bluuebunny/crossref_metadata_2025_split huggingface dataset:
Orientation of the brush‐border membranal proteinase which specifically splits the catalytic subunit of cAMP‐dependent protein kinase
Authors: DE JONGE Hugo , SCHMEEDA Hilary , SHALTIEL Shmuel 
Published: March 2005

The active site of the rat intestinal brush‐border membranal proteinase [Alhanaty E. and Shaltiel, S. (1979) Biochem. Biophys. Res. Commun. 89, 323–332], which splits the catalytic subunit (C) of cAMP‐dependent protein kinase with a remarkable specificity [Alhanaty E., Tauber‐Finkelstein, M., Schmeeda, H. and Shaltiel, S. (1985) Curr. Topics Cell. Regul. 27, 267–277], is shown to face predominantly the cell exterior; vesicles prepared from these brush‐borders (mostly sealed and right‐side‐out) fully express the proteinase activity as judged by the fact that there is no increase in activity upon rupture or solubilization of the vesicles.Although the brush‐border vesicles contain a cAMP‐dependent protein kinase, this membrane‐bound kinase is not likely to be the physiological target of the proteinase, since it appears to have an intracellular orientation and, at least in the vesicles, to be inaccessible to the proteinase. It is, therefore, suggested that the physiological substrate of the proteinase might be either an extracellular cAMP‐dependent protein kinase, which is lost (e.g. removed, inactivated or degraded) in the course of vesicle isolation, or a kinase domain in one of the family of proteins recently shown to have a considerable structural and conformational homology with C. Alternatively the physiological site of action of this kinase‐splitting proteinase might be an intracellular organelle to which it is translocated by endocytosis.
Purification and characterization of an 8‐hydroxy‐5‐deazaflavin‐reducing hydrogenase from the archaebacterium Methanococcus voltae
Authors: MUTH Erika , MÖRSCHEL Erhard , KLEIN Albrecht 
Published: March 2005

A methylviologen and 8‐hydroxy‐5‐deazaflavin(F420)‐reducing hydrogenase was purified over 800‐fold to near homogeneity from the archaebacterium Methanococcus voltae with 10 U mg−1 F420‐reducing activity. It is the only hydrogenase in this organism. The enzyme showed Km values of 16 μM for F420 and 1.2 mM for methylviologen. A turnover number of 1050 min−1 was calculated for the minimal active unit. The protein tends to aggregate. The molecular mass of the minimal active unit is 105 kDa. Larger molecules of 745 kDa were regularly observed. The enzyme was resolved into subunits with molecular masses of 55 kDa, 45 kDa, 37 kDa and 27 kDa by SDS/polyacrylamide gel electrophoresis. Reversible conversion of an anionic into an uncharged form was observed by DEAE‐cellulose chromatography with concomitant changes in substrate specifities. The methylviologen‐reducing activity was heat‐resistant up to 65°C and was not affected by antiserum raised against the native enzyme, while F420 reduction was inactivated by both treatments. Nickel and selenium contents were determined as 0.6–0.7 mol each, FAD content as 1 mol and iron as 4.5 mol/mol protein (105 kDa), respectively.Electron micrographs taken from the purified enzyme show ring‐shaped molecules of 18 nm diameter, which represent the high‐molecular‐mass species of the enzyme.
Enzyme‐catalysed peptide amidation
Authors: BRADBURY Alan F. , SMYTH Derek G. 
Published: March 2005

A series of hydrazones and semicarbazones of glyoxylic acid were shown to have a potent inhibitory effect on the enzyme‐catalysed conversion of D‐Tyr‐Val‐Gly to D‐Tyr‐Val‐NH2. Among the derivatives tested, the inhibitory activity was increased by the presence of hydrophobic substituents and decreased by polar substituents. The inhibition produced by glyoxylic acid phenylhydrazone was shown to be competitive. No inhibition was obtained with pyruvic acid phenylhydrazone, which possesses a methyl group in place of the α‐H of glyoxylic acid phenylhydrazone. The inhibitory potencies of these non‐peptide substances are in accord with the specificity exhibited by the amidating enzyme in its reaction with peptide substrates.The inhibition produced by the glyoxylic acid derivatives was shown to be due to their ability to act as substrates for the peptide‐amidating enzyme. The product formed from [14C]glyoxylic acid phenylhydrazone was identified as oxalic acid phenylhydrazide by co‐chromatography in three chromatographic systems. The results demonstrate that the enzyme catalysed oxidation of glyoxylic acid phenylhydrazone takes place by a mechanism involving hydroxylation. It is implicit that peptide amidation catalysed by the same enzyme proceeds by a similar mechanism.
Interactions of fatty acids with neutral fatty‐acid‐binding protein from bovine liver
Authors: SCHULENBERG‐SCHELL Helmut , SCHÄFER Petra , KEUPER Hermann J. K. , STANISLAWSKI Bernd , HOFFMANN Eberhard , RÜTERJANS Heinz , SPENER Friedrich 
Published: March 2005

Hepatic‐type fatty‐acid‐binding protein (hFABP) from the cytosol of bovine liver is a 14.4‐kDa neutral protein with a blocked N‐terminus and a disulfide system located on the surface of the protein. It binds two molecules of fatty acid in one binding site, apparent dissociation constants of the oleic acid/hFABP complex are 0.24 μM and 2.15 μM. Computer analysis of circular dichroic spectra predicts that hFABP contains about 12%α‐helix, 45%β‐structure, 15%β‐turn and 27% unordered structure. Ellipticities indicative of secondary structure are not affected by fatty acid binding.Cationic amino acid residues of hFABP (1 His, 15 Lys, 2 Arg) were screened for ionic fatty acid/protein interactions. His was excluded, as 1H‐NMR analysis of His‐C2 and His‐C4 protons indicated that binding of oleic acid shifts the pK of His from 6.9 to 7.1 only in hFABP with the disulfide system in the oxidized state; acylation of His with diethylpyrocarbonate does not affect the binding of the fatty acid. Acetylation of Lys reduces binding marginally, whereas modification of Arg with phenylglyoxal lowers the binding activity by 65%.From 1H‐NMR investigations, conformational changes within the protein, due to a sort of disaggregation of hFABP upon fatty acid binding, were derived. Most of the proton resonances sharpen up with ligand binding, and some of the methyl resonances shift positions, possibly because they are directly involved in the fatty acid/protein interaction.
Reconstitution of myoglobin from apoprotein and heme, monitored by stopped‐flow absorption, fluorescence and circular dichroism
Authors: KAWAMURA‐KONISHI Yasuko , KIHARA Hiroshi , SUZUKI Haruo 
Published: March 2005

The reconstitution reaction of ferric cyanomyoglobin from apomyoglobin and hemin dicyanide was investigated with a stopped‐flow apparatus by the use of five kinds of probes; (a) Soret absorption, (b) fluorescence quenching of tryptophan, (c) far‐ultraviolet CD, (d) near‐ultraviolet CD, and (e) Soret CD.After mixing of apomyoglobulin with equimolar amounts of hemin dicyanide, the Soret absorption band was shifted to longer wavelengths within 10 ms. The shifted band kept its shape for a few seconds, and then gradually shifted to shorter wavelengths. A rate constant of the slow reaction was 1.1 × 10−2 s−1. Time courses of fluorescence quenching followed a second‐order reaction with a rate constant of 9 × 107 M−1 s−1. Far‐ultraviolet CD recovered to the level of native state within the response time of an apparatus (= 64 ms). Near‐ultraviolet CD and Soret CD changed with first‐order rate constants of 5–30 s−1 and 5 × 10−3 s−1 respectively.On the basis of the kinetic results we propose the following reconstitution pathway of myoglobin. Apomyoglobin has essentially a highly folded structure similar to myoglobin, but there are some differences in the secondary structure between them. In the first step, heme enters the pocket‐like site of apomyoglobin and interacts with surrounding hydrophobic residues in the pocket, and then the interaction may give a complete ordered structure to the protein. Second, the tertiary structure of the heme pocket is partly constructed. Third, the iron‐proximal His bond occurs, followed by the attainment of the final conformation. This sequence of the events shows that the polypeptide chain is entirely folded before the completion of three‐dimensional structure of the heme pocket. The reconstitution pathway is fairly different from that of the α subunit of hemoglobin reported by Leutzinger and Beychok [Proc. Natl Acad. Sci. USA (1981) 78, 780–784], which described how a drastic recovery in helicity was observed on the heme‐binding, and that the recovery is introduced by the formation of the heme pocket structure. The difference in the results found for the α subunit and myoglobin suggests a difference in conformation: in apomyoglobin most of the helices are arranged and folded around a helix core to form a compact structure as a whole, while in apo‐α‐subunit some helices are not folded around the helix core. Helix D, which is absent in the α subunit, may play an important role in folding of the helices.
Ca2+ and Mg2+ as modulators of mitochondrial l‐glycerol‐3‐phosphate dehydrogenase
Authors: BELEZNAI Zsuzsa , SZALAY Lucia , JANCSIK Veronika 
Published: March 2005

Physiological concentrations of either Ca2+ or Mg2+stimulated l‐glycerol 3‐phosphate oxidation by intact mitochondria isolated from various mammalian tissues (hamster brown adipose tissue, rat brain, liver of normal and hyperthyroid rats). A higher cation concentration was required for stimulation by Mg2+ than by Ca2+. l‐glycerol‐3‐phosphate dehydrogenase was the target of the stimulation by both cations as revealed by measurements with intact mitochondria as well as with the solubilized enzyme. With different electron acceptors Ca2+ and Mg2+ stimulation occurred at significantly different cation concentrations.
Substrate activation of mitochondrial l‐glycerol‐3‐phosphate dehydrogenase was observed in intact mitochondria and with the solubilized enzyme isolated from hyperthyroid rats in the absence of Ca2+ and Mg2+. According to kinetic analysis two independent binding sites, functioning with different turnovers and with different affinities for the substrate, could account for the phenomenon. In the presence of Ca2+ or Mg2+ substrate activation could not be detected; the kinetic parameters apparently correspond to the tight substrate‐binding site functioning with high turnover.
Thiol group(s), which in the absence of Ca2+ and Mg2+ did not participate in the functioning of the enzyme, played an essential role in the binding of these cations to the enzyme, as shown by chemical modification studies.
From the solubilized mitochondrial proteins l‐glycerol‐3‐phosphate dehydrogenase was bound selectively to the hydrophobic phenyl‐Sepharose 4B matrix in the presence Ca2+, and the bound enzyme could be eluted with EDTA. This suggests that Ca2+ caused an alteration in the conformation of the enzyme.
Hydrogen peroxide (H2O2) induces actin and some heat‐shock proteins in Drosophila cells
Authors: COURGEON Anne‐Marie , ROLLET Emmanuelle , BECKER Jacqueline , MAISONHAUTE Claude , BEST‐BELPOMME Martin 
Published: March 2005

Drosophila cells of a clone derived from line Kc were treated with various concentrations of hydrogen peroxide (H2O2). The concentration of 10 mM was lethal, whereas concentrations of 1–100 μM did not affect cell viability, rate of multiplication or protein synthesis. The intermediate concentration of 1 mM H2O2 was used to study the response of the cells to an oxidative stress.We observed a transitory decrease of the global protein synthesis, which was accompanied by changes in the polypeptide pattern. There was a 2.5‐fold increase of the synthesis of the heat‐shock proteins 70–68 and 23. The most prominent response was a 6.5‐fold increase of actin synthesis 3 h after a 1 mM H2O2 treatment. When aminotriazole (an inhibitor of catalase) was added in association with H2O2, the increase of actin synthesis became 8.5‐fold. Experiments in which catalase was added at various times after H2O2 showed that a 10‐min treatment with H2O2 was sufficient to induce actin and heat‐shock protein synthesis 3 h later. H2O2 was shown to induce the transcriptional activation of an actin gene and of the heat‐shock protein genes 70 and 23 within minutes.These results are coherent with the hypothesis that the byproducts of O2 reduction (the superoxide ion and hydrogen peroxide) could be inducers of the heat‐shock response. Whether the increase of actin synthesis is a stress‐related response, and the mode of action of H2O2 are discussed
Control of enzyme synthesis in the lysine biosynthetic pathway of Saccharomyces cerevisiae
Authors: RAMOS Fernando , DUBOIS Evelyne , PIÉRARD André 
Published: March 2005

Several enzymes of the lysine pathway of Saccharomyces cerevisiae were found to respond to an induction mechanism mediated by the product of gene LYS14 in the presence of 2‐aminoadipate semialdehyde, an intermediate of this pathway. This novel regulatory mechanism appears independent of the specific repression by lysine and of the general control of amino acid biosynthesis. Genes LYS1, LYS9 and LYS14 have been cloned and their DNAs used to assay the corresponding messenger RNAs. The results suggest that the induction mechanism, as well as the specific and general regulations, operate at the transcriptional level.The synthesis of saccharopine dehydrogenase (glutamate‐forming), previously shown to require the unlinked genes LYS9 and LYS14, is also affected by the induction mechanism. The leaky auxotrophic behaviour of lys14 mutants is explained by the low basal level of expression of LYS9, the structural gene of this enzyme, in the absence of induction by 2‐aminoadipate semialdehyde.
Purification and characterization of a liver microsomal cytochrome P‐450 isoenzyme with a high affinity and metabolic capacity for coumarin from pyrazole‐treated D2 mice
Authors: JUVONEN Risto O. , SHKUMATOV Vladimir M. , LANG Matti A. 
Published: March 2005

Microsomal coumarin 7‐hydroxylase activity is regulated differently from several other monooxygenase enzymes, at least in mice [Wood, A. W. and Conney, A. H. (1974) Science (Wash. DC) 612–614]. Recently we found that in D2 mice this activity is strongly and selectively induced by pyrazole [Juvonen, R. O., Kaipainen, P. K. and Lang, M. A. (1985) Eur. J. Biochem. 152, 3–8]. This paper describes the purification of the pyrazole‐inducible cytochrome P‐450 isoenzyme. Because of the lability of the protein, a special procedure for the purification was developed. The procedure is based on a combination of hydrophobic and ion‐exchange chromatography and the presence of 100 μM coumarin in the preparations throughout the whole purification.Coumarin effectively protected the P‐450 from degradation and also converted the pyrazole‐inducible P‐450 to its high‐spin state. This enabled us to choose only those fractions for further purification where the P‐450(s) was in its high‐spin state (rather than measuring the content of the total P‐450). As a result the purified protein had an apparent molecular mass of 49.7 kDa, a specific content of 19.9 nmol/mg protein and a very high affinity and metabolic capacity for coumarin.
Distributions of retinoids, retinoid‐binding proteins and related parameters in different types of liver cells isolated from young and old rats
Authors: HENDRIKS Henk F. J. , BLANER William S. , WENNEKERS Herm M. , PIANTEDOSI Roseann , BROUWER Adriaan , de LEEUW A. Margreet , GOODMAN DeWitt S. , KNOOK Dick L. 
Published: March 2005

The levels of retinoids, retinol‐binding protein, cellular retinol‐binding protein, cellular retinoic‐acid‐binding protein, transthyretin and the activities of retinyl palmitate hydrolase and cholesteryl oleate hydrolase were determined in purified parenchymal, fat‐storing, endothelial and Kupffer cell preparations, and in liver homogenates from young adult (6‐month‐old) and old (36‐month‐old) rats. Retinoid levels were also determined in the plasma from young and old rats. Retinoid contents were determined by HPLC. The binding proteins and transthyretin were measured by specific radioimmunoassays; retinyl palmitate and cholesterol oleate hydrolases were measured by sensitive microassays. The retinoid content of both the liver homogenates and of the fat‐storing, and parenchymal cell preparations increased between 6 months and 36 months of age. The cellular distribution of retinoids was similar for the two age groups analyzed with the fat‐storing cells being the main retinoid storage sites in the rat liver.Concentrations of retinol‐binding protein and transthyretin were high in parenchymal cell preparations. Cellular retinol‐binding protein was enriched both in parenchymal and in fat‐storing cell preparations; the highest concentrations of cellular retinoic‐acid‐binding protein were present in fat‐storing cell preparations. No major differences were observed between the two age groups in the cellular concentrations and distributions of any of these binding proteins. High activity of cholesterol oleate hydrolase was measured in parenchymal and in Kupffer cell preparations; endothelial cell preparations also contained considerable activities. The distribution of this activity over the various cell types reflects their role in lipoprotein metabolism. Retinyl palmitate hydrolase activity was specifically enriched in parenchymal and in fat‐storing cell preparations, consistent with the roles of these cells in retinoid metabolism. No major differences were observed between the two age groups in the cellular distributions of the two hydrolase activities. This study indicates that no major changes occur in the retinoid‐related parameters analyzed with age, suggesting that rat liver retinoid metabolism does not change dramatically with age and that retinoid homeostasis is maintained.
The hopanoids of the purple non‐sulfur bacteria Rhodopseudomonas palustris and Rhodopseudomonas acidophila and the absolute configuration of bacteriohopanetetrol
Authors: NEUNLIST Serge , BISSERET Philippe , ROHMER Michel 
Published: March 2005

Five complex hopanoids have been detected in the purple non‐sulfur bacterium Rhodopseudomonas acidophila. Next to the polyfunctionalized methylcyclopentane bacteriohopanetetrol ether already isolated from Methylobacterium organophilum, 35‐carbamoylbacteriohopane‐32,33,34‐triol, 34,35‐dicarbamoylbacteriohopane‐32,33‐diol and two nucleoside analogues, (22R)‐30‐(5′‐adenosyl)hopane and (22S)‐30‐(5′‐adenosyl)hopane were isolated and identified by spectroscopic and chemical methods. In Rhodopseudomonas palustris, however, only 35‐amino‐bacteriohopane‐32,33,34‐triol was detected.Chemical correlation between adenosylhopane and bacteriohopanetetrol, as well as comparison of derivatives obtained from bacterial and synthetic hopanoids, permitted the determination of the configurations of all asymmetric centres of the side‐chain of bacteriohopanetetrol as 22R, 32R, 33R and 34S. According to the stereochemistry, this side‐chain could be a d‐ribose derivative linked through its C‐5 carbon atom to the hopane skeleton.
Elicitor‐induced biosynthesis of psoralens in Ammi majus L. suspension cultures
Authors: HAMERSKI Daria , MATERN Ulrich 
Published: March 2005

Suspension cultures of Ammi majus L. cells produce various linear furanocoumarins in response to treatment with elicitor preparations from either Alternaria carthami Chowdhury or Phytophthora megasperma f.sp. glycinea. Microsomes which were isolated from these cells 14 h after addition of the elicitor efficiently catalyzed the conversion of demethyl [3 –14C]suberosin into labelled (+)marmesin in the presence of NADPH and oxygen. In contrast to the chemical cyclization of demethylsuberosin by m‐chloroperoxybenzoic acid, the reaction catalyzed by the marmesin synthase proceeded rapidly and no intermediate demethylsuberosin epoxide could be recovered. Significant blue‐light‐reversible inhibition by carbon monoxide and inhibition by various chemicals known to inhibit reactions dependent on cytochrome P450 suggested that the marmesin synthase is a cytochrome‐P450‐dependent monooxygenase.Upon prolonged incubation, a subsequent major labelled product originated from (+)marmesin, which was identified as psoralen. The psoralen synthase was also characterized as a cytochrome‐P450‐dependent monooxygenase. Both the marmesin synthase and the psoralen synthase, as well as enzymes catalyzing the formation of demethylsuberosin and O‐prenylumbelliferone from umbelliferone and dimethylallyl diphosphate, were associated with the endoplasmic reticulum in Ammi majus cells and their activities were concomitantly induced by elicitor treatment of the cells. We propose that in vivo these enzymes are active in the lumen of the endoplasmic reticulum from where the furanocoumarin phytoalexins are excreted into the cell culture fluid.
Human erythrocyte glucose‐6‐phosphate dehydrogenase
Authors: CAMARDELLA Laura , CARUSO Carla , RUTIGLIANO Bruno , ROMANO Mario , DI PRISCO Guido , DESCALZI‐CANCEDDA Fiorella 
Published: March 2005

Human erythrocyte glucose‐6‐phosphate dehydrogenase contains a reactive lysyl residue, which can be labelled with pyridoxal 5′‐phosphate. The binding of one mole of pyridoxal 5′‐phosphate per mole of enzyme subunit produces substantial inactivation. The substrate glucose‐6‐phosphate prevents the loss of activity, suggesting that the reaction site is close to the substrate‐binding site. A tryptic peptide containing the pyridoxal‐5′‐phosphate‐binding lysyl residue has been isolated and characterised. The reactive lysyl residue has been identified in the glucose‐6‐phosphate dehydrogenase amino acid sequence. Comparison with glucose‐6‐phosphate dehydrogenase from other sources shows a high homology with a peptide containing a reactive lysyl residue, isolated from the enzyme from Saccharomyces cerevisiae; glucose‐6‐phosphate dehydrogenase from Leuconostoc mesenteroides also contains a region highly homologous with the sequence around the reactive lysyl residue in the human enzyme.The results of this communication provide the first direct evidence for the association of an essential catalytic function with a specific region of the molecule of human erythrocyte glucose‐6‐phosphate dehydrogenase.
Depression in gene expression for poly(ADP‐ribose) synthetase during the interferon‐γ‐induced activation process of murine macrophage tumor cells
Authors: TANIGUCH Taketoshi , YAMAUCHI Kiyoshi , YAMAMOTO Tadashi , TOYOSHIMA Kumao , HARADA Nobuhiro , TANAKA Hideaki , TAKAHASHI Seiichi , YAMAMOTO Hiroshi , FUJIMOTO Shigeyoshi 
Published: March 2005

A 2.7‐kb cDNA clone coding for bovine poly(ADP‐ribose) synthetase was isolated from a λgt11 expression library by direct immunological screening with an antiserum to the enzyme. The cDNA hybridizes to an approximately 3.8‐kb bovine thymus polyadenylated RNA, which translates an immunoprecipitable 120‐kDa protein with the antibody to the enzyme. The partial DNA sequence of the cDNA was determined and portions of the predicted amino acid sequence matched the sequence of 26 amino acids at the N terminal of the 41‐kDa α‐chymotryptic fragment and two cyanogen‐bromide‐cleaved peptides of the enzyme.A subcloned fragment from the coding region of the cDNA was used as a probe to estimate the level of mRNA for the enzyme during the interferon‐γ‐induced activation process of the murine macrophage tumor P388D1 cell line. The amount of mRNA for the enzyme decreased nearly completely within 24 h after incubation in a medium containing interferon‐γ, while mRNA of the Ia antigen, one of the major histocompatibility gene products, was increased in the macrophage tumor cells by interferon‐γ as confirmed by the I‐Aβ cDNA as a probe. These results suggest that the gene expression for poly(ADP‐ribose) synthetase is depressed during the interferon‐γ‐induced activation process of macrophage tumor cells.