bluuebunny/crossref_metadata_2025_split huggingface dataset:
Evidence that the Yb subunits of hepatic glutathione transferases represent two different but related families of polypeptides
Authors: BEALE Dennis , MEYER David J. , TAYLOR John B. , KETTERER Brian 
Published: March 2005

Three soluble rat liver glutathione (GSH) transferases A, C and one referred to as ‘D’, all of which are dimers of Yb subunits [Bass et al. (1977) Biochim. Biophys. Acta, 492, 163–175], have been compared with respect to C‐terminal amino acids and tryptic peptide maps. GSH transferases A and ‘D’ gave different tryptic peptide maps and different C‐terminal amino acids, Iysine and proline respectively. In each case the number of tryptic peptides is about half of that expected from their Iysine and arginine content, and there are 2 mol C‐terminal amino acid/mol enzyme. This indicates that GSH transferases A and ‘D’ represent two different Yb homodimers, which we refer to here as Y1bY1b and Y2bY2b respectively. GSH transferase C is the corresponding heterodimer Y1bY2b since it gives all the tryptic peptides which arise from GSH transferase A and GSH transferase ‘D’ and also contains both C‐terminal Iysine and proline. These results provide a structural basis to similar conclusions drawn by Mannervik and Jensson [(1980) J. Biol. Chem. 257, 9909–9912] based on enzymic and immunological comparisons.Tryptic peptide maps show that GSH transferases A and ‘D’ have considerable homology since there are 23 peptides common to both, 12 peptides unique to A and 8 peptides unique to ‘D’. Even so GSH transferase A is selectively induced by a phenobarbitone regime. It is, therefore, concluded that Y1b and Y2b are derived from separate but related genes. A similar conclusion has been drawn concerning the Ya and Yc subunits [Beale et al. (1982) Eur. J. Biochem. 126, 459–463], and a comparison of amino acid compositions, presented here, further suggests a genetic relationship between both pairs of subunits.
Synthesis and properties of NBD‐n‐acylcholines, fluorescent analogs of acetylcholine
Authors: MEYERS Hans‐Wilhelm , JÜRSS Rolf , BRENNER Hans R. , FELS Gregor , PRINZ Heino , WATZKE Heidrun , MAELICKE Alfred 
Published: March 2005

We have synthesized a homologous series of fluorescent analogs of acetylcholine, N‐7‐(4‐nitrobenzo‐2‐oxa‐1,3‐diazolyl)‐ω‐amino‐n‐alkanoic acid β(N,N,N‐trialkylammonium) ethylesters (NBD‐n‐acylcholines) and report here on their physiological and biochemical properties.(1) All NBD‐n‐acylcholines trimethylated at the cholinergic nitrogen are agonists of acetylcholine at the frog neuromuscular junction. Their potencies in depolarizing frog muscle cells decrease with decreasing chain length.(2) The affinities of binding to the purified receptor from Electrophorus electricus also decrease with decreasing chain length with a large drop in affinity for the derivatives n= 4 and n= 3.(3) The rate constants of association to acetylcholine receptor and to acetylcholine esterase are of the order of 108 M−1s−1 and do not vary significantly with the chain length of the NBD‐n‐acylcholines. In contrast, the dissociation rate constants decrease with increasing chain length.(4) The quenching of fluorescence of NBD‐n‐acylcholines accompanying binding to purified receptor and esterase from E. electricus appears to be due to the formation of a hydrogen bond between the ω‐amino group as donor and an unidentified acceptor group in a hydrophobic pocket of the protein.With their advantageous fluorescence properties, their simple pharmacology, and their clear structure‐function relationships, these compounds are useful tools for the study of cholinergic mechanisms.
Redox‐linked proton translocation in the b–c1 complex from beef‐heart mitochondria reconstituted into phospholipid vesicles
Authors: LORUSSO Michele , GATTI Domenico , BOFFOLI Domenico , BELLOMO Elisabetta , PAPA Sergio 
Published: March 2005

Possible involvement of polypeptides of b–c1 complex of beef‐heart mitochondria in its redox and protonmotive activity has been investigated, by means of chemical modification of amino acid residues in the soluble as well as in the phospholipid‐reconstituted b–c1 complex. Treatment of the enzyme with tetranitromethane (C(NO2)4) or with ethoxyformic anhydride (EFA), that modify reversibly tyrosyl and hystidyl residues respectively, resulted in a marked inhibition of electron transport from reduced quinols to cytochrome c. This was accompanied, in b–c1 reconstituted into phospholipid vesicles, by a parallel inhibition of respiratory‐linked proton translocation; the H+/e− stoichiometry remained unchanged. Treatment of b–c1 complex with DCCD, that specifically modifies carboxylic groups of glutammic or aspartic residues caused a marked depression of proton translocation in b–c1 vesicles, under conditions where the rate of electron flow in the coupled state, was enhanced. As a consequence the H+/e− stoichiometry was lowered.SDS gel electrophoresis and [14C]DCCD‐labelling of the polypeptides of the b–c1 complex showed a major binding of 14C‐DCCD to the 8‐kDa subunit of the complex and possible cross‐linking, induced by DCCD treatment, of polypeptide(s) in the 8‐kDa band and the 12‐kDa band, with the Fe‐S protein of the complex, with the appearance of a new polypeptide band with an apparent molecular mass of about 40 kDa.Involvement of polypeptides of low molecular mass, for which no functional role was so far described, and possibly of the Fe‐S protein in the redox‐linked proton translocation in b–c1 complex is suggested.
Interaction of a glutathione S‐conjugate with glutathione reductase Kinetic and X‐ray crystallographic studies
Authors: BILZER Manfred , KRAUTH‐SIEGEL R. Luise , SCHIRMER R. Heiner , AKERBOOM Theo P. M. , SIES Helmut , SCHULZ Georg E. 
Published: March 2005

S‐Conjugates of glutathione influence the glutathione/glutathione disulfide (GSH/GSSG) status of hepatocytes in at least two ways, namely by inhibition of GSSG transport into the bile [Akerboom et al. (1982) FEBS Lett. 140, 73–76] and by inhibition of the enzyme GSSG reductase (EC 1.6.4.2).
The interaction of GSSG reductase with a well‐studied conjugate, namely S‐(2,4‐dinitrophenyl)‐glutathione and its electrophilic precursor 1‐chloro‐2,4‐dinitrobenzene are described. For short exposures both compounds are reversible inhibitors of the enzyme, the Ki values being 30 μM and 22 μM respectively. After prolonged incubation, 1‐chloro‐2,4‐dinitrobenzene blocks GSSG reductase irreversibly, which emphasizes the need for rapid conjugate formation in situ.
As shown by X‐ray crystallography the major binding site of S‐(2,4‐dinitrophenyl)‐glutathione in GSSG reductase overlaps the binding site of the substrate, glutathione disulfide. However, the glutathione moiety of the conjugate does not bind in the same manner as either of the glutathiones in the disulfide.
Effect of nickel on activity and subunit composition of purified hydrogenase from Nocardia opaca 1b
Authors: SCHNEIDER Klaus , SCHLEGEL Hans G. , JOCHIM Karla 
Published: March 2005

The NAD‐reducing hydrogenase of Nocardia opaca 1b was found to be a soluble, cytoplasmic enzyme. N. opaca 1b does not contain an additional membrane‐bound hydrogenase. The soluble enzyme was purified to homogeneity with a yield of 19% and a final specific activity of 45 μmol H2 oxidized min−1 mg protein−1.NAD reduction with H2 was completely dependent on the presence of divalent metal ions (Ni2+, Co2+, Mg2+, Mn2+) or of high salt concentrations (0.5–1.5 M). The most specific effect was caused by NiCl2, whose optimal concentration turned out to be 1 mM. The stimulation of activity by salts was the greater the less chaotrophic the anion. Maximal activity was achieved in 0.5 M potassium phosphate. Hydrogenase was also activated by protons. The pH optimum in 50 mM triethanolamine/HCl buffer containing 1 mM NiCl2 was 7.8–8.0. In the absence of Ni2+, hydrogenase was only active at pH values below 7.0. The reduction of other electron acceptors was not dependent on metal ions or salts, even though an approximately 1.5‐fold stimulation of the reactions by 0.1–10 μM NiCl2 was observed. With the most effective electron acceptor, benzyl viologen, a 50‐fold higher specific activity was determined than with NAD.The total molecular weight of hydrogenase has been estimated to be 200000 (gel filtration) and 178000 (sucrose density gradient centrifugation, and sodium dodecyl sulfate electrophoresis) respectively. The enzyme is a tetramer consisting of non‐identical subunits with molecular weights of 64000, 56000, 31000 and 27000. It was demonstrated by electrophoretic analyses that in the absence of NiCl2 and at alkaline pH values the native hydrogenase dissociates into two subunit dimers. The first dimer was dark yellow coloured, completely inactive and composed of subunits with molecular weights of 64000 and 31000. The second dimer was light yellow, inactive with NAD but still active with methyl viologen. It was composed of subunits with molecular weights of 56000 and 27000.Immunological comparison of the hydrogenase of N. opaca 1b and the soluble hydrogenase of Alcaligenes eutrophus H16 revealed that these two NAD‐linked hydrogenases are partially identical proteins.
Cyclic changes of the rate of phospholipid synthesis during synchronous growth of Escherichia coli
Authors: JOSELEAU‐PETIT Danièle , KEPES François , KEPES Adam 
Published: March 2005

The problem of the coordination between cyclic events in the DNA assembly line and the cell envelope assembly line was approached with the technique of synchronized cultures. Escherichia coli strains ML 30, K12 3300, K12 PC2, K12 BB2014 and B/rF were synchronized by repeated cycles of mass doubling followed by short phosphate starvation periods. Steady‐state balanced growth was obtained by subsequent incubation in non‐limiting growth conditions for one or more generation times.Several successive cell cycles were monitored for mass increase and cell number, while the rate of DNA synthesis and the rate of phospholipid synthesis were usually measured with more than one method.In all strains, and in strain ML 30 in five different growth media giving doubling times from 20–110 min, a discontinuity in the rate of synthesis of phosphatidylethanolamine and of phosphatidylglycerol was observed. These two major phospholipid components of inner and outer membranes were synthesized at a constant rate per cell for a large portion of the cell cycle and the rate of synthesis of both increased twofold at the same time. This cyclic program was reproducible not only in successive cell cycles, but also in separate experiments with the same strain, in the same medium. In contrast, differences in timing were observed with different strains, and in the same strain with different carbon sources. In particular, the simultaneity of the increase in phospholipid synthesis either with DNA initiation or with cell division could not be observed as a rule.
Monoclonal anti‐glycoprotein antibody that blocks cell adhesion in Polysphondylium pallidum
Authors: TODA Katsumi , BOZZARO Salvatore , LOTTSPEICH Friedrich , MERKL Rainer , GERISCH Günther 
Published: March 2005

Polyclonal and monoclonal antibodies were prepared against a glycoprotein (gp 64) of Polysphondylium pallidum previously shown to act as a target site of adhesion‐blocking Fab prepared from antisera against whole membranes of aggregation‐competent cells. The purified glycoprotein, with a nominal Mr of 64000, could be fractionated into two subspecies, gp 64I and gp 64II with apparent Mr of 66000 and 60000, as determined in 7.5% sodium dodecyl sulfate/polyacrylamide gels.Rabbit antibodies against purified gp 64 reacted not only with the two subspecies but also with many other membrane proteins. Almost all the cross‐reactivity could be abolished by absorption of the antibodies with extensively purified gp 64. All monoclonal antibodies obtained by screening with gp 64 showed similar cross‐reactivity. One monoclonal antibody specifically precipitating gp 64 was selected by screening with antigen that had been pretreated with anhydrous hydrogen fluoride for removal of carbohydrates.Fab from polyclonal anti‐(gp 64) sera as well as one monoclonal Fab completely blocked cell adhesion of aggregation‐competent P. pallidum cells. A carbohydrate fraction prepared by treatment of gp 64 with proteases and hydrazine completely neutralized the adhesion‐blocking Fab. The product of hydrazinolysis contained less than 3% of the original peptide as based on the glucosamine recovered, but the specific neutralizing activity of the carbohydrate was essentially the same as that of the glycoprotein. In conclusion, monoclonal as well as polyclonal adhesion‐blocking Fab reacted with carbohydrates; gp 64 shared the relevant carbohydrate moieties with other membrane proteins.
Catabolite repression in yeasts is not associated with low levels of cAMP
Authors: ERASO Pilar , GANCEDO Juana M. 
Published: March 2005

The relationship between levels of cAMP and catabolite repression in yeasts has been investigated. Strains of Saccharomyces cerevisiae, Schizosaccharomyces pombe and Kluyieromyces fragilis were used. The yeasts were grown on different carbon sources to attain various degrees of repression. Galactose repressed as much as glucose, while maltose was less effective. Full derepression was achieved with ethanol.The enzymes tested were fructose‐bisphosphatase, malate dehydrogenase, glutamate dehydrogenase (NAD dependent), cytochrome oxidase and isocitrate lyase (this last enzyme was found to be absent in Schizosaccharomyces).The levels of cAMP were 2–3 times higher in the repressed conditions than in the derepressed ones. It is therefore concluded that in yeasts catabolite repression is not mediated by a lowering of the intracellular concentration of cAMP.
The regulatory properties of isocitrate dehydrogenase kinase and isocitrate dehydrogenase phosphatase from Escherichia coli ML308 and the roles of these activities in the control of isocitrate dehydrogenase
Authors: NIMMO Gillian A. , NIMMO Hugh G. 
Published: March 2005

Isocitrate dehydrogenase kinase can use ATP but not other nucleoside triphosphates as a phosphate donor. It responds hyperbolically to both ATP and isocitrate dehydrogenase.
The kinase is inhibited sigmoidally by low concentrations of DL‐isocitrate and hyperbolically by ADP, AMP, NADPH, phosphoenolpyruvate and several other effectors.
Isocitrate dehydrogenase phosphatase requires a nucleotide for activity; ADP and ATP are the best activators. The phosphatase responds hyperbolically to ADP or ATP, to Mg2+ ions and to phosphorylated isocitrate dehydrogenase.
The phosphatase is activated twofold to threefold by AMP, oxaloacetate, pyruvate, phosphoenolpyruvate, 2‐oxoglutarate and DL‐isocitrate. It is inhibited hyperbolically by NADPH.
The pH optima and the Km values for substrates of the kinase and the phosphatase are reported.
We propose that the role of the phosphorylation of isocitrate dehydrogenase during growth of Escherichia coli on acetate is to render this enzyme rate‐limiting in the citric acid cycle; this should cause an increase in the level of isocitrate and divert the flux of carbon through the glyoxylate bypass.
We suggest that the phosphorylation state of isocitrate dehydrogenase in intact cells is controlled by the levels of isocitrate, phosphoenolpyruvate, NADPH and the adenine nucleotides. This theory can explain many recent observations on the control of the activity of isocitrate dehydrogenase.
Characterization of the vacuolar ATPase activity of the crassulacean‐acid‐metabolism plantKalanchoë daigremontianaReceptor modulating
Authors: SMITH J. Andrew C. , URIBE Ernest G. , BALL Erika , HEUER Sabine , LÜTTGE Ulrich 
Published: March 2005

Plants performing crassulacean acid metabolism show a large nocturnal accumulation of malic acid in the vacuole of the photosynthetic cells. It has been postulated that an H+‐translocating ATPase energizes the transport of malic acid across the tonoplast into the vacuole. In the present work we have characterized the ATPase activity associated with vacuoles of the crassulacean‐acid‐metabolism plantKalanchoë daigremontianaand compare it with other phosphohydrolases.Vacuoles were isolated by polybase‐induced lysis of mesophyll‐cell protoplasts. The vacuoles had a high activity of unspecific acid phosphatase (pH optimum 5.3). The acid phosphatase was strongly inhibited by ammonium molybdate (with 50% inhibition at about 0.5 mmol m3‐), but was not completely inhibited even at much higher ammonium‐molybdate concentrations. In contrast, the vacuolar ATPase activity, assayed in the presence of 100 mmol m−3ammonium molybdate, had a pH optimum of 8.0. ATP was the preferred substrate, but GTP, ITP and ADP were hydrolyzed at appreciable rates. The mean ATPase activity at pH 8.0 was 14.5 mmol h−1(103vacuoles)−1an average 13% of which was attributable to residual acid‐phosphatase activity. Inorganic‐pyrophosphatase activity could not be demonstrated unambiguously.The vacuolar ATPase activity was M2+‐dependent, had an apparentKmfor MgATP2‐of 0.31 mol m−3, and was 32% stimulated by 50 mol m3‐KCl. Of the inhibitors tested, oligomycin slightly inhibited the vacuolar ATPase activity and diethylstilbestrol and NO−3were both markedly inhibitory. Dicyclohexylcarbodiimide and tributyltin were also strongly inhibitory. Tributyltin caused a 50% inhibition at about 0.3 mmol m−3. This is taken as evidence that the vacuolar ATPase might function as an H+‐translocating ATPase.It is shown that the measured activity of the vacuolar ATPase would be of the right order to account for the observed rates of nocturnal malic‐acid accumulation inK. daigremontiana.
Studies on the transmembrane disposition of the neural cell adhesion molecule N‐CAM
Authors: GENNARINI Gianfranco , ROUGON Geneviève , DEAGOSTINI‐BAZIN Hermine , HIRN Michel , GORIDIS Christo 
Published: March 2005

The N‐CAMs are a group of surface glycoproteins involved in adhesive interactions of neurones. Related molecules of the mouse nervous system, identified in our laboratory, have been called BSP‐2 and shown to act as ligands in adhesion of neuroblastoma cells. Results presented in this report show that they are immunochemically identical with N‐CAM. A monoclonal anti‐(N‐CAM) antibody, that recognized a determinant accessible only after permeabilization of intact cells, was used to define the mode of association of the N‐CAMs with the plasma membrane. This antibody bound a 35000‐Mr fragment in lysates of trypsin‐treated neuroblastoma cells. It is concluded that the antibody reacts with a transmembrane or cytoplasmic domain of the molecules.The same antibody recognized the Mr‐180000 and Mr‐140000 proteins but not the Mr‐120000 chain, which copurify from adult mouse brain. The latter polypeptide was detected in the cytosol and could be partially released from brain membranes by osmotic shock. Part or all of the Mr‐120000 protein may thus lack a transmembrane segment.Our conclusion that the N‐CAM forms of higher Mr are transmembrane proteins was further corroborated by our finding that they contain phosphoserine residues, which can be labeled with (32P)phosphate in intact neuroblastoma cells.
Content and localization of FMN, Fe‐S clusters and nickel in the NAD‐linked hydrogenase of Nocardia opaca 1b
Authors: SCHNEIDER Klaus , CAMMACK Richard , SCHLEGEL Hans G. 
Published: March 2005

By preparative polyacrylamide gel electrophoresis at pH 8.5, and in the absence of nickel ions, two types of subunit dimers of the NAD‐linked hydrogenase from Nocardia opaca 1b were separated and isolated, and their properties were compared with each other as well as with the properties of the native enzyme.The intact hydrogenase contained 14.3 ± 0.4 labile sulphur, 13.6 ± 1.1 iron and 3.8 ± 0.1 nickel atoms and approximately 1 FMN molecule per enzyme molecule. The oxidized hydrogenase showed an absorption spectrum with maxima (shoulders) at 380 nm and 420 nm and an electron spin resonance (ESR) spectrum with a signal at g= 2.01. The midpoint redox potential of the Fe‐S cluster giving rise to this signal was + 25 m V. In the reduced state, hydrogenase gave characteristic low‐temperature (10 – 20 K) and high‐temperature (>40 K) ESR spectra which were interpreted as due to [4Fe–4S] and [2Fe–2S] clusters, respectively. The midpoint redox potentials of these clusters were determined to be ‐420 mV and ‐285 mV, respectively.The large hydrogenase dimer, consisting of subunits with relative molecular masses Mr of 64000 and 31000. contained 9.9 ± 0.4 S2− and 9.3 ± 0.5 iron atoms per protein molecule. This dimer contained the FMN molecule, but no nickel. The absorption and ESR spectra of the large dimer were qualitatively similar to the spectra of the whole enzyme. This dimer did not show any hydrogenase activity, but reduced several electron acceptors with NADH as electron donor (diaphorase activity).The small hydrogenase dimer, consisting of subunits with Mr of 56000 and 27000, was demonstrated to have substantially different properties. For iron and labile sulphur average values of 3.9 and 4.3 atoms/dimer molecule have been determined, respectively. The dimer contained, in addition, about 2 atoms of nickel and was free of flavins. In the oxidized state this dimer showed an absorption spectrum with a broad band in the 400‐nm region and a characteristic ESR signal at g= 2.01. The reduced form of the dimer was ESR‐silent. The small dimer alone was diaphorase‐inactive and did not reduce NAD with H2, but it displayed high H2‐uptake activities with viologen dyes, methylene blue and FMN, and H2‐evolving activity with reduced methyl viologen. Hydrogen‐dependent NAD reduction was fully restored by recombining both subunit dimers, although the reconstituted enzyme differed from the original in its activity towards artificial acceptors and the, ESR spectrum in the oxidized state.
Two different aldolase A mRNA species in rat tissues
Authors: TSUTSUMI Reiko , TSUTSUMI Ken‐ichi , NUMAZAKI Masayoshi , ISHIKAWA Kiichi 
Published: March 2005

Double‐stranded DNA was synthesized with reverse transcriptase from size‐fractionated poly(A)‐containing RNA from rat ascites hepatoma cells. The cDNA was introduced into Escherichia coli HB101 using pBR 322 DNA as a cloning vector. Several plasmids containing aldolase A cDNA were identified by colony hybridization with 32P‐labeled cDNA prepared from immunologically purified aldolase A mRNA. The partial amino acid sequence of the cDNA sequence was determined, and found to coincide with that of rabbit aldolase A.Using aldolase A cDNA as a hybridization probe, the aldolase A mRNA concentrations in various rat tissues were analysed, and two aldolase A mRNA species differing in nucleotide length were found; the smaller mRNA (about 1550 nucleotides) in muscle, and the larger one (about 1650 nucleotides) in brain and hepatoma cells.