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D C Gautheron

Publications and source records attributed to D C Gautheron.

At least 19 recordsLinked to original sources
PubMed1994

Preparation of highly phosphorylating mitochondria from the yeast Schizosaccharomyces pombe.

Schizosaccharomyces pombe yeast cells grown on either fermentable or respiratory media were efficiently converted to stable spheroplasts by the alpha-(1-->3)-glucanase Novozym 234 in the presence of 1.2 M sorbitol. Lysis of spheroplasts by gentle homogenization in dilute sorbitol resulted in the preparation of mitochondria with a structure similar to that observed within the starting yeast cells. The isolated mitochondria exhibited high oxidation rates with various respiratory substrates, NADH being the most efficient. The mitochondria appeared well coupled since the second State 4 rate observed after ADP consumption was identical to the initial one. The State 3 rate in the presence of ADP was completely inhibited by low oligomycin concentrations, similarly to the concomitant ATP synthesis of 900 nmol/min x mg protein. These NADH oxidation and dependent ATP-synthesis activities are much higher than those previously described for mitochondria isolated from Schizosaccharomyces pombe, and similar to the highest values reported for Saccharomyces cerevisiae.

Adenosine Diphosphate↗
PubMed1992

Differential nucleotide binding to catalytic and noncatalytic sites and related conformational changes involving alpha/beta-subunit interactions as monitored by sensitive intrinsic fluorescence in Schizosaccharomyces pombe mitochondrial F1.

Mitochondrial F1 from the yeast Schizosaccharomyces pombe exhibits an intrinsic tryptophan fluorescence sensitive to adenine nucleotides and inorganic phosphate [Divita, G., Di Pietro, A., Deléage, G., Roux, B., & Gautheron, D.C. (1991) Biochemistry 30, 3256-3262]. The present results indicate that the intrinsic fluorescence is differentially modified by nucleotide binding to either catalytic or noncatalytic sites. Guanine or hypoxanthine nucleotides, which selectively bind to the catalytic site, produce a hyperbolic saturation monitored by fluorescence quenching at 332 nm, the maximal emission wavelength. On the contrary, adenine nucleotides, which bind to both catalytic and noncatalytic sites, exhibit a biphasic saturation. High-affinity ATP binding produces a marked quenching as opposed to the lower-affinity one. In contrast, ADP exhibits a sigmoidal saturation, with high-affinity binding producing no quenching but responsible for positive cooperativity of binding to the lower-affinity site. The catalytic-site affinity for GDP is almost 20-fold higher at pH 5.0 as compared to pH 9.0, and the high sensitivity of the method allows detection of the 10-fold lower-affinity GMP binding. In contrast, high-affinity binding of ADP, or AMP, is not pH-dependent. The selective catalytic-site saturation induces a F1 conformational change decreasing the Stern-Volmer constant for acrylamide and the tryptophan fraction accessible to iodide. ATP saturation of both catalytic and noncatalytic sites produces an additional reduction of the accessible fraction to acrylamide.

Adenine Nucleotides↗
PubMed1991

Large-scale purification and characterization of the five subunits of F1-ATPase from pig heart mitochondria.

A large-scale purification procedure was developed to isolate the five subunits of F1-ATPase from pig heart mitochondria. The previously described procedure (Williams, N. and Pedersen, P.L. (1986) Methods Enzymol. 126, 484-489) to dissociate the rat liver F1-ATPase by cold treatment followed by warming at 37 degrees C has been adapted for the pig heart enzyme. Removal of endogenous nucleotides from that enzyme before dissociation led to the efficient separation of the alpha and gamma subunits from beta, delta and epsilon subunits. The beta subunit was purified in the hundred-milligram range by anion-exchange chromatography in the absence of any denaturing agent. This subunit was free from any bound nucleotide and almost no ATPase and adenylate kinase-like activities were detected. The delta and epsilon subunits were purified by reversed-phase chromatography (RP-HPLC) in the milligram range. As recently reported (Penin, F., Deléage, G., Gagliardi, D., Roux, B. and Gautheron, D.C. (1990) Biochemistry 29, 9358-9364), these purified subunits kept biophysical features of folded proteins and their ability to reconstitute the tight delta epsilon complex. The alpha and gamma subunits remained poorly soluble and required dissociation by 8 M guanidinium chloride prior to their purification by RP-HPLC. In addition, characterizations of the five subunits by IEF and SDS-polyacrylamide gel electrophoresis are reported, as well as ultraviolet spectra and solubility properties of the beta, delta and epsilon subunits.

Animals↗
PubMed1991

Alteration of apparent negative cooperativity of ATPase activity by alpha-subunit glutamine 173 mutation in yeast mitochondrial F1. Correlation with impaired nucleotide interaction at a regulatory site.

The first described alpha-subunit mutation of yeast mitochondrial F1 has been recently identified as a single Gln173----Leu substitution in a strongly conserved sequence (Falson, P., Maffey, L., Conrath, K., and Boutry, M. (1991) J. Biol. Chem. 266, 287-293). This mutation is shown here to greatly modify the biphasic pattern of ATPase activity as a function of pH: (i) the shoulder observed at acidic pH is significantly increased; (ii) the main peak, at alkaline pH, is markedly lowered; (iii) the optimal pH is shifted from 8.8 to 7.7. The mutation lowers both apparent negative cooperativity and sensitivity to azide inhibition which concomitantly increase when the assay pH decreases. Azide partial inhibition produces apparent negative cooperativity which can be further abolished by bicarbonate. The mutation increases both activation energies determined from biphasic Arrhenius plots. The mutation decreases the inactivation rate by 5'-p-fluorosulfonylbenzoyladenosine and abolishes the protection by nucleotide binding at the adenine-specific regulatory site. On the contrary, it does not modify the reactivity of 5'-p-fluorosulfonylbenzoylguanosine at the less-selective catalytic site. In addition, partial inactivation by 5'-p-fluorosulfonylbenzoyladenosine, as opposed to 5'-p-fluorosulfonylbenzoylguanosine, produces apparent negative cooperativity under conditions where unmodified-enzyme kinetics are noncooperative. The results show that alpha-Gln173 participates in nucleotide interaction at a regulatory site which controls the negative cooperativity of F1-ATPase activity.

Adenosine↗
PubMed1991

Intrinsic tryptophan fluorescence of Schizosaccharomyces pombe mitochondrial F1-ATPase. A powerful probe for phosphate and nucleotide interactions.

Mitochondrial F1 from the yeast Schizosaccharomyces pombe, in contrast to the mammalian enzyme, exhibits a characteristic intrinsic tryptophan fluorescence with a maximal excitation at 291 nm and a maximal emission at 332 nm. Low values of Stern-Volmer quenching constants, 4.0 M-1 or 1.8 M-1, respectively, in the presence of either acrylamide or iodide, indicate that tryptophans are mainly buried inside the native enzyme. Upon subunit dissociation and unfolding by 6 M guanidine hydrochloride (Gdn.HCl), the maximal emission is shifted to 354 nm, a value very similar to that obtained with N-acetyltryptophanamide, a solute-tryptophan model compound. The tryptophan content of each isolated subunit has been estimated by fluorescence titration in the presence of Gdn.HCl with free tryptophan as a standard. Two tryptophans and one tryptophan are found respectively in the alpha and epsilon subunits, whereas none is detected in the beta, gamma, and delta subunits. These subunit contents are consistent with the total of seven tryptophans estimated for native F1 with alpha 3 beta 3 gamma 1 delta 1 epsilon 1 stoichiometry. The maximal emission of the isolated epsilon subunit is markedly blue-shifted to 310-312 nm by interaction with the isolated delta subunit, which suggests that the epsilon subunit tryptophan might be a very minor contributor to the native F1 fluorescence measured at 332 nm. This fluorescence is very sensitive to phosphate, which produces a marked blue shift indicative of tryptophans in a more hydrophobic environment. On the other hand, ADP and ATP quench the maximal emission at 332 nm, lower tryptophan accessibility to acrylamide, and reveal tryptophan heterogeneity.

Acrylamide↗
PubMed1991

A 28 kDa mitochondrial protein is radiolabelled by crosslinking with a 125I-labelled presequence.

A 13-residue peptide containing the first 12 amino acids of the N-terminal part of the signal sequence of yeast cytochrome c oxidase subunit IV is shown by chemical crosslinking to interact with a mitochondrial protein. This result is obtained with mitochondria from four different origins. Submitochondrial localization experiments suggest that the 28 kDa labelled component is present on the outer face of the inner membrane. Since such addressing peptides are imported into mitochondria through the same machinery as protein precursors, the 28 kDa protein might be a component of the translocation apparatus.

Amino Acid Sequence↗
PubMed1990

Interaction between delta and epsilon subunits of F1-ATPase from pig heart mitochondria. Circular dichroism and intrinsic fluorescence of purified and reconstituted delta epsilon complex.

A delta epsilon complex has been purified as a molecular entity from pig heart mitochondrial F1-ATPase. This delta epsilon complex has also been reconstituted from purified delta and epsilon subunits. Both isolated and reconstituted delta epsilon complexes have delta 1 epsilon 1 stoichiometry and are indistinguishable by their chromatographic behavior, their circular dichroism spectra (CD spectra), and their intrinsic fluorescence features. The content of secondary structures deduced from CD spectra of the delta epsilon complex appears to be the sum of the respective contributions of purified delta and epsilon subunits. All intrinsic fluorescence studies carried out on isolated epsilon subunit and delta epsilon complex show that the single tryptophan residue located on epsilon is involved in the interaction between delta and epsilon subunits. Results obtained with F1-ATPase are in favor of the same delta epsilon interaction in the entire enzyme.

Animals↗
PubMed1989

Purification from a yeast mutant of mitochondrial F1 with modified beta-subunit. High affinity for nucleotides and high negative cooperativity of ATPase activity.

Mitochondrial F1 containing genetically modified beta-subunit was purified for the first time from a mutant of the yeast Schizosaccharomyces pombe. Precipitation by poly(ethylene glycol) allowed us to obtain a very stable and pure enzyme from either mutant or wild-type strain. In the presence of EDTA, purified F1 retained high amounts of endogenous nucleotides: 4.6 mol/mol and 3.7 mol/mol for mutant and wild-type F1, respectively. The additional nucleotide in mutant F1 was ATP; it was lost in the presence of Mg2+, which led to a total of 3.4 mol of nucleotides/mol whereas wild-type F1 retained all its nucleotides. Mutant F1 bound more exogenous ADP than wild-type F1 and the same total nucleotide amount was reached with both enzymes. Kinetics of ATPase activity revealed a much higher negative cooperativity for mutant than for wild-type F1. Bicarbonate abolished this negative cooperativity, but higher concentrations were required for mutant F1. The mutant enzyme was more sensitive than the wild-type one to azide inhibition and ADP competitive inhibition; this indicated stronger interactions between nucleotide and F1 in the mutant enzyme. The latter also showed increased sensitivity to N,N'-dicyclohexylcarbodiimide irreversible inhibition.

Adenosine Diphosphate↗
PubMed1989

A yeast strain with mutated beta-subunits of mitochondrial ATPase-ATPsynthase: high azide and bicarbonate sensitivity of the ATPase activity.

A phenotypic revertant with modified beta-subunits of mitochondrial ATPase-ATP synthase has been obtained for the first time by selection from a beta-less mutant of the yeast Schizosaccharomyces pombe. Contrary to the parental mutant, the phenotypic revertant grows on glycerol, has normal respiratory activity and shows immunodetectable beta-subunits. However the kinetic properties of its submitochondrial particles ATPase activity differ markedly from those of the wild strain. The optimal pH is increased by about one unit. The maximal rate of the revertant ATPase activity at pH 8.5 is 4 to 5-fold lower than that of the wild strain, but it can be greatly increased upon addition of bicarbonate whereas the wild strain is completely insensitive to this anion. Furthermore the revertant ATPase activity is much more sensitive to azide inhibition. The results suggest that ADP dissociation is the rate-limiting step of ATP hydrolysis by the revertant.

Azides↗
PubMed1989

Structure-function relationships of mitochondrial ATPase-ATPsynthase using Schizosaccharomyces pombe yeast mutants with altered F1 subunits.

Phenotypic revertants have been selected from mutants of the yeast Schizosaccharomyces pombe devoid of either alpha or beta subunits of mitochondrial ATPase-ATPsynthase. In contrast to parental mutants, phenotypic revertants are able to grow on glycerol respiratory medium and show immunodetectable alpha and beta subunits. However, growth and cellular respiration are only partially restored as compared to the wild strain, indicating that the recovered subunits are mutated. ATPase activity of revertant submitochondrial particles shows markedly different parameters: more acidic optimal pH, absence of bicarbonate activation and decreased sensitivity to azide inhibition in the alpha subunit-modified R3.51. Opposite differences are observed in the beta subunit-modified R4.3: more alkaline optimal pH, much higher bicarbonate activation, and increased sensitivity to azide. The ITPase activity of R4.3 submitochondrial particles is also more sensitive to azide as compared to the wild strain. ATPase activity of purified F1 also exhibits marked differences: loss of bicarbonate-sensitive negative cooperativity, decreased sensitivity to both ADP and azide inhibitions in the R3.51 revertant. On the contrary, increased negative cooperativity and increased sensitivity to both ADP and azide inhibitions are observed for the R4.3 revertant enzyme which in addition exhibits a much lower maximal rate. The beta subunit-mutation of R4.3 also increases the sensitivity of ITPase activity to tripolyphosphate inhibition, whereas the alpha subunit-mutation of R3.51 is without any effect. Soluble F1 with beta subunit-mutation is very sensitive to high ammonium sulfate concentrations required for enzyme precipitation and concentration and known to partially deplete the enzyme from its endogenous nucleotides. On the contrary, poly(ethylene)glycol is very efficient for preparing from any strain a pure and very stable enzyme retain-ing high amounts of endogenous nucleotides. The R4.3 revertant F1 retains even more nucleotides than the wild-strain F1 and is much less sensitive to high iodide concentrations which favor enzyme dissociation and precipitation. The tryptophan intrinsic fluorescence of F1 is modified by both mutations that increase the maximal emission intensity. The most important effect is produced by beta subunit-mutation which decreases the quenchable fraction, one-third to one-half tryptophans being no longer accessible to iodide. The overall results suggest that both mutations modify enzyme-nucleotide interactions: the alpha subunit-mutation of R3.51 would favor ADP release by lowering interactions with the adenine moiety, whereas the beta subunit-mutation of R4.3 would lower ADP release by strengthening interactions with the phosphate chain moiety.

Adenosine Triphosphatases↗
PubMed1988

Fate of nucleotides bound to reconstituted Fo-F1 during adenosine 5'-triphosphate synthesis activation or hydrolysis: role of protein inhibitor and hysteretic inhibition.

The protein ATPase inhibitor entraps about five nucleotides in pig heart mitochondrial F1, one at least being a triphosphate [Di Pietro, A., Penin, F., Julliard, J.H., Godinot, C., & Gautheron, D.C. (1988) Biochem. Biophys. Res. Commun. 152, 1319-1325]. The fate of these nucleotides was studied during ATP synthesis driven by NADH oxidation in reconstituted inverted submitochondrial particles. Iodinated F1, containing 0.7 mol of endogenous nucleotides/mol, was first loaded with tritiated adenine nucleotides in the presence or absence of the protein inhibitor and then reassociated with F1-depleted submitochondrial particles (ASU particles) to reconstitute an efficient NADH-driven ATP synthesis. In the absence of the protein inhibitor, 1.7 mol of labeled nucleotides remained bound per mole of reassociated F1, 0.8-0.9 mol being rapidly exchangeable against medium ADP or ATP, as measured after rapid filtration through nitrocellulose filters. In the presence of the protein inhibitor, as many as 3.25 mol of labeled nucleotides remained bound per mole of reassociated F1. Under hydrolysis conditions where ATPase activity was highly inhibited, no release of tritiated nucleotide occurred. In contrast, under ATP synthesis conditions where the protonmotive force was generated by NADH oxidation, the progressive reversal of inhibition by the protein inhibitor was correlated to a concomitant release of tritiated nucleotide. When ATP synthesis became fully active, about one nucleotide was completely exchanged whereas more than three nucleotides remained tightly bound and did not appear to be directly involved in ATP synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Diphosphate↗
PubMed1988

Cyclic GMP, cyclic AMP, glucose at birth, and maturation of rat liver mitochondria.

The improvement in mitochondrial functions which normally occurs in newborn rat liver in vivo during the few hours following delivery is inhibited by a glucose injection at birth (Meister, R., Comte, J., Baggetto, L., G., Godinot, C. and Gautheron, D.C. (1983) Biochim. Biophys. Acta 722, 36-42). To test whether this improvement could be correlated to changes in cyclic nucleotides, the levels of cAMP and cGMP have been measured during the 2 h following birth. At birth, a short rise followed by a decrease of cAMP occurs, then a significant increase of cAMP level is observed between 45 min and 2 h. The cAMP level for animals injected at birth with glucose is lower than for control animals at each time studied. The cGMP level is not significantly affected in control animals, while in glucose-treated animals a significant decrease of cGMP is observed in the postnatal 2 h. The present work shows also that the glucose-induced inhibition of mitochondrial maturation is mimicked by injection at birth of either 8-Br-cGMP or nitroprusside. The latter transiently increases intracellular cGMP. In contrast, the glucose-induced inhibition is prevented by the injection at birth of either dbcAMP or alkylxanthines together with glucose (Comte, J., Meister, R., Baggetto, L.G., Godinot, C. and Gautheron, D.C. (1986) Biochem. Pharmacol. 35, 2411-2416). It is concluded that the postnatal improvement of mitochondrial functions is stimulated by cAMP and inhibited by cGMP, and that glucose-induced inhibition of the maturation is at least partly supported by a decrease in cAMP but not correlated to an increase in cGMP.

Adenosine Diphosphate↗
PubMed1988

IF1 inhibition of mitochondrial F1-ATPase is correlated to entrapment of four adenine- or guanine-nucleotides including at least one triphosphate.

This paper demonstrates that the inhibition of F1 ATPase activity by the natural inhibitor protein IF1 is correlated to triphosphate nucleotide entrapment in F1. The complete balance of nucleotides bound after preincubation with Mg-[alpha-32P]GTP or Mg-[alpha-32P]ATP, used to promote IF1 inhibition, has been established on purified F1 containing 0.7 mol of non-exchangeable endogenous nucleotides. As many as 4 mol of labelled guanine- or adenine- nucleotides are trapped in F1; at least one of these nucleotides is a triphosphate. On the contrary, in the absence of IF1, no triphosphate nucleotide is significantly retained and the diphosphate nucleotides bound are mainly exchangeable.

Adenine Nucleotides↗
PubMed1988

Evidence from immunological studies of structure-mechanism relationship of F1 and F1F0.

Monoclonal and polyclonal antibodies directed against peptides of F1-ATPase of F1F0-ATPase synthase provide new and efficient tools to study structure-function relationships and mechanisms of such complex membrane enzymes. This review summarizes the main results obtained using this approach. Antibodies have permitted the determination of the nature of subunits involved in the complex, their stoichiometry, their organization, neighboring interactions, and vectorial distribution within or on either face of the membrane. Moreover, in a few cases, amino acid sequences exposed on a face of the membrane or buried inside the complex have been identified. Antibodies are very useful for detecting the role of each subunit, especially for those subunits which appear to have no direct involvement in the catalytic mechanism. Concerning the mechanisms, the availability of monoclonal antibodies which inhibit (or activate) ATP hydrolysis or ATP synthesis, which modify nucleotide binding or regulation of activities, which detect specific conformations, etc. brings many new ways of understanding the precise functions. The specific recognition by monoclonal antibodies on the beta subunit of epitopes in the proximity of, or in the catalytic site, gives information on this site. The use of anti-alpha monoclonal antibodies has shown asymmetry of alpha in the complex as already shown for beta. In addition, the involvement of alpha with respect to nucleotide site cooperativity has been detected. Finally, the formation of F1F0-antibody complexes of various masses, seems to exclude the functional rotation of F1 around F0 during catalysis.

Antibodies↗
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↗