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D C Gautheron

Publications and source records attributed to D C Gautheron.

At least 37 records · Page 2Linked to original sources
PubMed1988

ANTHEPROT: a package for protein sequence analysis using a microcomputer.

A simple microcomputer package is described to make the theoretical analysis of protein sequences. Several methods designed to compare two sequences, to model proteolytic reactions and to predict the secondary structure, the hydrophobic/hydrophilic regions and the potential antigenic sites of proteins have been included in an Apple II microcomputer software. The package comprises 21 programs as well as the secondary structure database of Kabsch and Sander (1983).

Algorithms↗
PubMed1987

Revertant of the yeast Schizosaccharomyces pombe with modified alpha subunits of mitochondrial ATPase-ATPsynthase: impaired nucleotide interactions with soluble and membrane-bound enzyme.

A partial revertant from a mutant with modified alpha subunits of mitochondrial ATPase-ATPsynthase has been obtained for the first time from the yeast Schizosaccharomyces pombe. The purified F1 contains a lower amount of endogenous nucleotides as compared to the wild-strain enzyme. In contrast to the wild-type, the F1 ATPase activity from the revertant does not exhibit bicarbonate-sensitive negative cooperativity. The revertant Michaelis constant for Mg-ATP is very similar to that of normal F1 in the presence of bicarbonate while the Vm is slightly lower. The revertant enzyme is much less sensitive to inhibitions by ADP and by azide. It is proposed that the lack of negative cooperativity of revertant F1 ATPase activity is due to lower affinity for ADP, the release of which is no longer the rate-limiting step.

Adenine Nucleotides↗
PubMed1986

Efficient reconstitution of mitochondrial energy-transfer reactions from depleted membranes and F1-ATPase as a function of the amount of bound oligomycin sensitivity-conferring protein (OSCP).

Pig heart mitochondrial membranes depleted of F1 and OSCP by various treatments were analyzed for their content in alpha and beta subunits of F1 and in OSCP using monoclonal antibodies. Membrane treatments and conditions of rebinding of F1 and OSCP were optimized to reconstitute efficient NADH- and ATP-dependent proton fluxes, ATP synthesis and oligomycin-sensitive ATPase activity. F1 and OSCP can be rebound independently to depleted membranes but to avoid unspecific binding of F1 to depleted membranes (ASUA) which is not efficient for ATP synthesis, F1 must be rebound before the addition of OSCP. The rebinding of OSCP to depleted membranes reconstituted with F1 inhibits the ATPase activity of rebound F1, while it restores the ATP-driven proton flux measured by the quenching of ACMA fluorescence. The rebinding of OSCP also renders the ATPase activity of bound F1 sensitive to uncouplers. The rebinding of OSCP alone or F1 alone, does not modify the NADH-dependent proton flux, while the rebinding of both F1 and OSCP controls this flux, inducing an inhibition of the rate of NADH oxidation. Similarly, oligomycin, which seals the F0 channel even in the absence of F1 and OSCP, inhibits the rate of NADH oxidation. OSCP is required to adjust the fitting of F1 to F0 for a correct channelling of protons efficient for ATP synthesis. All reconstituted energy-transfer reactions reach their optimal value for the same amount of OSCP. This amount is consistent with a stoichiometry of two OSCP per F1 in the F0-F1 complex.

Adenosine Triphosphatases↗
PubMed1986

Role of phosphate on the ADP-induced hysteretic inhibition of mitochondrial adenosine 5'-triphosphatase. Effects of the natural protein inhibitor.

Preincubation of F1-ATPase with ADP and Mg2+ leads to ADP binding at regulatory site inducing a hysteretic inhibition of ATP hydrolysis, i.e., an inhibition that slowly develops after Mg-ATP addition (Di Pietro, A., Penin, F., Godinot, C. and Gautheron, D.C. (1980) Biochemistry 19, 5671-5678). It is shown here that inorganic phosphate (Pi) together with ADP during preincubation abolishes the time-dependence of the inhibition after the addition of the substrate Mg-ATP. This preincubation in the presence of both Pi and ADP slowly leads to a conformation of the enzyme immediately inhibited after the addition of the substrate Mg-ATP. The Pi effect is half-maximal at 35 microM and pH 6.6, whereas a limited effect is induced at pH 8.0. The preincubation of F1-ATPase with Pi and ADP must last long enough (t1/2 = 5 min). The effects can be correlated to the amount of Pi bound to the enzyme, 1 mol Pi per mol (apparent KD of 33 microM) at saturation. Pi neither modifies the ADP binding nor the final level of the concomitant inhibition. When Pi is not present in the preincubation, the final stable rate of ADP-induced hysteretic inhibition is always reached when a near-constant amount of Pi has been generated during Mg-ATP hydrolysis. Kinetic experiments indicate that preincubation with ADP and Pi decreases both Vmax and Km which would favor a conformational change of the enzyme. Taking into account the Pi effects, a more precise model of hysteretic inhibition is proposed. The natural protein inhibitor IF1 efficiently prevents the binding of Pi produced by ATP hydrolysis indicating that the hysteretic inhibition and the IF1-dependent inhibition obey different mechanisms.

Adenosine Diphosphate↗
PubMed1986

Reversal of glucose-induced inhibition of newborn rat liver mitochondrial maturation by administration of alkylxanthines at birth.

A glucose injection given immediately after birth delays the maturation which normally occurs in rat liver mitochondria and which increases the rate of ATP synthesis coupled to succinate oxidation from a low value at birth to the adult value a few hours after birth [R. Meister, J. Comte, L. Baggetto, C. Godinot and D. C. Gautheron, Biochim. biophys. Acta 722, 36 (1983)]. Alkylxanthine (pentoxifylline, HWA 285) administration at birth has no effect on the maturation of mitochondria prepared from 2-hr-old rat livers while DBcAMP administration increases their RCR and their rate of ATP synthesis. On the contrary, both alkylxanthines and DBcAMP reverse the glucose-induced inhibition of mitochondrial maturation. This DBcAMP effect cannot be mimicked by butyrate and is therefore related to cAMP. The cAMP content of rat liver increases during this postnatal period in both control and glucose-treated rats, although glucose administration tends to decrease the level of cAMP. Alkylxanthine administration restores after 2 hr the cAMP level in glucose-treated animals. The variations of RCR could not be completely correlated with the level of cAMP. The possible involvement of other factors in the mitochondrial maturation and the glucose effect is discussed.

Animals↗
PubMed1986

Chemical modification of thiol groups of mitochondrial F1-ATPase from the yeast Schizosaccharomyces pombe. Involvement of alpha- and gamma-subunits in the enzyme activity.

Mitochondrial F1-ATPase from the yeast Schizosaccharomyces pombe has been prepared under a stable form and in relatively high amounts by an improved purification procedure. Specific chemical modification of the enzyme by the thiol reagent N-ethylmaleimide (NEM) at pH 6.8 leads to complete inactivation characterized by complex kinetics and pH dependence, indicating that several thiols are related to the enzyme activity. A complete protection against NEM effect is afforded by low concentrations of nucleotides in the presence of Mg2+, with ADP and ATP being more efficient than GTP. A total binding of 5 mol of [14C]NEM/mol of F1-ATPase is obtained when the enzyme is 85% inactivated: 3 mol of the label are located on the alpha-subunits and 2 on the gamma-subunit. Two out of the 3 mol on the alpha-subunits bind very rapidly before any inactivation occurs, indicating that the two thiols modified are unrelated to the inactivation process. Complete protection by ATP against inactivation by NEM prevents the modification of three essential thiols out of the group of five thiols labeled in the absence of ATP: one is located on a alpha-subunit and two on the gamma-subunit. These two essential thiols of the gamma-subunit can be differentiated by modification with 6,6'-dithiodinicotinic acid (CPDS), another specific thiol reagent. A maximal binding of 4 mol of [14C]CPDS/mol of enzyme is obtained, concomitant to a 25% inhibition. Sequential modification of the enzyme by CPDS and [14C]NEM leads to the same final deep inactivation as that obtained with [14C]NEM alone. One out of the two thiols of the gamma-subunit is no longer accessible to [14C]NEM after CPDS treatment. When incubated at pH 6.8 with [3H]ATP in the presence of Mg2+, F1-ATPase is able to bind 3, largely exchangeable, mol of nucleotide/mol of enzyme. Modification of the three essential thiols by NEM dramatically decreases the binding of 3H-nucleotide down to about 1 mol/mol of enzyme. Partial modification modifies the cooperative properties, the enzyme being no longer sensitive to anion activation.

Adenosine Triphosphate↗
PubMed1986

Topography of oligomycin sensitivity conferring protein in the mitochondrial adenosinetriphosphatase-ATP synthase.

The topographical organization of oligomycin sensitivity conferring protein (OSCP) in the mitochondrial adenosinetriphosphatase (ATPase)-ATP synthase complex has been studied. The accessibility of OSCP to monoclonal antibodies has been qualitatively visualized by using the protein A-gold electron microscopy immunocytochemistry or quantitatively estimated by immunotitration of OSCP in depolymerized or intact membranes. Besides, OSCP cannot be labeled by 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine ([125I]TID) which selectively labels the hydrophobic core of membrane proteins. These observations demonstrate an external location of OSCP on the inner face of the inner mitochondrial membrane. The position of OSCP relative to other peptides of the complex has been analyzed by cross-linking experiments using either zero length N-(ethoxycarbonyl)-2-ethoxydihydroquinoline or 11-A span dimethyl suberimidate cross-linkers in the ATPase-ATP synthase complex. The OSCP cross-linked products were identified either by immunocharacterization with anti-alpha, anti-beta, or anti-OSCP monoclonal antibodies or by their molecular weight. OSCP was cross-linked with either the alpha- or beta-subunits of F1 or to a subunit of Mr 24 000. Other types of cross-linking were obtained by the labeling of OSCP with [cysteamine-35S]-N-succinimidyl 3-[[2-((2-nitro-4-azidophenyl)amino)ethyl]dithio]propionate ([35S]SNAP) and reconstitution of SNAP-OSCP with F1 in urea-treated submitochondrial particles. Under these conditions, OSCP is found to be adjacent to two other peptides of molecular weight close to 30 000. A comparison is made between the topology and the organization of the b-subunit of Escherichia coli and OSCP, suggesting an analogy between OSCP and the hydrophilic part of the b-subunit.

Adenosine Triphosphatases↗
PubMed1986

Chemical modification of essential carboxyl group and histidine residue in the plasma-membrane 5'-nucleotidase.

An investigation, using specific chemical reagents, of the amino acids involved in the catalytic activity of the purified 5'-nucleotidase (5'-ribonucleotide phosphohydrolase, EC 3.1.3.5) from bovine liver plasma membranes, was carried out. The enzyme was irreversibly inactivated by N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ). The inhibition kinetics were of the first-order type and decreased partially in the presence of nucleotides and divalent cations. These results indicate for the first time that a carboxyl group is essential for the catalytic process of 5'-nucleotidase. Moreover, chemical modification by diethylpyrocarbonate also produced inactivation of the enzyme and showed a differential spectrum with a peak at 240 nm characteristic of N-carbethoxyhistidine residues. This inactivation was efficiently released upon decarbethoxylation by hydroxylamine only when the extent of inactivation, due to low concentration of diethylpyrocarbonate, was limited. The time-dependent inactivation followed first-order kinetics and nucleotides afforded significant protection against diethylpyrocarbonate modification. The results indicate the involvement of the histidine residue in catalysis.

5'-Nucleotidase↗
PubMed1986

Immobilization of factor XIII on collagen membranes.

Factor XIII from placenta was successfully grafted onto collagen membranes by the acyl-azide procedure. The transamidase activity retained on collagen membranes was determined by measuring the increase of fluorescence resulting from dansylcadaverine incorporation into casein. We studied the effect of different factors on the grafting: concentration and composition of the factor XIII preparation in the coupling solution and influence of the preactivation of factor XIII by thrombin. Stability studies have shown that the activity of factor XIII grafted on collagen membranes was almost constant over a period of 8 months. Sterilization by gamma-irradiation of factor XIII revealed a drastic loss of activity while the use of high-energized electron bombardment caused a reduced loss of activity. The potential of such a material for biomedical use is presently under investigation.

Animals↗
PubMed1985

Coupled reaction of immobilized aspartate aminotransferase and malate dehydrogenase. A plausible model for the cellular behaviour of these enzymes.

To study the effect of facilitated diffusion of the intermediate metabolite, oxaloacetate, on the coupled reaction of aspartate aminotransferase (L-aspartate: 2-oxoglutarate aminotransferase, EC 2.6.1.1) and malate dehydrogenase (L-malate:NAD+ oxidoreductase, EC 1.1.1.37), these enzymes were co-immobilized on the surface of a collagen film. The kinetic properties of the immobilized enzymes were compared with those observed with the enzymes in solution. Since the reactions correspond to the cytosolic enzymes, they have been studied in the direction aspartate aminotransferase toward malate dehydrogenase. Coupled enzymes in solution showed classical behaviour. A lag-time was observed before they reached a steady state and this lag-time was dependent on the kinetic properties of the second enzyme, malate dehydrogenase. The same lag-time was observed when malate dehydrogenase in solution was coupled with aspartate aminotransferase bound to the film. When aspartate aminotransferase in solution was coupled with malate dehydrogenase bound to the collagen film, a very long lag-time was observed. Theoretical considerations showed that in the latter case, the lag-time was dependent on the kinetic properties of the second enzyme and the transport coefficient of the intermediate substrate through the boundary layer near the surface of the film. Then both enzymes were co-immobilized on the collagen film. The coupled activity of aspartate aminotransferase and malate dehydrogenase was compared for films with an activity ratio of 5 and 0.8. In both cases, a highly efficient coupling was observed. In the former case, where malate dehydrogenase was rate-limiting, 81% of this limiting activity was observed. In the latter case, aspartate aminotransferase was rate-limiting and 82% of its rate was obtained for the final product formation. The linear increase of product formation with time corresponded fairly well to the theoretical equations developed in the paper. To interpret these rate equations, one should assume that the intermediate substrate oxaloacetate formed by aspartate aminotransferase was used by malate dehydrogenase in the diffusion layer near the film, before diffusing in the bulk solution.

Animals↗
PubMed1985

Modified properties of hexokinase from heart mitochondria prepared using proteolytic enzyme.

Isolation of muscle mitochondria is made easier by using proteolytic treatment of the tissue before homogenization. Normally, the proteolytic enzyme is discarded with the supernatant of the first centrifugation. However, our results show that a fraction of enzyme activity remains associated with mitochondria. As shown in experiments described in this paper, mitochondrial hexokinase from tissue treated or not with the proteolytic enzyme exhibits similar properties except that the solubilized enzyme from protease treated tissue is no longer able to rebind to mitochondrial membrane. This modification of the binding ability of the enzyme results from a partial hydrolysis of hexokinase during solubilization experiments by the proteolytic enzyme. Since, as pointed out here, proteolytic enzyme can remain associated with mitochondria, [either absorbed on mitochondrial membrane or included in the mitochondrial pellet] its use for the isolation of muscle mitochondria should be avoided.

Adenosine Triphosphate↗
PubMed1984

Monoclonal antibodies to mitochondrial F1-ATPase and oligomycin sensitivity conferring protein (OSCP). Tools for recognition of well conserved and essential antigenic sites.

The preparation of anti-OSCP monoclonal antibodies is described for the first time. One of these antibodies prevents the activating effect of OSCP in reconstitution experiments. These antibodies and antibodies previously obtained against the alpha- and beta-subunits of pig heart mitochondrial F1-ATPase have been used to look for well conserved epitopes in various species. One anti-beta antibody can recognize all species tested while the anti-OSCP antibodies only recognize the pig or beef enzyme. The above anti-beta antibody inhibits ATP synthesis without modifying the rate of ATP hydrolysis. This antibody also prevents the ADP-induced hysteretic inhibition of F1-ATPase.

Adenosine Triphosphatases↗
PubMed1984

Two-dimensional gel electrophoresis of membrane proteins using anionic and cationic detergents. Application to the study of mitochondrial F0-F1-ATPase.

Polyacrylamide gel electrophoresis in the presence of a cationic detergent, tetradecyltrimethylammonium bromide (TDAB) has been compared to electrophoresis in the presence of an anionic detergent, sodium dodecyl sulfate (SDS). Although, in both systems, the peptides generally migrated as a function of their molecular weight, the TDAB electrophoresis permitted us to obtain a much better resolution of several peptides of the mitochondrial F0-F1-ATPase, especially for the alpha and beta subunits and for the oligomycin sensitivity conferring protein (OSCP). The differences between the two electrophoretic profiles have been used to devise a new technique of two-dimensional electrophoresis using successively anionic and cationic detergents. This method could be very useful in the case of membrane proteins, which are generally soluble only in the presence of powerful ionic detergents. It has been particularly successful in resolving the small peptides of the F0-F1-ATPase which were difficult to differentiate by other techniques in one- or two-dimensional polyacrylamide gel electrophoresis.

Adenosine Triphosphatases↗
PubMed1984

Effects of ATP on various steps controlling the rate of oxidative phosphorylation in newborn rat liver mitochondria.

Preincubation of newborn rat liver mitochondria with ATP increases their state 3 respiration rate [J. K. Pollak (1975) Biochem. J. 150, 477-488; J. R. Aprille, and G. K. Asimakis (1980) Arch. Biochem. Biophys. 201, 564-575]. To determine which reactions contribute to control the rate of succinate oxidation with and without prior exposure to ATP, the effects of inhibitors specific for various reactions were studied. The adenine nucleotide translocator does not control the respiration in newborn more than in the adult mitochondria. The supply of reducing equivalents to the respiratory chain is an important step controlling the rate of oxidative phosphorylation by mitochondria from newborn rat liver, especially after preincubation with ATP. On the contrary, titrations with oligomycin show that the preincubation with ATP markedly decreases the control exerted by the ATPase-ATP synthase complex. That the rate of ATP synthesis is one of the steps controlling the rate of oxidative phosphorylation in newborn rat liver mitochondria is in striking contrast to the behavior of adult rat liver mitochondria. Other differences include a greater permeability to protons and a marked increase in sensitivity to mersalyl, indicating an easier accessibility of the proteins involved in oxidative phosphorylation to the thiol reagent.

Adenosine Triphosphate↗