bluuebunny/crossref_metadata_2025_split huggingface dataset:
Purification, characterization and redox properties of hydrogenase from Methanosarcina barkeri (DSM 800)
Authors: FAUQUE Guy , TEIXEIRA Miguel , MOURA Isabel , LESPINAT Paul A. , XAVIER António V. , DER VARTANIAN Daniel V. , PECK Harry D. , LE GALL Jean , MOURA José G. 
Published: March 2005

A soluble hydrogenase from the methanogenic bacterium, Methanosarcina barkeri (DSM 800) has been purified to apparent electrophoretic homogeneity, with an overall 550‐fold purification, a 45% yield and a final specific activity of 270 μmol H2 evolved min−1 (mg protein)−1. The hydrogenase has a high molecular mass of ∼ 800 kDa and subunits with molecular masses of ∼ 60 kDa. The enzyme is stable to heating at 65°C and to exposure to air at 4°C in the oxidized state for periods up to a week. The overall stability of this enzyme is compared with other hydrogenase isolated from strict anaerobic sulfate‐reducing bacteria. Ms. barkeri hydrogenase shows an absorption spectrum typical of a non‐heme iron protein with maxima at 275 nm, 380 nm and 405 nm. A flavin component, identified as FMN or riboflavin was extracted under acidic conditions and quantified to approximately one flavin molecule per subunit. In addition to this component, 8–10 iron atoms and 0.6–0.8 nickel atom were also detected per subunit.The electron paramagnetic resonance (EPR) spectrum of the native enzyme shows a rhombic signal with g values at 2.24, 2.20 and ∼ 2.0. probably due to nickel which is optimally measured at 40 K but still detectable at 77 K. In the reduced state, using dithionite or molecular hydrogen as reductants, at least two types of g= 1.94 EPR signals, due to iron‐sulfur centers, could be detected and differentiated on the basis of power and temperature dependence. Center I has g values at 2.04, 1.90 and 1.86, while center II has g values at 2.08, 1.93 and 1.85. When the hydrogenase is reduced by hydrogen or dithionite the rhombic EPR species disappears and is replaced by other EPR‐active species with g values at 2.33, 2.23, 2.12, 2.09, 2.04 and 2.00. These complex signals may represent different nickel species and are only observable at temperatures higher than 20 K. In the native preparation, at high temperatures (T > 35 K) or in partially reduced samples, a free radical due to the flavin moiety is observed. The EPR spectrum of reduced hydrogenase in 80% Me2SO presents an axial type of spectrum only detectable below 30 K.
Expression of aldolase isozyme mRNAs in fetal rat liver
Authors: NUMAZAKI Masayoshi , TSUTSUMI Ken‐ichi , TSUTSUMI Reiko , ISHIKAWA Kiichi 
Published: March 2005

The regulation of aldolase isozyme expression during development was studied by measuring the concentrations of mRNAs coding for aldolase A and B subunits in fetal and adult rat liver. Poly(A)‐containing RNAs were extracted from livers at various stages of development of fetal rats, and the aldolase A and B subunits in the in vitro translation products of these RNAs were analyzed immunologically. The content of aldolase B mRNA in 14‐day fetal liver, measured quantitatively as translational activity, was somewhat smaller than that of aldolase A mRNA; immunologically precipitable aldolase B and A amounted to 0.06% and 0.25%, respectively, of the total products. Similar experiments using RNAs from fetuses at later stages, however, showed that aldolase B mRNA increased during development, whereas aldolase A mRNA decreased. In newborn rat liver, aldolase B constituted 0.56% of the total translation products of mRNA, but there was little detectable aldolase A (0.03%).The changes of aldolase mRNA levels were analyzed further by northern blot and dot‐blot hybridization experiments using cloned aldolase A and B cDNAs. The content of aldolase B mRNA increased in the fetal stage, and that in newborn rat liver was about 12 times that in 14‐day fetal liver. In contrast, the aldolase A mRNA content decreased during gestation and that in newborn rat liver was about one‐eighth of that in 14‐day fetal liver. These observations suggest that the switch of aldolase isozyme expression in fetal liver is controlled by the levels of the respective mRNAs.
A cold‐labile acetyl‐coenzyme‐A hydrolase from the supernatant fraction of rat liver
Authors: ISOHASHI Fumihide , NAKANISHI Yoko , MATSUNAGA Takaharu , SAKAMOTO Yukiya 
Published: March 2005

An acelyl‐coenzyme‐A hydrolase from the supernatant fraction of rat liver is known to be rapidly inactivated at low temperature. Loss of catalytic activity is accompanied by apparent dissociation of tetrameric and dimeric forms of the enzyme into monomers. It was found that rewarming under appropriate conditions almost completely reversed the cold‐induced inactivation and dissociation of the enzyme: At a protein concentraion of 14 μg/ml, simple rewarming only partially restored the enzyme activity (less than 3% of the original activity), but at a higher concentration of the enzyme or in the presence of 1 mg/ml bovine serum albumin, the reactivation by warming was greater. Warming at 37°C appeared to be optimal for reactivation; warming at 25°C or at 43°C was less effective. Longer exposure to cold did not affect reactivation on rewarming, but on repeated inactivation and reactivation the reactivation decreased to some extent, especially at lower concentrations of enzyme protein. Among various nucleotides tested, ATP greatly enhanced the restoration of the activity, while ITP, UTP and ADP were less effective and AMP, GTP, TTP and CTP had little effect. At an enzyme‐protein concentration of 14 μg/ml, 2 mM ATP restored the enzyme activity to about 70% of that before cold treatment, while acetyl‐CoA (0.5 mM) restored the activity about 50% High concentrations of phosphate (0.92 M) and pyrophosphate (0.45 M) restored about 80% and 95%, respectively, of the original activity.Sucrose density gradient centrifugation of the active dimer at high enzyme concentration at 4°C for 20 h produced a monomeric form without catalytic activity. Gel filtration showed that simple rewarming mostly converted the monomeric enzyme obtained in this way to the dimeric form, whereas on rewarming with ATP the monomer was mostly converted to a tetrameric form. The dimeric and tetrameric forms both had catalytic activity.
Analysis of the control of citrulline synthesis in isolated rat‐liver mitochondria
Authors: WANDERS Ronald J. A. , VAN ROERMUND Carlo W. T. , MEIJER Alfred J. 
Published: March 2005

The amount of control of the various steps involved in the citrulline synthesizing pathway in isolated rat‐liver mitochondria incubated with saturating concentrations of ammonia and ornithine has been measured.

The flux control coefficient of carbamoyl‐phosphate synthase (ammonia) was close to one. Control exerted by other steps in the pathway, including ornithine transcarbamoylase, carbonic anhydrase, ATP production and transport of ornithine, was negligible.
The high flux control coefficient of carbamoyl‐phosphate synthetase is due to the low elasticity coefficient of this enzyme towards its product, intramitochondrial carbamoyl phosphate, in combination with a high elasticity coefficient of ornithine transcarbamoylase towards carbamoyl phosphate.
Inhibitory action of certain cyclophosphate derivatives of cAMP on cAMP‐dependent protein kinases
Authors: DE WIT René J. W. , HEKSTRA Doeke , JASTORFF Bernd , STEC Wojciech J. , BARANIAK Janina , VAN DRIEL Roel , VAN HAASTERT Peter J. M. 
Published: March 2005

A series cAMP derivatives with modifications in the adenine, ribose and cyclophosphate moiety were screened for their binding affinity for the two types of cAMP‐binding sites in mammalian protein kinase type I. In addition, the activation of the kinase by these analogs was monitored. The binding data indicate that cAMP is bound to both sites in a comparable manner: the adenine appears to have no hydrogen‐bond interactions with the binding sites, whereas the ribose may be bound by three hydrogen bonds involving the 2′, 3′ and 5′ positions of cAMP. The binding data are not conclusive about the nature of the interaction with the exocyclic oxygen atoms on phosphorus, though a charge interaction seems to be absent. The cAMP molecule seems to be bound in the syn conformation.The results of activation experiments show that modifications in the adenine and ribose moiety do not affect the maximal activation level, while alteration of the two exocydic oxygen atoms may result in a reduced maximal activation level and in one case, (Rp)‐adenosine 3′, 5′‐monophosphorothioate [Rp−cAMPS], in total absence of activation even at concentrations, at which the analog saturates both binding sites. Since occupancy of the cAMP‐binding sites by this derivative apparently did not lead to activation of the enzyme, we examined whether this compound could antagonize the activation by cAMP. Indeed (Rp)‐cAMPS was found to inhibit cAMP stimulated kinase activity at concentrations compatible to its binding affinity. Also with mammalian protein kinase type II (Rp)‐cAMPS showed antagonistic activity, while with a cAMP‐dependent protein kinase from Dictyostelium discoideum partial agonistic activity was observed.Previously a mechanism for activation of protein kinase type I was proposed involving a charge interaction between the equatorial exocyclic oxygen atom and the binding site [De Wit et al. (1982) Eur. J. Biochem. 122, 95‐99]. This was based on measurements with impure preparations of (Rp)‐cAMPS and the Rp and Sp isomers adenosine 3′, 5′‐monophosphodimethylamidate, cAMPN(CH3)2. The present work using highly purified compounds suggests the absence of a charge interaction, since the uncharged analog (Sp)‐cAMPN(CH3)2 activates the kinase effectively. The data seem compatible with an activation model involving the formation of a covalent bond with phosphorus in both cAMP binding sites.
Sidedness of native membrane vesicles of Escherichia coli and orientation of the reconstituted lactose :H+ carrier
Authors: SECKLER Robert , WRIGHT J. Keith 
Published: March 2005

The orientation of the lactose:H+ carrier of Escherichia coli in various preparations of native and reconstituted vesicles is determined with two impermeant, macromolecular probes: antibodies directed against the C‐terminal decapeptide of the carrier and carboxypeptidase A (EC 3.4.17.1). Two methods are employed. Method I is based upon the digestion of all accessible and, therefore, presumably external, C termini of the carrier with carboxypeptidase A and detection of the remaining, internal C termini with 125I‐labelled anti‐(C‐terminus) antibody after electrophoresis of the carrier in the presence of sodium dodecyl sulfate and transfer to nitrocellulose filters. Method II is based upon the binding of 125I‐labelled anti‐(C‐terminus) antibody to the external C termini of the carrier in vesicles and the subsequent isolation of bound antibody by centrifugation. The labelled antibodies are calibrated using a preparation of inside‐out vesicles prepared by high‐pressure lysis of strain T206. The carrier content is determined by substrate binding. Because the C terminus of the carrier is known to reside on the cytoplasmic side of the membrane, these methods can also be used to determine the sidedness of various preparations of membrane vesicles.Spheroplasts are confirmed to contain carrier molecules of a single orientation, corresponding to that in right‐side‐out vesicles. In contrast, in purified cytoplasmic membrane vesicles and in crude membrane preparations obtained by sonication or by high‐pressure lysis, 96% of the C termini are accessible to carboxypeptidase A, even after repeated sonication. This implies that nearly all carrier molecules in these preparations possess an orientation opposite to that in the cell or in right‐side‐out vesicles.In proteoliposomes containing carrier reconstituted or purified and reconstituted by two different methods, only 48% of the carrier molecules are oriented in the same way as in the cell. Subjecting such proteoliposomes to cycles of freezing and thawing or to sonication results in a reshuffling of carrier molecules between the inside‐out and right‐side‐out populations while maintaining 41% in the right‐side‐out orientation.Digestion of the C terminus of the carrier with carboxypeptidase A does not alter either galactoside binding or countertransport. Thus carrier molecules of the inside‐out orientation cannot be selectively inactivated. Additionally, an antiserum directed against the purified carrier is demonstrated to contain nearly exclusively anti‐(C‐terminus) antibodies, which can, in principle, be used in Method I. Modification of Method II permits the determination of carrier levels in vesicles prepared from cells by high‐pressure lysis with as little as 0.1 pmol of carrier to an accuracy of 30–35%.The functional symmetry of certain aspects of carrier‐catalyzed facilitated diffusion and active transport is discussed in the context of the nonpreferential, bimodal insertion of the carrier into proteoliposomes.
Topological aspects of microsomal N‐acetyltransferase, an enzyme responsible for the acetylation of cysteine S‐conjugates of xenobiotics
Authors: OKAJIMA Kenji , INOUE Masayasu , MORINO Yoshimasa , ITOH Kazunobu 
Published: March 2005

Acetylation of cysteine S‐conjugates of xenobiotics by microsomal N‐acetyltransferase is the final step of detoxicative metabolism leading to mercapturic acid biosynthesis. To elucidate the subcellular site of N‐acetylation and the effective mechanism by which the final metabolites are eliminated from the organisms, topological aspects and catalytic properties of microsomal N‐acetyltransferase and mercapturic acid biosynthesis in vivo were investigated. Intravenous administration of radioactive S‐benzyl‐L‐cysteine, a model compound of cysteine S‐conjugates, resulted in rapid acetylation of the conjugate in liver and kidney to a similar extent. The acetylation was followed by a rapid excretion of the metabolite, a mercapturic acid, into the urine; about 60% of the injected dose appeared in urine within 60 min of administration.Limited proteolysis of microsomal vesicles obtained from liver and kidney by chymotrypsin or trypsin inactivated the transferase by 49–62% and 62–73%, respectively. Proteolytic inactivation of the transferase was not significantly affected by the presence of 0.04% sodium deoxycholate by which the vesicles became permeable to macromolecules due to its detergent action.To determine the sidedness of the active site of N‐acetyltransferase on the microsomal membranes, two S‐acetyldextran polymer derivatives (Mt 500000) of cysteine and N‐acetylcysteine which represent an nonpermeant substrate and product for this enzyme, respectively, were examined for their effects on the vesicle‐associated enzyme activity. Both derivatives inhibited the transferase activity in a dose‐dependent fashion; maximum inhibition of the enzyme activity was 40% by the former and 60% by the latter.Sulfobromophthalein strongly inhibited the enzyme activity and, this inhibition was completely reversed by adding an equimolar amount of hepatic glutathione S‐transferases (ligandins). In contrast to the strong inhibition by sulfobromophthalein itself, its glutathione S‐conjugate did not inhibit the enzyme activity.These results indicate that the active site and the protease‐sensitive domain(s) of the microsomal N‐acetyltransferase are localized on the outer surface (cytoplasmic side) of endoplasmic reticulum and that the ligandin(s) might protect membranous N‐acetyltransferase‐from inhibition by organic anions by binding them and catalyzing the conjugation with glutathione.
Increased stability and antiviral activity of 2′‐O‐phosphoglyceryl derivatives of (2′‐5′)oligo(adenylate)
Authors: BAYARD Bernard , BISBAL Catherine , SILHOL Michele , CNOCKAERT Joël , HUEZ Georges , LEBLEU Bernard 
Published: March 2005

Metabolically stable analogues of (2′‐5′)oligo(adenylate), (2′‐5′)(A)n. might constitute a new class of antiviral agents as they mimic some of the effects of interferons. 2′‐O‐phosphoglyceryl derivatives of (2′‐5′)(A)n oligomers, (2′‐5′)(A)n‐PGro have been synthesized by chemical modification of their terminal ribose residue. Such analogues are resistant to degradation by phosphodiesterases but remain sensitive to phosphatase activity, at least in cell‐free extracts.In line with its increased stability, (2′‐5′)(A)n‐PGro has a powerful antiviral activity against an RNA virus when microinjected with micropipettes into the cytoplasm of intact cells. This antiviral activity remains transient however, possibly as a consequence of degradation in intact cells.Since (2′‐5′)(A)n and its derivatives do not easily cross cell membranes, their possible use in antiviral chemotherapy is tightly linked with the development of vectors suitable for their administration in vivo.
Cytochrome b of cob revertants in yeast
Authors: BURGER Gertraud , HOFNER Eva 
Published: March 2005

22 revertants of Saccharomyces cerevisiae with intragenic suppressors (sup3) of cob exon mutations (G. Burger. Mol. Gen. Genet., in the press) were analyzed. They display either a reduced amount of cytochrome b, or a shifted maximum absorption wavelength of total cytochrome b or a reduced growth rate on glycerol.The relationship of physico‐chemical properties (content, light absorption and midpoint potential of cytochromes bK and bT) and functional properties (electron transport and energy yield) has been examined.In seven of eight revertants with a shifted maximum absorption wavelength of cytochrome b neither growth rate nor electron transfer activity was affected.In 13 of 14 revertants, reduced content of cytochrome b corresponds to a reduced electron transport rate through the cytochrome bc1 segment.A lower enzymatic activity, which is not due to a quantitative but to a qualitative alteration of cytochrome b was found in two revertants.Two revertants show electron transport rates of wild‐type level concomitant with a reduced growth rate on glycerol, obviously due to a less efficient energy coupling. All revertants were shown to contain a high and a low potential cytochrome b, referred to as bK and bT.Those cob−/supa mutations which shift the maximum absorption wavelength or diminish the content of cytochrome b affect both b cytochromes in all cases. The results support that electron transport and energy conservation are catalyzed by the unity of cytochrome bK and bT and that both heme centers are bound to an identical apoenzyme.Comparing electron flow rates of succinate:cytochrome c oxidoreductase and NADH:cytochrome c oxido‐reductase in cob mutants and two revertants provides evidence that ubiquinone does not constitute a homogeneous pool, suggested by the dissimilar interaction of both dehydrogenases with the bc1 segment.
Nucleotide sequence of the pyr D gene of Escherichia coli and characterization of the flavoprotein dihydroorotate dehydrogenase
Authors: LARSEN Jørgen Nedergaard , JENSEN Kaj Frank 
Published: March 2005

Dihydroorotate dehydrogenase (EC 1.3.3.1) was purified to near electrophoretic homogeneity from the membranes of a strain of Eschrichia coli carrying the pyr D gene on a multicopy plasmid. The preparation had a specific activity of 120 μmol min−1 mg−1 and contained flavin mononucleotide (FMN) in amounts stoichiometric to the dihydroorotate dehydrogenase subunit (Mr= 37000). The flavin group was reduced when dihydroorotate was added in the absence of electron acceptors.The complete sequence of 1357 base pairs of an EcoRI‐EcoRI DNA fragment containing the pyr D gene was established. Dihydroorotate dehydrogenase is encoded by a 336‐triplets open reading frame. The molecular mass (Mr= 36732), the amino acid composition and the N‐terminal sequence of the predicted polypeptide agree well with the data obtained by analysis of the purified protein. A region of the amino acid sequence (residues 292–303, i. e. Ile‐Ile‐Gly‐Val‐Gly‐Gly‐Ile‐Asp‐Ser‐Val‐Ile‐Ala) shows distinct homology to the cofactor binding site of other flavoproteins. No hydrophobic regions large enough to span the cytoplasmic membrane were observed.By the S 1‐nuclease technique an mRNA start was mapped 34 ± 2 nucleotide residues upstream of the beginning of the coding frame of pyr D. The leader region contains no similarity to the attenuators of the pyr B and pyr E genes of E. coli.
Solubilization and purification of the ATPase from the tonoplast of Hevea
Authors: MARIN Bernard , PREISSER Joachim , KOMOR Ewald 
Published: March 2005

The tonoplast‐bound ATPase of Hevea brasiliensis (caoutchouc tree) was solubilized with dichloromethan and purified 100‐fold with two ammonium sulfate precipitation steps and a G‐200 gel filtration step. The resulting ATPase activity eluted according to a molecular mass of approximately 200 kDa and chromatographed at an isoelectric pH of 5.3. Subunits of molecular mass 100 kDa, 68 kDa, 24 kDa and 12 kDa appeared after treatment with 1% sodium dodecyl sulfate or spontaneously during storage of the solubilized ATPase. Dodecyl sulfate/polyacrylamide gel electrophoresis yielded four polypeptides of molecular mass 54 kDa, 66 kDa, 23 kDa and 13 kDa. From protein determination by ultraviolet absorption and Coomassie stain it appears that the 54‐kDa and the 66‐kDa polypeptides exist in multiple copies. No close resemblance to the membrane‐bound ATPase of mitochondria, plastids, plasmalemma, chromaffin granules and synaptic vesicles is seen. No antibody crossreaction to F1 of bacteria is observed. Therefore it is concluded that the vacuolar ATPase represents a novel type of ATPase.Many properties of the tonoplast‐bound ATPase such as pH‐dependence, substrate specificity, ion‐dependence and inhibitor sensitivity did not change when the enzyme had been solubilized and purified. The phosphatase activity was lost during the purification procedure. The stimulation of ATP‐hydrolysis in tonoplast vesicles by uncouplers and ionophores was absent in the solubilized ATPase, and also the stimulation by chloride was significantly reduced. Anion channel blockers, such as triphenyltin and 4,4′‐diisothiocyano‐2,2′‐disulfonic acid stilbene, which are strong inhibitors of membrane‐bound ATPase, fully or partly lost their inhibiting effect after solubilization of the ATPase. These results are interpreted to indicate that ionophores do not directly affect the ATPase molecule, whereas chloride might have a small direct effect on the ATPase besides its effect as a permeating anion.
Amino acid sequence of histone H1 at the ADP‐ribose‐accepting site and ADP‐ribose · histone‐H1 adduct as an inhibitor of cyclic‐AMP‐dependent phosphorylation
Authors: USHIROYAMA Takahisa , TANIGAWA Yoshinori , TSUCHIYA Mikako , MATSUURA Ryoji , UEKI Minoru , SUGIMOTO Osamu , SHIMOYAMA Makoto 
Published: March 2005

The ADP‐ribosylation site of histone H1 from calf thymus by purified hen liver nuclear ADP‐ribosyltransferase was determined and effects of the ADP‐ribose · histone‐H1 adduct on cAMP‐dependent phosphorylation of the histone H1 were investigated.ADP‐ribosylated histone H1 was prepared by incubation of histone H1, 1 mM [adenylate‐32P]NAD and the purified ADP‐ribosyltransferase. N‐Bromosuccinimide‐directed bisection of ADP‐ribosylated histone H1 showed that the NH2‐terminal fragment (Mr= 6000) was modified and contained serine residue 38, the site of phosphorylation by cAMP‐dependent protein kinase. Digestion of the NH2‐terminal fragment with cathepsin D and trypsin, and purification of this fragment, using high‐performance liquid chromatography, yielded a radiolabelled single peptide corresponding to residues 29–34 of histone H1, containing the arginine residue as the ADP‐ribosylation site. These results indicate that ADP‐ribosylation of histone H1 occurs at the arginine residue 34, sequenced at the NH2‐terminal side of the phosphate‐accepting serine residue 38.Phosphorylation of histone H1 from calf thymus by cAMP‐dependent protein kinase was markedly reduced when histone H1 was ADP‐ribosylated. Kinetic studies of phosphorylation revealed that ADP‐ribosylated histone H1 was a linear competitive inhibitor of histone H1 and a linear non‐competitive inhibitor of ATP.