (l‐Lactate Cytochrome c Oxidoreductase)
Authors: Lederer Florence , Jacq Claude
Published: March 2005

1.1 to 1.4 residues of lysine were found as N‐terminal amino acid in crystallized cytochrome b2, 1 residue of alanine and variable amounts of lysine as C‐terminal amino acids. Examination of the end groups of the purified S‐carboxymethylated subunits showed that, besides lysine at the N‐terminus in both chains, alanine at the C‐terminal of the light chain and lysine at the C‐terminal of the heavy chain, other end groups were detected in minor amounts. Digestion of the native protein with carboxypeptidase B led to the finding that a protease still contaminates preparations of type II cytochrome b2. Phenylmethylsulfonyl fluoride is an inhibitor of this protease, the action of which can explain the observed microheterogeneity.
The Cholesterol Side‐Chain Cleavage Enzyme System in Mitochondria of Human Term Placenta
Authors: Mason James I. , Boyd George S.
Published: March 2005

The cholesterol side‐chain cleavage enzyme system of human term placental mitochondria has been studied. Native mitochondria were capable of converting [4‐14C]cholesterol to [4‐14C]‐progesterone in the presence of NADPH and oxygen.

Disruption of mitochondrial structure resulted in an inhibition of the enzymic activity. However, active enzyme could be extracted from the disrupted mitochondria by high speed centrifugation, the endogenous inhibitor remaining associated with the particulate material.

The nature of the endogenous inhibitor has been determined. It appears to involve the mitochondrial phospholipid, especially lecithin. Investigations with authentic phospholipids confirm these studies.

Separation of the enzyme system from the bulk of the mitochondrial material permitted investigation of the characteristics of the enzyme.

Light reversibility studies of the carbon monoxide inhibition of the enzymic activity showed the involvement of cytochrome P450 in the enzyme system.

Fractionation of the enzyme using gel exclusion and ion—exchange chromatography revealed the involvement of a heam iron protein (presumably cytochrome P450), a component having NADPH‐diaphorase activity (flavoprotein) and a non‐haem iron ‘protein in the enzyme system.
On the Role of Quinones in Bacterial Electron Transport
Authors: Kröger Achim , Dadák Vladimír , Klingenberg Martin , Diemer Friederike
Published: March 2005

The individual roles of ubiquinone and menaquinone in the electron transport of Proteus rettgeri have been investigated and their functional positions with respect to the cytochromes and dehydrogenases elucidated.

A method has been developed for the parallel determination of both ubiquinone and menaquinone in the nanomole range in the same extract.

Ubiquinone and menaquinone are not separated by various methods of cell disintegration and remain associated with the cytochromes.

The relative contents of ubiquinone and menaquinone are independent of the hydrogen source and depend on the redox acceptor offered in the growth medium. As in Escherichia coli, ubiquinone is preferentially formed in cutures with maximal oxygen supply and menaquinone in anaerobic cultures. The activity of fumarate reduction by NADH and by formate parallels the content of menaquinone.

On extraction of the quinones the electron transport is inhibited. Ubiquinone reactivates the respiration with succinate, NADH and formate and menaquinone restores the fumarate reduction. In anaerobically grown P. rettegri the respiration with NADH and formate is partly restored by menaquinone, too.

In the anaerobic states, ubiquinone is reduced with succinate, NADH and formate, and menaquinone with NADH and formate only. Ubiquinone is partly oxidized in the steady state of respiration and completely reduced in the steady state of fumarate reduction, where menaquinone is partly oxidized.

2‐n‐Heptyl‐4‐hydroxyquinoline‐N‐oxide inhibits the respiration of succinate, NADH and formate at a common site located on the oxygen side of ubiquinone.

Two b‐type cytochromes (besides cytochrome o) are differentialed: (1) Cytochrome bF is situated on the substrate side of 2‐n‐heptyl‐4‐hydroxyquinoline‐N‐oxide action and is reduced by formate only. (2) Cytochrome b1 is located on the oxygen side of this action and is reducible by all substrates.
Nicotinamide Mononucleotide Determination of its Enzymatic Formation in vitro and its Physiological Rôle for the Biosynthesis of Nicotinamide‐Adenine Dinucleotide in Mice
Authors: Streffer Christian , Beneš Jiri
Published: March 2005

A method is described to measure the activity of the NMN pyrophosphorylase (NMN: pyrophosphate phosphoribosyltransferase) in crude extracts of mouse tissues. The homogenate is incubated with 5‐phosphoribose‐1‐pyrophosphate, ATP, Mg2+ and [14C]nicotinamide in the presence of unlabelled NMN. Optimal conditions for the enzymatic assay were determined.

The NMN pyrophosphorylase activity was measured in several organs of mice by this method. The observed activites were found to be considerably higher than reported in the literature.

The formation of 14C‐labelled pyridine nucleotides in mouse liver after intraperitoneal injection of [14C]nicotinamide was studied. After the application of a high nicotinamide dose (1 mmole/kg) the biosynthesis of NAD was mainly via nicotinate. However, after the injection of a small nicotinamide dose within the physiological range the pathway via NMN is utilized to a large extent. Under these conditions a partial inhibition of the metabolic pathway of nicotinate to NAD has no influence on the NAD formation from nicotinamide. It is concluded that NMN is the physiological intermediate for NAD biosynthesis from nicotinamide.
Essential Fatty Acid Deficiency and the Activity of the Sarcoplasmic Calcium Pump
Authors: Seiler Dieter , Hasselbach Wilhelm
Published: March 2005

The fatty acid composition of the lipids of the sarcoplasmic membranes isolated from rats is changed when the animals are kept on a diet free of essential fatty acids. The fatty acids of the oleic acid family are increased and the fatty acids of the linoleic acid family are decreased.

The calcium permeability of the modified membranes is reduced giving rise to a higher rate of calcium uptake, a reduction of the extra ATPase activity and a higher calcium concentrating ability.
Brain Glucosyl Ceramides Containing 2‐Hydroxy Acids
Authors: Hammarström Sven
Published: March 2005

Glucosyl ceramides were isolated from young mouse brains by a combination of column chromatography and thin layer chromatography. Analysis by gas‐liquid chromatography—mass spectrometry of the trimethylsilyl ether derivatives revealed that about 20% of these compounds contained 2‐hydroxy acids (mainly cerebronic acid, 2‐hydroxy behenic acid, and 2‐hydroxy stearic acid). The rest contained non‐hydroxy acids (stearic acid was most abundant; arachidic acid, lignoceric acid and behenic acid came next). Unsaturated acids were virtually absent. The major long chain base was C18‐sphingenine. Small amounts of C18‐sphinganine were also present. Of interest was the finding that C20‐sphingenine constituted about 20% of the total long chain bases.