bluuebunny/crossref_metadata_2025_split huggingface dataset:
Modified Plasma‐Membrane ATPase in Mutants of Saccharomyces cerevisiae
Authors: ULASZEWSKI Stanislaw , GRENSON Marcelle , GOFFEAU André 
Published: March 2005

Mutations affecting the plasma membrane ATPase of Saccharomyces cerevisiae were obtained by selecting mutants resistant to Dio‐9.In a plasma‐membrane‐enriched fraction of the mutant MG2130, the ATPase activity was resistant to vandate (50% inhibition by 26 μM in the mutant compared to 1.3 μM in the parental strain).Several catalytic properties of the membrane‐bound ATPase were modified by 60–120% in the mutant which had a higher Km for MgATP and was more heatstable, less sensitive to mercurials, and more stimulated by monovalent cations than the parental type.A single mutation is responsible for the phenotypes of four independent allelic mutants. Resistance to Dio‐9 in vivo and resistance to vanadate in vitro segregated together in three tetrads issued from a cross between the wild type and mutant. The mutation is semi‐dominant as shown by expression of the mutant phenotype in a heterozygous diploid resulting from the cross between the wild type and mutant.It is concluded that the pma locus, affected by these mutations, is the structural gene either for the 100000‐Mr subunit of plasma membrane ATPase or for a protein which tightly controls the conformation of the plasma‐membrane ATPase within the membrane.
Proteolytic Cleavage of Hansenula anomala Flavocytochrome b2 into Its Two Functional Domains
Authors: GERVAIS Michel , RISLER Yanick , CORAZZIN Sylvie 
Published: March 2005

In a previous work, we have described the tryptic cleavage of yeast flavocytochrome b2 into its two functional domains: a cytochrome b2 core and a flavodehydrogenase. The lactate dehydrogenase efficiency of the latter was, however, dramatically low, only about 1% that of intact flavocytochrome b2. Our present study concerns a new flavodehydrogenase derivative of Hansenula anomala flavocytochrome b2 which spontaneously dissociates from the cytochrome domain when the polypeptide bridge connecting them is cleaved by Staphylococcus aureus V8 protease I.This flavodehydrogenase was purified and some of its functional and structural properties were studied. It presents an exceptionally high lactate dehydrogenase activity, about 80% that of flavocytochrome b2. This result clearly demonstrates that the cytochrome domain is not necessary for the lactate dehydrogenase function and suggests an autonomous folding for both domains.Our results are discussed in terms of ‘gene fusion’.
Variation of (2′‐5′)Oligo(adenylate) Synthetase Activity during Rat‐Liver Regeneration
Authors: SMEKENS‐ETIENNE Martine , GOLDSTEIN Jacques , OOMS Henri A. , DUMONT Jacques E. 
Published: March 2005

The interferon‐induced double‐stranded RNA‐activated (2′‐5′)oligo(adenylate) synthetase converts ATP into (2′‐5′)oligo(adenylate) [(2′‐5′)oligo(A)] and pyrophosphate. In turn, (2′‐5′)oligo(A) activates a latent endoribonuclease which cleaves single‐stranded RNA.(2′‐5′)Oligo(A) synthetase activity has been characterized in liver cells of normal and germ‐free rats. This enzyme is predominantly nuclear. After partial hepatectomy, (2′‐5′)oligo(A) synthetase activity decreased rapidly (after 10 h) and markedly to a minimum of 25% of the control after 20 h. This decrease was followed by a slow restoration of the activity. No such change was observed in sham‐operated animals. This important decrease of enzymatic activity occurred during the first few hours of liver regeneration (6–24 h). This period corresponds also to the prereplicative and replicative (18–24 h after hepatectomy) phases of DNA. Detailed kinetics indicated that the loss of (2′‐5′)oligo(A) synthetase activity preceded the onset of the incorporation of tritiated thymidine in DNA and was minimal when the rate of DNA synthesis was maximal. These results and those obtained in culture of cells in vitro are compatible with the hypothesis that the (2′‐5′)oligo(A) system participates in the negative control of cell proliferation.
α1 Acid Glycoprotein: a Small‐Angle Neutron Scattering Study of a Human Plasma Glycoprotein
Authors: LI Zong‐Qi , PERKINS Stephen J. , LOUCHEUX‐LEFEBVRE Marie H. 
Published: March 2005

Small‐angle neutron scattering experiments on α1 acid glycoprotein showed that it has a molecular weight of 37000 and a matchpoint of 44.7%2H2O. The molecular weight, the matchpoint and a v̄ of 0.704 ml/g are in agreement with the primary sequence and standard residue volumes for amino acids and carbohydrates.The radius of gyration RG of α1 acid glycoprotein was found to be independent of concentration in the range 2–11 mg/ml, but increases on going from a buffer containing 0.2 M NaCl to one containing 1 M NaCl. A contrast variation study showed that the RG at infinite contrast is 2.47 nm for the expanded form and 2.19 nm for the contracted form, and that the two Stuhrmann α values are similar at 27 × 10−5. The latter is greater than that expected for globular proteins and are explained by the surface disposition of the five glycan chains on a core of protein in α1 acid glycoprotein. Modelling calculations account for the two RG values in which for the expanded form the glycan chains extend out into the solvent and for the contracted form they either fold back or are splayed out such that they are able to interact with the surface of the protein core.
Two Proteolytic Degradation Products of Calf‐Thymus Poly(ADP‐ribose) Polymerase Are Efficient ADP‐ribose Acceptors
Authors: HOLTLUND Jostein , JEMTLAND Rune , KRISTENSEN Tom 
Published: March 2005

Two polypeptides with molecular masses of 76 and 59 kDa were found to copurify with poly(ADP‐ribose) polymerase from calf thymus, and to be as efficient acceptors of ADP‐ribose as the polymerase itself. Analysis of their CNBr fragments by sodium dodcylsulfate/polyacrylamide gel electrophoresis revealed that the polypeptides were derived from the 112‐kDa polymerase. Isolation of poly(ADP‐ribose) polymerase in the absence of protease inhibitors resulted in a loss of more than 90% of the polymerase activity and an increased proportion of the 76‐kDa and 59‐kDa polypeptides in the final polymerase preparation.When the polymerase and the two polypeptides were separated by gel filtration or polyacrylamide gel electrophoresis in 5% acetic acid, no polymerase activity was found associated with the two fragments.Analysis of the CNBr fragments of the three polypeptides after incubation of the enzyme preparation with [32P]NAD showed that most of the fragments were radioactive, indicating multiple ADP‐ribosylation sites. Several ADP‐ribosylated fragments were found to be common to all three polypeptides, or to two of them.
A Stereochemical and Positional Isotope‐Exchange Study of the Mechanism of Activation of Methionine by Methionyl‐tRNA Synthetase from Escherichia coli
Authors: LOWE Gordon , SPROAT Brian S. , TANSLEY Gaynor 
Published: March 2005

Methionyl‐tRNA synthetase from Escherichia coli catalyses the activation of [18O2]methionine by adenosine 5′‐[(R)‐α17O]triphosphate with inversion of configuration at Pα. Furthermore methionyl‐tRNA synthetase does not catalyse positional isotope exchange in adenosine 5′‐[β‐18O2]triphosphate in the absence of methionine or in the presence of the competitive inhibitor, methioninol, which eliminates the possibility of either adenylyl‐enzyme or adenosine metaphosphate intermediates being involved. These observations require that methionyl‐tRNA synthetase catalyses the activation of methionine by an associative ‘in‐line’ nucleotidyl transfer mechanism.A kinetic study of positional isotope exchange in adenosine 5′‐[β‐18O2]triphosphate in the presence of methionine, Mg2+ and methionyl‐tRNA synthetase showed that torsional equilibration (18O exchange into the Pα—O—Pβbridge) occurs faster than tumbling (18O exchange into P7 by rotation about the C2 axis of Mg[18O2]PPi), demonstratings that the positional isotope exchange occurs at least in part in the E · Met‐AMP · Mg[18O2]PPi complex.
Coordinate and Non‐coordinate Estrogen‐Induced Expression of A and B Groups of Vitellogenin Genes in Male and Female Xenopus Hepatocytes in Culture
Authors: WOLFFE Alan P. , TATA Jamshed R. 
Published: March 2005

The concentration of mRNA transcribed from A and B groups of vitellogenin genes, induced by estrogen added to primary cultures of hepatocytes from male and female adult Xenopus, was measured by a technique of filter disc hybridization with cloned Xenopus vitellogenin cDNA probes.In cells from naive male Xenopus (i.e. not previously exposed to hormone in vivo or in vitro), the two groups of the multigene family were non‐coordinately expressed during the early stages of response to estradiol in vitro. Only B group transcripts could be detected for the first 2–3 h. At later times, or upon successive additions of estradiol to the male cell cultures, both A and B group mRNAs accumulated at the same rate and to the same extent. In female hepatocytes both groups of mRNAs accumulated in parallel at all stages of their response to estrogen, reaching levels 10‐fold higher than in naive male cells similarly exposed to the hormone. However, hepatocytes from male Xenopus that had received a single injection of estradiol 5 weeks before the cells were prepared, now exhibited identical rates and extent of accumulation of A and B groups of vitellogenin mRNAs to those observed in female cells.Pulse‐labeling of RNA in cultured male Xenopus hepatocytes confirmed that the coordinate and non‐coordinate accumulation of mRNAs were largely a function of differential or equal transcription of the A and B groups of vitellogenin genes. A phenomenon analogous to the hormone‐induced shift from non‐coordinate to coordinate expression of two groups of genes of the same multigene family has not been described previously, and we discuss possible mechanisms underlying the transition.
6‐Phospho‐d‐gluconate Dehydrogenase from Pseudomonas fluorescens
Authors: STOURNARAS Christos , MAURER Peter , KURZ Gerhart 
Published: March 2005

The 6‐phospho‐d‐gluconate dehydrogenase (decarboxylating) (EC 1.1.1.44) from Pseudomonas fluorescens, a B‐side stereospecific enzyme, is active with both NAD+ and NADP+, having a specific activity of the homogeneous enzyme of 121 μmol NADH and 23 μmol NADPH, respectively, formed min−1 mg protein−1. The pI of the native enzyme is 4.62, the pH optimum is about 8.2.
The molecular weight of the native enzyme has been determined to be 126000 by sedimentation equilibrium studies. The molecular weight of the polypeptide chains composing the enzyme has been found to be 32000 bydodecylsulfate/polyacrylamide gel electrophoresis and 31000 by sedimentation equilibrium studies in presence of 6 M guanidine hydrochloride. The native enzyme is composed of four polypeptide chains.
Reacting enzyme centrifugation studies gave at pH 8.2 a sedimentation coefficient s20, w of 8.04 S and a diffusion coefficient D20, w of 6.56 F, resulting in a molecular weight of 115000 for the catalytically active form. Thus, the enzyme is active as the tetramer. So far the enzyme from P. fluorescens is the sole 6‐phospho‐d‐gluconate dehydrogenase (decarboxylating) composed of four polypeptide chains.
The Major Proteins from HeLa Cells
Authors: LENSTRA Johannes A. , BLOEMENDAL Hans 
Published: March 2005

Subcellular fractionation allows a classification of the most abundant proteins from HeLa cells, detected after two‐dimensional gel electrophoresis. The positions of the spots belonging to actin, α‐tubulin, β‐tubulin and vimentin were indentified on the basis of literature data. Most other relatively abundant proteins could be identified, i.e. the cytokeratins, membrane proteins, elongation and initiation factors of protein biosynthesis.
From the latter group, the elongation factors eEF‐Tu and EF‐2 and the initiation factor eIF‐4A were identified separately.
The intensity of the spot corresponding to eEF‐Tu, described as one of the most abundant proteins in all eucaryotic cells [L. I. Slobin (1980) Eur. J. Biochem. 110, 555–563], was affected strongly by aggregation during isoelectric focusing. A method is described for extracting this protein from cultured cells by using its particular aggregation properties.
Primary Structure of Histone H2A from Gonads of the Starfish Asterias rubens
Authors: MARTINAGE Arlette , BELAICHE Denise , DUPRESSOIR Thierry , SAUTIERE Pierre 
Published: March 2005

The complete amino acid sequence (124 residues) of histone H2A from gonads of the starfish Asterias rubens has been established from automated sequence analyses of large fragments obtained by staphylococcal protease digestion of histone H2A and by limited hydrolysis of H2A‐H2B complex with mouse submaxillary gland protease and from structural studies of peptides generated by enzymatic hydrolyses of these fragments or of the protein.By comparison with calf homologus histone, the starfish histone H2A shows 5 deletions and 12 substitutions. Half of the substitutions are non‐conservative.Microheterogeneities were found at positions 18, 40 and 50 and result in the existence of at least two variants of starfish gonad histone H2A.
The Proof by 13C‐NMR Spectroscopy of the Predominance of the C5 Pathway over the Shemin Pathway in Chlorophyll Biosynthesis in Higher Plants and of the Formation of the Methyl Ester Group of Chlorophyll from Glycine
Authors: PORRA Robert J. , KLEIN Otto , WRIGHT Peter E. 
Published: March 2005

The 13C‐NMR spectrum of chlorophyll a formed when excised etiolated maize leaves were greened in the presence of [1‐13C]glutamate showed that the four methine bridge carbons (C‐5, C‐10, C‐15 and C‐20) and four pyrrole α‐carbons (C‐4, C‐9, C‐14 and C‐16) were considerably enriched relative to the natural‐abundance spectrum thus providing unequivocal proof of the operation of the C5 pathway of 5‐aminolaevulinate formation during chlorophyll biosynthesis in maize. [The carbon numbering system used is that recommended by IUB, see Pure Appl. Chem. 51, 2251–2304 (1979).]The 13C‐NMR spectrum of chlorophyll a formed during greening in the presence of [2‐13C]glycine showed that little or no enrichment of the above‐mentioned methine bridge or pyrrole α‐carbons had occurred thus indicating that the Shemin pathway makes little or no contribution to chlorophyll formation in greening maize. There was, however, considerable enrichment of the methoxyl carbon of the methoxycarbonyl group attached to isocyclic ring carbon C‐132. This shows that the C‐2 carbon of glycine is used to label the C1 pool and subsequently the methyl ester carbon of magnesium‐protoporphyrin IX‐monomethyl ester which is an intermediate in chlorophyll biosynthesis.The 13C‐NMR spectrum of chlorophyll a obtained when greening occurs in the presence of [5‐13C]glutamate showed considerable enrichment of the two carboxyl carbons of chlorophyll a (C‐133 and C‐173). This spectrum also showed that enrichment, equivalent to 15% of that occurring at the carboxyl carbons, occurred at the four methine bridge carbons and four relevant pyrrole α‐carbons. This is consistent with the conversion of [5‐13C]glutamate to [1‐13C]2‐oxoglutarate (or glutamate) via [5‐13C]2‐oxoglutarate and recycling in the tricarboxylic acid cycle: the soformed 1‐13C‐labelled substrate would then be incorporated into chlorophyll by the C5 pathway. This assessment of the amount of [5‐13C]glutamate incorporated into chlorophyll after recycling as 2‐oxoglutarate in the tricarboxylic acid cycle is used not only to confirm the view that the C5 pathway is the major, possibly sole, route for chlorophyll biosynthesis in higher plants but is also used to negate previous assertions of the involvement of the Shemin pathway based on comparison of the incorporation of 2‐oxoglutarate 14C‐labelled at C‐1 or C‐5.In these studies we also found that chlorophyll a purified by silica‐gel thin‐layer chromatography contained allomerization products and was unsuitable for 13C‐NMR spectroscopy.
The Fate of Xenopus and Locust Vitellogenins Made in Xenopus Oocytes
Authors: LANE Charles D. , CHAMPION Janet , COLMAN Alan , JAMES Tharappel C. , APPLEBAUM Shalom W. 
Published: March 2005

Xenopus liver or locust fat body RNA injected into Xenopus oocytes directs the formation of a high‐molecular‐weight species which is found transiently within the oocyte in a membranous vesicle fraction. The conversion of such internally generated Xenopus vitellogenin to yolk platelet proteins is reduced by the presence in the incubation medium of anti‐vitellogenin antibodies, or of tunicamycin, or of a mixture of colchicine and cytocholasin. but neither export nor conversion is blocked by removal of the follicular layers which surround the oocyte. Moreover, surrounding mRNA injected defolliculated oocytes with an excess of small defolliculated oocytes leads to a reduction in lipovitellin accumulation in the cells making vitellogenin and the appearance of lipovitellin in the platelets of the uninjected feeder cells.We propose that newly‐made locust and Xenopus vitellogenins are sequestered in vesicles and are then secreted by the oocyte, the locust species being further modified by cleavage just before export. Thus processing of heterologous yolk precursors follows the pathway characteristic of the cell type used to prepare the donor RNA. We suggest that the import mechanism discriminates between the exported locust and frog proteins, and that only the Xenopus vitellogenin subsequently enters the oocyte, where it is converted to lipovitellin and phosvitin by an enzyme present in the yolk platelets. Thus we explain the apparently paradoxical observations that internally generated and externally supplied frog vitellogenin are both converted to yolk platelet proteins, whilst injected vitellogenin is not.
A Novel One‐Carbon Carrier (Carboxy‐5,6,7,8‐tetrahydromethanopterin) Isolated from Methanobacterium thermoautotrophicum, and Derived from Methanopterin
Authors: KELTJENS Jan T. , DANIELS Lacy , JANNSEN Henny G. , BORM Paul J. , VOGELS Godfried D. 
Published: March 2005

During short‐term labeling experiments, cells of Methanobacterium thermoautotrophicum incorporated a substantial part of 14CO2 in a compound with a bright yellow fluorescence on dry thin‐layer chromatography plates and called yellow fluorescent compound (YEC) [Daniels, L. and Zeikus, J. G. (1978) J. Bacteriol. 136, 75–84]. This compound was extracted and purified by ion‐exchange column chromatography with formic acid gradients up to 0.3 M. Out of 325 g wet cells of M. thermoautotrophicum about 4 mg of the compound were isolated. This material and some degration products obtained from it were studied by means of chemical decomposition, ultraviolet‐visible‐light spectroscopy and preliminary 1H‐NMR spectroscopy. It has structural elements in common with methanopterin (see preceding paper in this journal); these elements are a pterin group, glutamate, a hexosamine.The pterin in this compound is present in reduced form, presumably as 5,6,7,8‐tetrahydromethanopterin, and the additional one‐carbon unit is probably present as a carboxy group.Probably the first step of methanogenesis implies a carboxylation of methanopterin and a concomitant reduction of the pterin. The trivial name carboxy‐5,6,7,8‐tetrahydromethanopterin is introduced for the compound.
Interaction of Dicyclohexylcarbodiimide with the Proton‐Conducting Pathway of Mitochondrial H+‐ATPase
Authors: KOPECKY Jan , GUERRIERI Ferruccio , PAPA Sergio 
Published: March 2005

In this paper a study is presented of the interaction of N,N′‐dicyclohexylcarbodiimide (DCCD) with the H+‐ATPase of mitochondria. Inhibition of proton conduction by the membrane sector (Fo) of the H+‐ATPase has been characterized by kinetics analysis of the effect of DCCD on the anaerobic decay of ΔμH+ set up by respiration in submitochondrial particles containing (ESMP) or deprived (USMP) of the catalytic sector (F1).The inhibitory effect of DCCD was directly related to the amount of DCCD bound independently of the time of incubation and of the concentration applied. Both in ESMP or USMP 2—3 nmol DCCD/mol Fo were required for the maximal inhibition of proton conduction.In ESMP the anaerobic relaxation of ΔμH+ is biphasic. Both phases were inhibited by DCCD and the titration curve indicated that the slow phase is directly related to the hydrolytic activity of H+‐ATPase. In USMP the anaerobic relaxation of ΔμH+ is monophasic. DCCD, at low concentrations, restored the biphasic pattern with the appearance of a transient fast phase having a kinetic constant higher than that of the control. Whilst the slow phase was immediately inhibited by DCCD the fast phase started to be inhibited only at concentrations higher than 1 nmol DCCD bound/mg protein.The data reported support the view that the fast phase of proton conduction reflects a conformational state of Fo, with higher proton conductivity, which is induced by high aerobic ΔμH+.Electrophoretic analysis of the labeling by [14C]DCCD of membrane polypeptides indicated that the inhibition of the state of low proton conductivity of Fo can be correlated with binding to the 8000‐Mr form of DCCD‐binding proteolipid, whilst inhibition of the state of high proton conductivity of Fo can be correlated with binding to the 16000‐Mr polypeptide.
Mitochondrial Pantetheinephosphate Adenylyltransferase and Dephospho‐CoA Kinase
Authors: SKREDE Sverre , HALVORSEN Ola 
Published: March 2005

Pantetheinephosphate adenylyltransferase and dephospho‐CoA kinase, which are last in the sequence leading to the biosynthesis of CoA, are localized in mitochondria (about 30% of the activity in the cell) and cytosol. The first three enzymes in the pathway are only found in cytosol [Skrede, S. and Halvorsen, O. (1979) Biochem. Biophys. Res. Commun. 91, 1536–1542].
Both enzymes are present in the mitochondrial matrix, but dephospho‐CoA kinase also has a more ‘external’ pool and is less easily removed from the membrane fractions of the mitochondria.
In accordance with the submitochondrial fractionation studies, pantetheinephosphate adenylyltransferase is to a larger extent activated by detergents or mechanical disruption of the mitochondria than is dephospho‐CoA kinase.
In intact mitochondria, the biosynthesis of CoA from 4‐phosphopantetheine was stimulated by uncoupling agents, probably because of facilitated penetration of the substrate through the inner mitochondrial membrane and or of leakage of a regulator.
Intact mitochondria could increase the synthesis of CoA 5–10 times, when conditions were chosen to promote biosynthesis from 4‐phosphopantetheine and to remove CoA by acylation and/or ‘leakage’ out of the mitochondria.
The kinetic properties of both mitochondrial enzymes are quite similar to the cytosolic ones (but our reinvestigation of the latter enzymes only partially confirm previous studies). Km for 4‐phosphopantetheine and dephosplio‐CoA were 0.015 and 0.01 mmol/1, respectively, for the mitochondrial enzymes.
Mitochondrial biosynthesis of CoA was strongly inhibited by dephospho‐CoA at the pantetheinephosphate adenylyltransferase level and more slightly by CoA at the dephospho‐CoA kinase level