bluuebunny/crossref_metadata_2025 huggingface dataset:
Sulfite Binding to a Flavodehydrogenase, Cytochrome b2 from Baker's Yeast
Authors: LEDERER Florence
Published: March 2005

Baker's yeast l‐lactate dehydrogenase (flavocytochrome b2) is a typical flavodehydrogenase, in that it accepts two electrons from the substrate but has a monoelectronic acceptor. Yet it forms a red semiquinone [Capeillère Blandin et al. Eur. J. Biochem. 54, 549‐566 (1975)] and it is shown in this paper that it forms a reversible covalent complex with sulfite (Kd= 1.4 μM). This complex can be observed by difference spectroscopy and provides a convenient tool for visualizing the flavin chromophore, usually hidden behind the intense heme absorbance. A number of anions (d‐lactate, oxalate and pyruvate) are inhibitors of the enzymatic reaction and induce spectral perturbations of the flavin spectrum. It is concluded that probably two positive charges exist at the active site: one which stabilizes the red semiquinone and one which attracts organic anions and sulfite. It is also concluded that the correlation between reactivity with sulfite and reactivity with oxygen among flavo‐proteins may not be as general as previously proposed [Massey et al. J. Biol. Chem. 244, 3999‐4006 (1969)].
Alanine Metabolism in Rat Liver Mitochondria
Authors: DIETERLE Peter , BRAWAND Fritz , MOSER Ulrich K. , WALTER Paul
Published: March 2005

In the presence of 2‐oxoglutarate, bicarbonate, Pi and ATP, rat liver mitochondria were found to metabolize alanine. The main products were glutamate, aspartate, malate and citrate. Pyruvate did not accumulate but reached a low steady‐state concentration. Addition of octanoate caused a strong stimulation of alanine metabolism and a concomitant decrease in pyruvate concentration. NO inhibition of alanine metabolism was obtained in the presence of inhibitors of mitochondrial pyruvate transport, α‐cyano‐4‐hydroxy‐cinnamate and α‐cyano‐cinnamate, showing that alanine was not transaminated by a contamination of the mitochondria by cytosolic alanine aminotransferase. In the presence of l‐cycloserine, alanine metabolism in mitochondria was strongly inhibited indicating that in contrast to earlier reports l‐cycloserine can enter the mitochondria.

In isolated liver cells, addition of α‐cyano‐cinnamate caused a strong pyruvate accumulation and a nearly complete inhibition of gluconeogenesis with serine as substrate, whereas with alanine no pyruvate increase and a weak inhibition of gluconeogenesis was observed.

From a comparison of the employed substrate concentrations and of the observed rates of alanine transamination in intact mitochondria with those under physiological conditions in cellular systems, it is concluded that cellular conversion of alanine to pyruvate most likely occurs to a great extent within the mitochondria as has been proposed earlier by DeRosa and Swick (1975) J. Biol. Chem. 250, 7961‐7967.
Incorporation Rates of [1‐14C]Glycerol into the Molecular Species of Alkyl Ether Phospholipids of Ehrlich Ascites Tumor Cells in vivo
Authors: WAKU Keizo , NAKAZAWA Yasuo
Published: March 2005

[1‐14C]Glycerol was injected into the peritoneal cavity of mice bearing Ehrlich ascites tumor cells and the lipids were extracted from the cells after selected periods. 1‐O‐Alkyl‐2‐acyl‐3‐acetyl‐glycerols and 1,2‐diacyl‐3‐acetylglycerols were prepared from the ethanolamine and choline phosphoglycerides and fractionated by AgNO3‐impregnated thin‐layer chromatography into seven molecular species. Bands 1‐7 obtained in this way can be designated as saturated, monoene, diene (11), diene (02), triene, tetraene and pentaene + hexaene species, respectively. Band 7, composed mainly of hexaene species, was predominant among those derived from ether phospholipids. The predominant species was band 4 (saturated/linoleate) in both diacyl ethanolamine and choline phospholipids.

The specific radioactivities of the fractionated species were determined. In diacyl or alkyl ether compounds of ethanolamine and choline phosphoglycerides, disaturated species turned over very rapidly, while in alkyl ether ethanolamine phosphoglyceride, hexaene + pentaene molecular species turned over rapidly. In diacyl compounds of the ethanolamine phosphoglyceride, hexaene molecular species also turned over at a high rate. In diacyl ethanolamine or choline phosphoglycerides, dienoic species turned over rather slowly.
Evidence for Multiple Reactive Forms of Papain
Authors: POLGÁR László , HALÁSZ Piroska
Published: March 2005

The pH‐dependence of the second‐order rate constants of acylation and alkylation reactions of the –SH group of papain were determined by using neutral and charged reactants under identical conditions. From these pH rate profiles, in contrast to previous claims, different pKa values were obtained with different groups of reactants. In the case of charged reactants, like chloroacetate (pKa= 3.6) and arginine derivatives (pKa= 4.3), the pKa differences can be attributed to electrostatic effects. However, the fact that a pKa of 3.25 was found with methyl and ethyl bromoacetate, and a pKa of 4.0 was obtained with bromoacetamide and a number of neutral substrates, is inconsistent with the theories put forth hitherto for the meaning of such pKa values, because they all consider only one reactive enzyme form. The different pKa values obtained here with neutral reactants are explained in terms of various reactive papain forms.

The perturbation of pKa by electrostatic effects was examined by reacting simple thiol compounds containing different charges, like 2‐mercaptoacetate, 2‐mercaptoethylamine, 2‐mercaptoethanol and glutathione, with the neutral chloroacetamide and with the negatively charged chloroacetate. Differences in pKa can be interpreted in terms of intramolecular and intermolecular electrostatic interactions.
Cryoenzymologic Studies on Arginine Kinase: Solvent, Temperature and pH Effects on the Overall Reaction
Authors: TRAVERS Franck , BERTRAND Raoul , ROSEAU Gilbert , van THOAI Nguyen
Published: March 2005

The overall reaction catalyzed by the phosphotransferase arginine kinase was studied at normal and subzero temperatures. Ethylene glycol was used as the antifreeze and its effects on the Km values of substrates, kcat and pH profiles were investigated in detail.

a) The Km values for the substrate (2 mM for ATP and 0.6 mM for arginine) were little affected by the solvent composition or temperature of the reaction mixture.

b) At concentration of ethylene glycol higher than 40% there was a sharp drop of enzyme activity.

c) Ethylene glycol induces a large shift in the enzymic pK

d) At ‐5 °C in 40% of solvent there was a break in the Arrhenius plot suggesting a change of the rate‐limiting step.

The relevance of these results to the reaction pathway of arginine kinase is discussed. In addition, controlled perturbations induced by cosolvent and temperature appear as useful tools for further kinetic investigations.
Lipid‐Bound Oligosaccharides in Insects
Authors: QUESADA ALLUE Luis A. , BELOCOPITOW Enrique
Published: March 2005

Membrane preparations from immature stages of the fruit fly Ceratitis capitata catalyze the transfer of mannose from GDP‐[14C]mannose into lipid‐linked oligosaccharides. These compounds behave as polyprenyl derivatives and their formation is stimulated by the addition of an acidic glyco‐lipid fraction isolated from insects.

The mannose‐labeled oligosaccharides are attached to the poly‐isoprenol by a pyrophosphoryl linkage and can be released by mild acid hydrolysis. The trisaccharide lipid has been partially characterized. The results indicate that the compound is polyprenyl‐pyrophosphate‐N,N′‐diacetylchitobiose‐mannose.

Incubation of dolichyl phosphate [14C]mannose or lower 14C‐labeled oligosaccharide lipids with unlabeled GDP‐mannose and the insect enzyme leads to the labeling of a higher lipid‐bound oligosaccharide.

When UDP‐N‐acetyl[14C]glucosamine was incubated with insect membranes a 14C‐labeled chitobiosyl lipid was synthesized. If unlabeled GDP‐mannose was also present, the 14C label appeared in the trisaccharide and higher oligosaccharide lipids.

Preliminary evidence indicates that the insect polyprenyl oligosaccharides described here might participate in glycoprotein biosynthesis.
Structure of Antigenic Determinants in the Amino‐Terminal Region of Bovine Fibrinogen Aα Chain
Authors: TANSWELL Paul , Hildegard Hildegard , TIMPL Rupert
Published: March 2005

A radioimmunoassay was developed for peptide F‐CB1α from the amino end of bovine fibrinogen Aα chain, isolated after reduction and carboxymethylation of the multichain disulfide‐linked cyanogen bromide peptide F‐CB1. Seven out of twelve different rabbit antisera produced against fibrinogen, peptide F‐CB1 or Aα chain showed distinct binding to 125I‐labelled F‐CB1α. Thrombin cleavage of F‐CB1α yielded two fragments: fibrinopeptide A (residues 1‐19) and the carboxy‐terminal fragment Th2 (residues 20‐54). Antisera could be classified into three groups, according to whether they recognized antigenic determinants on fibrinopeptide A, on peptide Th2 or as they showed diminished reactions with both fragments. Only little or no cross‐reaction was observed with the amino‐terminal cyanogen bromide peptides of Bβ and γ chain.

Proteolytic fragments of fibrinopeptide A were isolated and tested for inhibitory activity with two antisera. One antiserum contained antibodies binding selectively to the amino‐terminal sequence (residues 4‐11) and did not cross‐react with human fibrinopeptide A. Another antiserum showed a specific binding restricted to the carboxy‐terminal sequence (residues 11‐18) and cross‐reacted completely with human fibrinopeptide A. These results correlate well with the primary structures of the two fibrinopeptides. The antigenic activity of the peptide fragment Th2 was localized on a 15‐residue tryptic peptide derived from the central portion of the sequence. These and further data indicate that at least six different antigenic determinants are present in peptide F‐CB1α.
Ribosomal Genes in Physarum polycephalum: Transcribed and Non‐Transcribed Sequences Have Similar Base Compositions
Authors: STEER Wendy M. , MOLGAARD Harald V. , BRADBURY E. Morton , MATTHEWS Harry R.
Published: March 2005

The transcribed and non‐transcribed sequences in Physarum polycephalum ribosomal DNA (rDNA) were separated by restriction nuclease digestion of pure rDNA and the products fractionated by zone sedimentation in sucrose gradients. The base compositions of the fragments were determined by analytical centrifugation in CsCl or in CsCl with netropsin. All the fragments had dA + dT contents in the range 44‐48%. From the known sequence arrangement and transcription pattern of Physarum rDNA it was concluded that coding sequences, transcribed but non‐coding sequences, and non‐transcribed sequences all possess similar base compositions, contrary to the situation in many other systems. The thermal denaturation profile of Physarum rDNA is reported. It suggests the rDNA sequence is complex and supports the above conclusion of limited heterogeneity of base composition.
Production of NADPH in the Mannitol Cycle and Its Relation to Polyketide Formation in Alternaria alternata
Authors: HULT Karl , GATENBECK Sten
Published: March 2005

The enzymes mannitol‐1‐phosphate dehydrogenase, mannitol‐1‐phosphatase, mannitol dehydrogenase and hexokinase participate in an enzymatic cycle in the fungus Alternaria alternata. One turn of the cycle gives the net result:

The cycle alone can meet the total need of NADPH formation for fat synthesis in the organism. A polyketide producing strain of A. alternata shows a lower mannitol oxidation as well as a lower fat synthesis than a nonproducing mutant, supporting the hypothesis that polyketide formation is favoured at limiting NADPH production. It is further suggested that the mannitol cycle is regulating the glycolytic flux by substrate withdrawal from phosphofructokinase.
Factors Modifying the Equilibrium between Activated and Non‐Activated Forms of Steroid‐Receptor Complexes
Authors: BAILLY Alain , SAVOURET Jean‐François , SALLAS Nicole , MILGROM Edwin
Published: March 2005

Steroid‐receptor complexes formed in concentrated cytosol at low temperature, low ionic strength and neutral pH are unable to bind to nuclei. Various procedures are known to promote their ‘activation’. In the present work it is shown that an increase in temperature only enhances the rate of the reaction whereas no change in the equilibrium between activated and non‐activated complexes is observed. On the contrary an increase in ionic strength or pH, as well as a removal of a low‐molecular‐weight inhibitor, not only accelerate the reaction but also increase the concentration of activated complexes at equilibrium. Using two steroids differing 3‐fold in their affinity for the receptor, no difference was seen in the effect of the bound steroid on receptor activation.

When combining various activation procedures it was observed that they acted independently of each other and additively. In all cases they retained their property of either modifying only the rate of the reaction or both its rate and equilibrium.

Using changes in pH, it was also possible to induce shifts in the equilibrium between activated and non‐activated complexes. After activation at pH 6.5, a first equilibrium was attained. When the pH was increased to 8 the equilibrium was displaced towards higher concentrations of activated complexes. A lowering of the pH resulted in a reversal of steroid‐receptor complexes from the activated to the non‐activated state. To clearly establish that this was not due to irreversible damage of the receptor, which would render it unable to bind to nuclei, it was shown that the complexes which had reverted to the non‐activated to the non‐activated state were still susceptible to activation.

Regulatory events may thus exist which, for a given level of hormone and receptor, modulate the concentration of activated steroid‐receptor complexes.
Adaptive Responses of Enzymes of Carbohydrate and Lipid Metabolism to Dietary Alteration in Genetically Obese Zucker Rats (fa/fa)
Authors: SPYDEVOLD Sverre O. , GREENBAUM A. Leslie , BAQUER Najma Zaheer , MCLEAN Patricia
Published: March 2005

Measurements have been made of the activities of enzymes of the glycolytic route, the pentose phosphate pathway, the tricarboxylic acid cycle and lipogenesis in liver and adipose tissue from genetically obese (fa/fa) rats and their lean litter mates (fa/‐). The effect of food restriction for a period of three weeks on the enzyme profile of liver and adipose tissue of the obese rat was also studied.

The most striking increases in enzyme activity in livers from obese rats were:(a) among enzymes of lipogenesis; ATP‐citrate lyase, acetyl‐CoA carboxylase, fatty acid synthetase, malate dehydrogenase (decarboxylating) and cytoplasmic glycerolphosphate dehydrogenase; (b) within the pentose phosphate pathway; glucose‐6‐phosphate dehydrogenase and 6‐phosphogluconate dehydrogenase; (c) within the glycolytic pathway; glucokinase, pyruvate kinase and lactate dehydrogenase. All of these enzymes showed a significant increase in activity on the basis of U/g liver and U/mg DNA. In adipose tissue all the enzymes of lipogenesis, of the glycolytic route, of the oxidative segment of the pentose phosphate pathway and of the tricarboxylic acid cycle were increased when expressed as U/2 fat pads or as U/mg DNA.

The restriction of the food intake of obese rats to that consumed by their lean litter mates for periods of three weeks did not produce the expected adaptive decrease in enzymes of lipogenesis; in adipose tissue, only ATP‐citrate lyase and malate dehydrogenase (decarboxylating) showed a marked decrease; no significant change was found in adipose tissue or liver of the activities of acetyl‐CoA carboxylase and fatty acid synthetase, when expressed on a cell basis (U/mg DNA). The nonoxidative enzymes of the pentose phosphate pathway and enzymes involved in glycerogenesis (pyruvate carboxylase, malate dehydrogenase and phosphoenolpyruvate carboxylkinase) all increased in adipose tissue from limit‐fed obese rats.

The rate of conversion of specifically labelled glucose to [14C]O2 and 14C‐labelled lipid by pieces of adipose tissue and by liver slices was also measured. Insulin caused an increase in the conversion of [1‐14C]glucose to [14C]O2 and 14C‐labelled lipid in obese rats fed ad libitum, limit‐fed rats and in their lean litter mates.

The results are discussed in relation to the raised insulin and hypothyroid state of the obese rat. The effect of this altered hormonal status on the activity of cyclic nucleotide phosphodiesterases and cellular levels of adenosine 3′:5′ ‐monophosphate and guanosine 3′ :5′ ‐monophosphate in relation to the obese syndrome is considered.
Interaction between Membrane Functions and Protein Synthesis in Reticulocytes
Authors: WRESCHNER Daniel , MELLOUL Danielle , HERZBERG Max
Published: March 2005

An inhibitor of protein synthesis has been isolated from reticulocyte membranes by solubilization with Triton X‐100; it has been purified using heat treatment, filtration on Amicon filters, DEAE‐cellulose ion‐exchange chromatography and Sephadex G‐75 gel chromatography. A final purification of 120‐fold was achieved.

The purified inhibitor was found to be 95% homogenous when run on a dodecylsulfate/polyacrylamide gel system. Three independent methods were used to estimate the molecular weight of the purified inhibitor: Sephadex G‐75 gel chromatography, dodecylsulfate/polyacrylamide gel electrophoresis and sucrose gradient all confirmed that the purified inhibitor was a small molecule with a sedimentation coefficient of 0.7 S and a molecular weight ranging between 5000 and 8000.

The purified inhibitor was shown to possess a specific endonucleolytic activity, degrading the 28‐S species of ribosomal RNA to species sedimenting between 10 and 14S. Due to its membrane localisation the name RNase M is proposed.

The purified inhibitor's endonucleolytic activity was characterized with regard to its kinetics, concentration dependence, pH optimum and its requirements for divalent cations.

Kinetics showed that RNase M retained its specificity after 60 min of incubation with the RNA substrate. Specificity was also demonstrated by incubating the polysomal RNA with high concentrations of purified enzyme. The pH optimum was found to be between pH 6 and pH 7, and the enzyme did not require divalent cations for its activity. Pancreatic RNase B used at a similar protein synthesis inhibitory concentration as the RNase M caused a complete breakdown of ribosomal RNA to oligonucleotides and mononucleotides.

The possible biological significance of the purified inhibitor in regulating protein synthesis in the maturing reticulocyte is discussed.
The Amino‐Acid Sequence of S‐100 Protein (PAP I‐b Protein) and Its Relation to the Calcium‐Binding Proteins
Authors: ISOBE Toshiaki , OKUYAMA Tsuneo
Published: March 2005

The amino acid sequence of a brain‐specific protein, S‐100, has been determined by the analysis of four cyanogen bromide peptides and fourteen tryptic peptides derived from the protein. The protein chain consists of 91 amino acid residues and has a molecular weight of 10 507. The sequence shows regions of strong clustering of hydrophobic, basic and acidic amino acids, with an apparent calcium‐binding site in the acidic cluster.
Ribosomal Subunits from Tetrahymena pyriformis
Authors: RODRIGUES‐POUSADA Claudina , HAYES Donal H.
Published: March 2005

Tetrahymena pyriformis ribosomal subunits were obtained by incubation of post‐mitochondrial supernatant in the presence of 0.2 mM GTP and 0.1 mM puromycin for 45 min at 28 °C, followed by sucrose density gradient centrifugation. Isolated 40‐S and 60‐S subunits were able to reassociate in vitro in the presence of 5 mM MgCl2 and 50 mM KCl and to perform poly(U)‐dependent protein synthesis. The 60‐S subunit carries the peptidyl transferase activity. The number of proteins in T. pyriformis ribosomal subunits was determined by two‐dimensional polyacrylamide gel electrophoresis. The 40‐S subunit contains 30 different protein species (including two acidic proteins). The 60‐S subunit contains 35 different protein species (including two acidic proteins). The proteins were numbered following the system of Kaltschmidt and Wittmann.
The Complete Amino‐Acid Sequence of Histone H2B(3) from Sperm of the Sea Urchin Parechinus angulosus
Authors: STRICKLAND Marie , STRICKLAND Walter N. , BRANDT Wolf F. , VON HOLT Claus , WITTMANN‐LIEBOLD Brigitte , LEHMANN Arnold
Published: March 2005

The primary structure of a third H2B histone isolated from sperm of the sea urchin Parechinus angulosus has been determined.

H2B(3) consists of a polypeptide chain of the following 148 amino acid residues: Pro‐Arg‐Ser‐Pro‐Ala‐Lys‐Thr‐Ser‐Pro‐Arg‐Lys‐Gly‐Ser‐Pro‐Arg‐Lys‐Gly‐Ser‐Pro‐Arg‐Lys‐Gly‐Ser‐Pro‐Ser‐Arg‐Lys‐Ala‐Ser‐Pro‐Lys‐Arg‐Gly‐Gly‐Lys‐Gly‐Ala‐Lys‐Arg‐Ala‐Gly‐Lys‐Gly‐Gly‐Arg‐Arg‐Arg‐Arg‐Val‐Val‐Lys‐Arg‐Arg‐Arg‐Arg‐Arg‐Arg‐Glu‐Ser‐Tyr‐Gly‐Ile‐Tyr‐Ile‐Tyr‐Lys‐Val‐Leu‐Lys‐Gln‐Val‐His‐Pro‐Asp‐Thr‐Gly‐Ile‐Ser‐Ser‐Arg‐Ala‐Met‐Ser‐Val‐Met‐Asn‐Ser‐Phe‐Val‐Asn‐Asp‐Val‐Phe‐Glu‐Arg‐Ile‐Ala‐Ser‐Glu‐Ala‐Ser‐Arg‐Leu‐Thr‐Ser‐Ala‐Asn‐Arg‐Arg‐Ser‐Thr‐Val‐Ser‐Ser‐Arg‐Glu‐Ile‐Gln‐Thr‐Ala‐Val‐Arg‐Leu‐Leu‐Leu‐Pro‐Gly‐Glu‐Leu‐Ala‐Lys‐His‐Ala‐Val‐Ser‐Glu‐Gly‐Thr‐Lys‐Ala‐Val‐Thr‐Lys‐Tyr‐Thr‐Ser‐Arg.

H2B(3)Parechinus closely resembles H2B(2)Parechinus but has one additional repeating pentapeptide in the amino‐terminal region and a serine replacing glycine at position 98.
Structure of an (A‐Blood‐Group)‐Active Glycolipid Isolated from Human Erythrocytes
Authors: GARDAS Andrzej
Published: March 2005

An (A‐blood‐group)‐active glycolipid possessing 23 sugars has been isolated, from human erythrocyte membrane. Smith degradation and methylation studies allow a structure to be proposed of the nonreducing end of the oligosaccharide chain.
Carboxylic and Polyethyleneimine‐Bound FAD Derivatives
Authors: ZAPPELLI Piergiorgio , PAPPA Rosario , ROSSODIVITA Antonio , RE Luciano
Published: March 2005

Alkylation at N‐1 of the FAD adenine ring with 3,4‐epoxybutanoic acid gave 1‐(2‐hydroxy‐3‐carboxypropyl)‐FAD. Dimroth rearrangement of the latter afforded N6‐(2‐hydroxy‐3‐carboxypropyl)‐FAD. Coupling of the two carboxylic FAD derivatives to polyethyleneimine gave the corresponding soluble macromolecular FAD analogues.

Fluorescence and circular dichroism determinations at neutral pH showed for the N‐1 carboxylic derivative an open conformation, while for the corresponding N6 derivative a stacked one as for FAD.

All the synthesized derivatives have been shown to reactivate Aspergillus niger glucose oxidase and hog kidney d‐amino acid oxidase apoenzymes, affording holoenzymes whose activity relative to that of the corresponding natural holoenzymes ranged from 3 to 15% for the N‐1 derivatives and from 20 to 97% for the N6 derivatives.

Investigations by ultraviolet difference spectra on the interaction with apo‐d‐amino acid oxidase showed substantial similarities between the N6 carboxylic derivative and FAD in the binding to the apoenzyme, while a completely different behaviour was observed for the N‐1 carboxylic derivative. The d‐amino acid oxidase apoenzyme, the holoenzyme and the glutaraldehyde‐treated holoenzyme were entrapped each in cellulose triacetate fibres together with the polyethyleneimine N6 derivative and beef liver catalase; the operational stability of the three recycling systems with dl‐alanine as substrate was shown to be respectively 6, 5 and 12 times higher than the one found for the entrapped natural holoenzyme system.
The 5‐S RNA · Protein Complex from an Extreme Halophile, Halobacterium cutirubrum
Authors: SMITH Nancy , YAGUCHI Makoto , WILLICK Gordon E. , NAZAR Ross N. , MATHESON Alastair T.
Published: March 2005

A 5‐S RNA · protein complex has been isolated from the 50‐S ribosomal subunit of an extreme halophile, Hulobacterium cutirubrum. The 50‐S ribosomal subunit from the extreme halophile requires 3.4 MK+ and 100 mM Mg2+ for stability. However, if the high K+ concentration is maintained but the Mg2+ concentration lowered to 0.3 mM, the 5‐S RNA · protein complex is selectively extracted from the subunit. After being purified on an Agarose 0.5‐m column the complex had a molecular weight of about 80000 and contained 5‐S RNA and two proteins, HL13 and HL19, with molecular weights (by sedimentation equilibrium) of 18700 and 18000, respectively. No ATPase or GTPase activity could be detected in the 5‐S RNA · protein complex. The amino acid composition and electrophoretic mobility on polyacrylamidc gels indicated both proteins were much more acidic than the equivalent from Escherichia coli or Bacillus stearothermophilus. Partial amino acid sequence data suggest HL13 is homologous to EL18 and HL19 to EL5.
Slow Association‐Dissociation Equilibrium of NADP‐Linked Isocitrate Dehydrogenase from Beef Liver in Relation to Catalytic Activity
Authors: CARLIER Marie‐France , PANTALONI Dominique
Published: March 2005

In this paper experiments are reported which show evidence for a relation between quaternary structure and catalytic activity of cytoplasmic NADP‐linked isocitrate dehydrogenase from beef liver. The inactivation of the enzyme occurring upon dilution and the plots of the catalytic activity versus the enzyme concentration indicate that the monomeric species is catalytically inactive and that the monomer‐dimer equilibrium is shifted towards the dimer upon binding of the substrate magnesium isocitrate complex. The association of the enzyme following binding of the substrate takes place at a rate comparable with that of the enzymatic reaction, which results in a ‘hysteretic’ behaviour of the enzyme. The possibility is discussed that slow changes in quaternary structure can give rise to a physiological regulation of the enzymatic activity.
Biochemical Characterization of the Restriction‐Modification System of Bacillus sphaericus
Authors: KONCZ Csaba , KISS Antal , VENETIANER Pál
Published: March 2005

A type II restriction endonuclease (endo R ·Bsp) has been purified from Bacillus sphaericus to electrophoretic homogeneity. The enzyme appears to be a single polypeptide chain with a molecular weight of 35000. Its pH optimum is around 8.2, it requires 20 mM Mg2+ for optimal activity and it is inhibited by Zn2+. The yield of the enzyme is higher than that of any type II restriction endonuclease so far reported.

The enzyme also cleaves single‐stranded DNA, albeit at a slower rate. It seems likely that single‐stranded DNA is cleaved at the same sequences as double‐stranded DNA.

Bacillus sphaericus also contains a modification methylase (meth M ·Bsp) which completely protects the cell's own DNA against cleavage by its restriction endonuclease. The methylase activity has been partially purified, it copurifies with the nuclease until the next to the last step. The enzyme does not require ATP or Mg2+, it transfers the methyl group of S‐adenosyl‐methionine to cytosine residues of DNA. As the action of this methylase completely protects any DNA from endo R ·Bsp cleavage, it seems likely that the methylase recognizes and methylates the same sequence (dG‐dG‐dC‐dC) as the nuclease.
Modification of Elongation‐Factor‐Tu · Guanine‐Nucleotide Interaction by Kirromycin
Authors: FASANO Ottavio , BRUNS Wolfgang , CRECHET Jean‐Bernard , SANDER Gernot , PARMEGGIANI Andrea
Published: March 2005

Kirromycin activates functions of elongation factor Tu (EF‐Tu) that normally require the presence of specific effectors [Wolf et al. (1974) Proc. Natl Acad. Sci. U.S.A. 71, 4910–4914]. As a result, the EF‐Tu GTPase activity is uncoupled from aminoacyl‐tRNA and ribosomes. For a better understanding of the action of the antibiotic, we have studied its effect on the interaction between EF‐Tu and guanine nucleotides and compared this with the action of the physiological effectors aminoacyl‐tRNA and elongation factor Ts (EF‐Ts). Kirromycin affects both association (k′+1) and dissociation (k′−1) rates of EF‐Tu and GTP, but in opposite ways, the k′+1 being strongly increased, whereas the k′‐1 is even more strongly decreased. This causes a lowering of the apparent K′ of this complex by two orders of magnitude, i.e. approaching that of EF‐Tu · GDP. By contrast, the k′+1 and k′‐1 are both increased to the same extent and consequently the apparent K' of this complex is unchanged in the presence of the antibiotic. Thus the action of kirromycin resembles that of EF‐Ts opening the EF‐Tu site for GDP, thereby increasing the rate of the EF‐Tu · GDP/GDP exchange, but in contrast to EF‐Ts kirromycin binds preferentially to EF‐Tu · GTP. Like aminoacyl‐tRNA, the antibiotic induces a specific conformation that locks GTP in its site on EF‐Tu. The regeneration of EF‐Tu · GTP from EF‐Tu · GDP does not limit the rate of GTP hydrolysis induced by kirromycin, even when aminoacyl‐tRNA or ribosomes stimulate this reaction. By contrast, the turnover activity occurring in the absence of the antibiotic and depending on aminoacyl‐tRNA plus ribosomes is limited by the dissociation rate of the EF‐Tu · GDP complex. Our results indicate that kirromycin induces a conformation of EF‐Tu that shares features with the conformations evoked sequentially by EF‐Ts, aminoacyl‐tRNA and ribosomes during the elongation cycle.
Purification and Characterization of an Endonuclease from Micrococcus luteus that Acts on Depurinated and Carcinogen‐Modified DNA
Authors: HECHT Reinhardt , THIELMANN Heinz Walter
Published: March 2005

An endonuclease which is active with regard to depurinated, alkylated, arylated, and arylamidated DNA has been purified 500‐fold from Micrococcus luteus. In this purification, separation from the pyrimidine‐dimer‐specific ultraviolet‐endonuclease has been achieved. The enzyme has a molecular weight of 30000 on the basis of gel filtration; its activity is not absolutely dependent upon the presence of Mg27plus;, but 5–30 mM Mg27plus; produces a five‐fold stimulation. Potassium chloride concentrations of less than 100 mM are optimal, while concentrations exceeding 100 mM inhibit. The enzyme has no effect on native DNA, but introduces single‐strand breaks into DNA containing apurinic/apyrimidinic sites produced by heating at an acidic pH. DNA treated with such carcinogens as N‐alkyl‐N‐nitrosoureas, alkyl methanesulfonates, alkyl sulfates, nitrogen mustard, β‐propiolactone, 7‐bromomethyl‐benz[a]anthracene, N‐acetoxy‐2‐acetylaminofluorene, and 7,12‐dimethyl‐benz[a]anthracene‐5,6‐oxide also becomes susceptible to enzymic action.

The activity of the enzyme has been detected by making use of the difference in mobility between supercoiled closed‐circular DNA of Pseudomonas phage PM2 and its nicked form in agarose gel electrophoresis. Even depurinated or carcinogen‐modified supercoiled PM2 DNA migrated faster than the respective relaxed nicked forms.

A comparison of the number of enzyme‐catalyzed single‐strand breaks with the number of alkali‐labile (i.e. apurinic) sites in carcinogen‐modified PM2 DNA showed that the enzyme preparation introduced approximately twice as many breaks into the substrates as the number of apurinic sites present. We conclude that the enzyme preparation either recognizes both apurinic sites and DNA bases carrying carcinogenic residues or contains DNA glycosidase activity in addition to the endonuclease activity. Exposure of ultraviolet‐irradiated PM2 DNA to the endonuclease preparation showed that pyrimidine dimers were not substrates. The yield of enzyme‐catalyzed single‐strand breaks found in ultraviolet‐irradiated DNA was five times the number of alkali‐labile sites present suggesting that minor photoproducts, possibly 5,6‐saturated pyrimidine residues, were recognized in addition to apurinic sites.
Mannosylation of Endogenous Proteins of Rough and Smooth Endoplasmic Reticulum and of Golgi Membranes
Authors: NILSSON Olle S. , DE TOMÁS Maria Eléna , PETERSON Elisabeth , BERGMAN Anders , DALLNER Gustav , HEMMING Frank W.
Published: March 2005

Mannosylation of the proteins of microsomal and Golgi membranes was investigated both after incubation in vitro of the isolated subfractions with GDP‐[14C]mannose and after injection of [3H]mannose into rats followed by separation of these subfractions. Mannosylation of endogenous and added exogenous dolichol phosphate and also of dolichol pyrophosphate‐oligosaccharide occurs in all three fractions. It was essential to inhibit antagonistic enzymes during incubation and to centrifuge after incubation. The presence of detergent in the incubation mixture influences the incorporation pattern of the different fractions in very different ways. In a system in vitro predominantly membrane proteins and not secretory proteins are mannosylated. Trypsin treatment of intact vesicles removes components from the outer surface only; such treatment liberates about one third of the radioactive mannose associated with lipid, releases radioactivity from the protein acceptor to the same extent and causes some inactivation of the transferase activities. It appears that a part of the mannosyl transferase system in rough and smooth endoplasmic reticulum and in Golgi membranes is localized at the cytoplasmic side of these membranes. This activity is probably involved in the glycosylation of proteins localized at the cytoplasmic surface of the endoplasmic reticulum.
The Reaction of Pyruvate with Saccharopine Dehydrogenase
Authors: SUGIMOTO Keiko , FUJIOKA Motoji
Published: March 2005

The preceding paper in this journal has reported that pyruvate could be substituted for 2‐oxoglutarate as a substrate of saccharopine dehydrogenase [ɛ‐N‐(l‐glutaryl‐2)‐l‐lysine: NAD oxidoreductase (l‐lysine‐forming)] in the direction of reductive condensation. In the present communication, the kinetic mechanism of saccharopine dehydrogenase reaction with NADH, l‐lysine and pyruvate as reactants is reported. The results of initial velocity study, inhibition studies with lysine analogs and a reaction product, NAD+, are consistent with an ordered mechanism with the coenzyme binding first and pyruvate last. The reaction mechanism is at variance with that of the normal reaction in which 2‐oxoglutarate is the substrate, in that the order of addition of the amino and oxo acid substrates is reversed. This fact suggests that there exists a small degree of randomness in the binding of amino and oxo acid substrates. From a product inhibition study, NAD+ was shown to be the last reactant released. Saccharopine [ɛ‐N‐(l‐glutaryl‐2)‐l‐lysine] was found to act as a potent dead‐end inhibitor of the condensation reactions (of lysine and 2‐oxoglutarate, and of lysine and pyruvate) by forming an abortive E · NADH · saccharopine complex.
Studies on the Structure of Protein L7/L12 from Escherichia coli Ribosomes
Authors: GUDKOV Anatoli T. , KHECHINASHVILI Nikolai N. , BUSHUEV Valeri N.
Published: March 2005

Circular dichroism, infrared and proton magnetic resonance spectroscopy as well as microcalorimetry methods were used to investigate the intact proteins L7/L12 in solution and their different derivatives (L7 with oxidized residues of methionine, fragments 27–120, 1–73 and 74–120). On the basis of the data obtained the following conclusions have been drawn: (a) there is no β structure in the protein L7, (b) the N‐terminal region of L7 forms a long α helix, (c)the Phe‐30 residue within the N‐terminal region of L7 takes part in the dimerization, (d) the C‐terminal region of L7 is globular and (e) the Phe‐54 residue is included in the hydrophobic core of the globular C‐terminal region.
Purification and Properties of a Ribonuclease Induced during the Early Larval Development of Artemia salina
Authors: SEBASTIÁN Jesús , HEREDIA Claudio F.
Published: March 2005

Dormant gastrulae and developing embryos of the brine shrimp Artemia salina contain very low levels of nuclease activity. During early larval development, there is an induction of a ribonuclease which has been partially purified and characterized. The enzyme catalyzes an endonucleolytic cleavage of RNA and has no detectable activity on native or denatured DNA. Among a series of synthetic polynucleotides, poly(U) is hydrolyzed with the highest efficiency and poly(G) is not cleaved by the enzyme. The activity on poly(U) is 100 times higher than on RNA. The enzyme requires Mg2+ or Mn2+ and is inactivated by treatment with a chelating agent. The inactive preparations can be reactivated by Ca2+ and Mn2+ but not by Mg2+. The ribonuclease is thermosensitive and has maximal activity at pH 7.5. These properties distinguish the Artemia salina ribonuclease from other eukaryotic ribonucleases already reported. The high activity and specificity of this ribonuclease on poly(U) may suggest a role for this enzyme in the processing of the messenger RNA.
Native and Latent Forms of Liver Phosphorylase Phosphatase
Authors: LALOUX Monique , STALMANS Willy , HERS Henri‐Géry
Published: March 2005

The directly measurable (native) phosphorylase phosphatase present in a fresh mouse liver extract is bound to particulate glycogen and is not inhibited by heat‐stable inhibitors. Treatment of the extract with trypsin or ethanol at room temperature caused a more than 10‐fold increase in phosphorylase phosphatase activity. This increased activity stems from the activation of completely inactive (latent) enzyme, the major part of which is present in the high‐speed supernatant. The trypsin‐revealed activity can be completely blocked by heat‐stable inhibitors. Treatment of the animal with glucocorticoids increases, and fasting decreases the activity of the native phosphorylase phosphatase. The level of latent enzyme, however, is unaffected by these treatments. The major portion of synthase phosphatase in the fresh liver extract is bound to glycogen. This enzyme is inhibited by the heat‐stable inhibitor‐2 and inactivated by trypsin or ethanol as well as by several treatments that have little effect on phosphorylase phosphatase.

Upon DEAE‐cellulose chromatography at 0°C of a fresh liver extract, phosphorylase phosphatase and synthase phosphatase were resolved as separate, single peaks. If the preparation was not kept at 0°C during the entire procedure, two peaks of each enzyme were observed. Under these conditions the first peak of phosphorylase phosphatase and of synthase phosphatase coincided. From these findings it is concluded that synthase phosphatase and phosphorylase phosphatase, in their native form, are distinct enzymes.
Purification and Physicochemical Properties of Fatty Acid Synthetase and Acetyl‐CoA Carboxylase from Lactating Rabbit Mammary Gland
Authors: HARDIE D. Grahame , COHEN Philip
Published: March 2005

Fatty acid synthetase was purified 13‐fold from lactating rabbit mammary glands by a procedure which involved chromatography on DEAE‐cellulose, ammonium sulphate precipitation and gel filtration on Sepharose 4B. The preparation was completed within two days and over 100 mg of enzyme was isolated from 100–150g of mammary tissue, which represented a yield of over 40%. The preparation was homogeneous by the criteria of polyacrylamide gel electrophoresis and ultracentrifugal analysis. The sedimentation constant, s20, w was 13.3S, the absorption coefficient, A280nm1%, measured refractometrically was 10.0 ± 0.1, and the amino acid composition was determined.

The subunit molecular weight determined by gel electrophoresis in the presence of sodium dodecyl sulphate was 252 000 ± 6 000, and the molecular weight of the native enzyme measured by sedimentation equilibrium was 515 000. These experiments indicate that at the concentrations which exist in mammary tissue (2–4 mg/ml) fatty acid synthetase is a dimer. The purified enzyme did however show a tendency to dissociate to a monomeric 9‐9S species on storage for several days or following exposure to a low ionic strength buffer at pH 8.3. There was only a small quantity of alkali labile phosphate (0.2 molecules per subunit) bound covalently to the purified enzyme.

Acetyl‐CoA carboxylase was purified 300‐fold in a 50% yield within 24h by ammonium sulphate and polyethylene glycol precipitations [Hardie, D. G. and Cohen, P. (1978) FEBS Lett. 91, 1–7]. The preparation was in a state approaching homogeneity as judged by polyacrylamide gel electrophoresis, gel filtration on Sepharose 4B and ultracentrifugal analysis. The sedimentation constant, s20, w, was 50.5S, the absorption index, A280nm1%, was 14.5 ± 0.7, and the amino acid composition was determined.

The subunit molecular weight of acetyl‐CoA carboxylase determined by gel electrophoresis in the presence of sodium dodecyl sulphate was identical to that of fatty acid synthetase (252 000) as shown by electrophoresis of a mixture of the two proteins. The preparations also contained two minor components of molecular weight 235 000 and 225 000, which appear to be derived from the major species of mol. wt 252 000. A large amount of phosphate (3.2 molecules per subunit) was found to be bound covalently to the purified enzyme.

The properties of fatty acid synthetase and acetyl‐CoA carboxylase are compared to those obtained by other workers.
Structure and Replication of Echovirus Type 12
Authors: ROSENWIRTH Brigitte , EGGERS Hans J.
Published: March 2005

The propagation of echovirus 12 in green monkey kidney (GMK) cells, a continuous cell line, is described. Virions and empty capsids are purified to homogeneity by CsCl density and by velocity centrifugation. The protein composition of the particles as compared to poliovirus 2 particles is analyzed by dodecyl sulfate/polyacrylamide gel electrophoresis. Infectious virions were found to be composed of four different polypeptide chains of molecular weights 33 000, 31 000, 24 000 and 7000. Some additional polypeptide bands at low concentrations are also visible. The empty capsids lack the 31 000‐Mr and 7000‐Mr components and contain instead a polypeptide of the molecular weight 46 000. The molecular weights of the structural polypeptides are the same regardless of whether the virus has been synthesized in primary African green monkey kidney cells or in the continuous GMK cell line. The sedimentation behavior of echovirus 12 RNA is shown to be similar to that of poliovirus 2 RNA.
Complete Assignment of Aromatic 1H Nuclear Magnetic Resonances of the Tyrosine Residues of Hen Lysozyme
Authors: DOBSON Christopher M. , FERGUSON Stuart J. , POULSEN Flemming M. , WILLIAMS Robert J. P.
Published: March 2005

The complete assignment of the aromatic proton nuclear magnetic resonances of the three tyrosine residues in hen lysozyme is reported. These assignments were made using double resonance techniques, specific chemical modifications of one residue (Tyr‐23), and by interpretation of the effects of paramagnetic lanthanide ions. Some aspects of the behaviour of the tyrosine residues are reported, including pK values, reactivity towards modifying agents and conformational mobility.
The Mechanism of Substrate Activation of Pyruvate Decarboxylase: A First Approach
Authors: HÜBNER Gerhard , WEIDHASE Rosemarie , SCHELLENBERGER Alfred
Published: March 2005

The sigmoidal shape of the curve for v/[S], characteristic of pyruvate decarboxylase, indicates that the catalytic activity of this enzyme is regulated by the substrate. The enzyme, which is inactive in the absence of its substrate, is activated not only by 2‐oxo acids but also by 2‐oxo acid amides, which cannot act as a substrate of the enzyme. Whilst the dissociation constant of the enzyme‐activator complex depends on the electrophilic nature of the carbonyl group, the catalytic activity reached at saturation concentrations of the activator species is indepedent of the structure of the activator molecules.

The mechanism of activation which proceeds via two reversible steps could be evaluated exactly by stopped‐flow techniques. The kinetic parameters of the activation and deactivation reaction were estimated and the validity of the equations derived which describe the activation kinetics could be proved by comparing them with the measured data.

Using glyoxylic acid as an irreversibly binding active‐site marker, it could be shown that addition of the substrate to the enzyme‐bound thiamin diphosphate is the step of the catalytic mechanism whose rate is controlled by the substrate (activator) molecule.
β‐Adrenergic Receptors in Brown‐Adipose Tissue. Characterization and Alteratios during Acclimation of Rats to Cold
Authors: BUKOWIECKI Ludwik , FOLLEA Nicole , VALLIERES Julien , LEBLANC Jacques
Published: March 2005

The capacity of brown adipose tissue to respond calorigenically to catecholamines increases markedly during cold‐acclimation of adult rats. To investigate this phenomenon, we have used the potent radioactive ligand (‐)‐[3H]dihydroalprenolol to directly estimate the number, the density and the affinity of β‐adrenergic receptors in brown adipose tissue membranes from control, cold‐exposed and cold‐acclimated rats.

Binding of (−)‐[3H]dihydroalprenolol to unfractionated membranes was rapid, stable, saturable and reversible. It displayed the affinity, specificity and stereoselectivity expected of binding to adenylate cyclase‐coupled β‐adrenergic receptors. β‐Adrenergic agonists competed for binding sites with an order of potency typical of the β1 subtype of adrenergic receptors: (−)‐Isoproterenol > (−)‐norepinephrine ≥ (−)‐epinephrine. Binding exhibited a remarkable stereoselectivity, the (−)‐isomers of β‐adrenergic agonists and antagonists being 34 to 280 times more potent than the (+)‐isomers in competing for (−)‐[3H]dihydroalprenolol binding sites.

Total interscapular brown adipose tissue of the adult, warm‐acclimated rat contained 1.12 ± 0.08 pmol of (−)‐[3H]dihydroalprenolol binding sites. During cold‐acclimation, growth of the tissue was accompanied by a 4–5‐fold increase in the total number of recptor sites. However, this increase did not keep pace with the increse in brown adipose tissue cellularity (as estimated by total tissue DNA content), resulting in a 40–50% reduction in receptor density. The decrease in receptor density was associated with cold‐exposure rather than with cold‐acclimation.

The affinity of (−)‐[3H]dihydroalprenolol receptor sites was not significantly altered by cold‐acclimation.

The results of this study are interpreted as indicating that catecholamines released via activation of the sympathetic nervous system regulate both the density and the number of their own receptors in brown adipose tissue of cold‐exposed animals. The development of the hyperadrenergic response of this thermogenic tissue during cold‐acclimation may result from a marked organ hyperplasia associated with an increased number of β‐adrenergic receptor sites and cannot be explained by alterations in receptor density or affinity.