Inhibition of the vacuolar ATPase of Acer pseudoplatanus cells by vanadate.

Unlike most tonoplast ATPases, the vacuolar ATPase of Acer pseudoplatanus cells (Km = 0.4 mM) was strongly inhibited by vanadate (I50 = 10 microM). The inhibition was non-competitive. Chemicals usually added in the reaction mixture either increase (NH+4, K+) or decrease (Na+, EDTA) the ATPase inhibition. However, these results do not explain the insensitivity to vanadate of most tonoplast ATPases. We suggest that the tonoplast contains 2 classes of ATPases, one sensitive to vanadate, the other insensitive; each class should be more or less abundant (or active) according to the plant species studied or its physiological state of growth. It appears from this study that sensitivity or insensitivity of an ATPase to vanadate is not really a good criterion to distinguish between plasmalemma and tonoplast.

Involvement of tyrosyl residues in the structure-function relationships of D-beta-hydroxybutyrate dehydrogenase: a phospholipid-requiring enzyme.

The involvement of tyrosyl residues in the function of D-beta-hydroxybutyrate dehydrogenase, a lipid-requiring enzyme, has been investigated by using several tyrosyl modifying reagents, i.e., N-acetylimidazole, a hydrophilic reagent, and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole and tetranitromethane, two hydrophobic reagents. Modification of the tyrosyl residues highly inactivates the derived enzyme: Treatment of the enzyme with 7-chloro-4-nitro[14C]benzo-2-oxa-1,3-diazole leads to an absorbance at 380 nm and to an incorporation of about 1 mol of 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole per polypeptide chain for complete inactivation. Inactivation by N-acetylimidazole induces a decrease in absorbance at 280 nm which can be reversed by hydroxylamine treatment. On the other hand, the ligands of the active site, such as methylmalonate, a pseudosubstrate, and NAD+ (or NADH), do not protect the enzyme against inactivation. In contrast, the presence of phospholipids strongly protects the enzyme against hydrophobic reagents. Finally, previous modification of the enzyme with N-acetylimidazole does not affect the incorporation of 7-chloro-4-nitro[14C]benzo-2-oxa-1,3-diazole while modification with tetranitromethane does. These results indicate the existence of two classes of tyrosyl residues which are essential for enzymatic activity, and demonstrate their location outside of the active site. One of these residues appears to be located close to the enzyme-phospholipid interacting sites. These essential residues may also be essential for maintenance of the correct active conformation.

The essential cationic charge of phospholipid polar head in the reactivation of D-beta-hydroxybutyrate apodehydrogenase revealed by cationic surfactants.

Attempts to reactivate purified D-beta-hydroxybutyrate apodehydrogenase, a lecithin-requiring enzyme, have been carried out using neutral, anionic, cationic and zwitterionic surfactants. Cationic and zwitterionic compounds exclusively are able to partially replace phosphatidylcholine, the reactivating phospholipid. The extent of reactivation depends on the steric hindrance of the polar head and on the hydrophobic tail length. A molecule bearing a positive charge and an aliphatic chain is the sole structure absolutely required for activity. However the presence of a negative charge is important for enzyme binding to amphiphilic structures and for the efficiency of reactivation.

Evidence for the presence of one carboxyl group in the catalytic center of D-beta-hydroxybutyrate dehydrogenase. Inactivation and binding studies with carbodiimide reagents.

D-beta-Hydroxybutyrate dehydrogenase D-3-hydroxybutyrate: NAD+ oxidoreductase, EC 1.1.1.30), a phosphatidylcholine-requiring enzyme, was irreversibly inactivated by a water-soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC) or a hydrophobic carbodiimide, N,N'-dicyclohexylcarbodiimide (DCCD). The inactivation is pseudo-first-order with a kinetic stoichiometry of about 1. Phospholipid-free apoenzyme was more sensitive towards these reagents than reconstituted phospholipid-enzyme or membrane-bound enzyme forms. Reduced coenzyme (NADH) protected the enzyme against the inactivation, while oxidized coenzyme (NAD+) in presence of 2-methylmalonate (a pseudo-substrate) gave a better protection. It was found that the phospholipid-free apoenzyme bound about 1 mol [14C]DCCD. This incorporation was prevented by EDAC, indicating that both reagents react at the same site. [14C]Glycine ethyl ester, a nucleophilic compound which reacts specifically with the carboxylcarbodiimide derivative was incorporated to the enzyme (1 mol [14C]glycine ethyl ester per polypeptide chain), whatever its form, in the presence of DCCD or EDAC. These results indicate the presence of one carboxyl group probably located at or near the coenzyme-binding site and near the interacting domain of the enzyme with phospholipid.

Limited proteolysis of D-beta-hydroxybutyrate dehydrogenase: relationships between phospholipid-protein interactions and catalytical activity.

Different forms of D-beta-hydroxybutyrate dehydrogenase were submitted to various proteases in order to get information on enzyme molecular structure and on phospholipid -enzyme interaction. Except for leucinaminopeptidase, all proteases tested inactivated the phospholipid-free enzyme, while no inactivation was observed with the lecithin-enzyme complex. However, non-reactivating phospholipid gave very poor protection against proteases. After endopeptidase treatment, a new band of 25,000 Mr appeared instead of the 32,000 Mr band (apodehydrogenase). Surprisingly, the so-called protected form of the enzyme (lecithin-complex) was also proteolysed while still enzymatically active. Carboxypeptidase A inactivated quite thoroughly the enzyme although the 32,000 Mr band appeared unaffected. These results demonstrate that the configuration of the phospholipid-free apodehydrogenase is quite vulnerable to most proteases, in contrast to the configuration of the lecithin-complexed enzyme. The N-terminal end is probably blocked while the C-terminal end looks quite important for enzymatic activity.

[Enzymatic and immunologic study of the role of the thyroid hormone in the formation of the internal mitochondrial membrane during postnatal development of the rat]. / Etude enzymatique et immunologique du rôle de l'hormone thyroïdienne sur la genèse de la membrane interne mitochondriale au cours du développement post-natal chez le rat.

Hypothyroidism induces an increase of liver D-beta-hydroxybutyrate dehydrogenase activity. Injection of thyroid hormone reverses the phenomena. The use of monospecific antibody raised against the purified enzyme indicates that there was not an increase of apoenzyme biosynthesis. The thyroid hormone negative control is due to a metabolism alteration of the membrane phospholipids which are directly involved in the apoenzyme activity. The highest difference is observed with 20 days old rats. Opposite effects were obtained on succinate cytochrome c reductase.

[Quantitative approach to the interactions of D-beta-hydroxybuturate dehydrogenase with phospholipids]. / Approche quantitative des interactions de la D-beta-hydroxybutyrate déshydrogénase avec les phospholipides.

Depending on the basis of the technique used, an estimation of 2 to 6 lecithin molecules are required for reactivation of purified D-beta-hydroxybutyrate apodehydrogenase (apoBDH). 60 to 70 mitochondrial phospholipid molecules are necessary for insertion of one BDH polypeptide chain where 16 molecules would be in direct interaction with the enzyme. Results show off the cofactor role of lecithin and also the requirement of an amphiphilic environment, essential for the enzyme function.

[Sensitivity of D-beta-hydroxybutyrate dehydrogenase to proteolytic enzymes; influence of phospholipids]. / Sensibilité de la D-bêta-hydroxybutyrate déshydrogénase aux enzymes protéolytiques; influence des phospholipides.

Controlled proteolysis of D-beta-hydroxybutyrate dehydrogenase in different forms were carried out using several proteases with different and well known specificities. The results obtained were the following: Purified apoBDH (phospholipid-free) was rapidly and strongly inactivated by all proteases tested except leucine aminopeptidase , in contrast with non-membrane enzymes which were unaffected by all proteases. BDH activity was completely preserved when proteases were incubated with either native BDH (membrane linked) or reconstituted BDH with reactivating-phospholipids (lecithins of total mitochondrial phospholipids), while non-reactivating-phospholipids gave no protection against proteases. C-terminal part of the enzyme was found to be essential for enzymatic activity while the N-terminal aminoacid is N-substituted. Controlled proteolysis whatever the protease used (except leucine aminopeptidase ) was followed by strong inactivation of the enzyme.

[Structural requirements of mitochondrial D-beta-hydroxybutyrate dehydrogenase with respect to its coenzyme]. / Exigences structurales de la D-beta-hydroxybutyrate déshydrogénase de mitochondries vis-à-vis du coenzyme.

The purpose of this work was to test structural analogs of NAD+ in order to know enzyme requirements of chemical structure of coenzyme to get catalytic activity and, in a other hand to see which chemical parts of the coenzyme were involved in the coenzyme binding to the active site. The binding of the coenzyme analog at the catalytic site requires an adenosine diphosphoribose structure without any additional phosphate group on the ribose linked to adenine.

Evidence for the existence of two classes of sulfhydryl groups essential for membrane-bound succinate dehydrogenase activity.

Kinetics of the inhibition of activated membrane-bound dehydrogenase by N-substituted maleimides were studied. Three maleimide derivatives having a different hydrophobic character (N-ethyl-, N-butyl-, and N-benzylmaleimide) were tested. The method developed by Ray & Koshland (Ray, W. J., Jr., & Koshland, D. E., Jr. (1961) J. Biol, Chem. 236, 1973-1979) was used for analyzing experimental data. The results showed that two classes of sulfhydryl groups, with quite different reactivities, were essential for catalytic activity. The most reactive sulfhydryl groups were located in the substrate site as revealed by the fact that they were protected against alkylation in the presence of succinate or a competitive inhibitor, malonate. However, ligands of the catalytic site did not completely prevent inactivation of succinate dehydrogenase. Analysis of the kinetics of the inhibition observed in the presence of substrate indicated that the slow-reacting sulfhydryl groups did not belong to the active site. Rate constant values of the reaction of each set of sulfhydryl groups with the three maleimide derivatives showed that the most reactive thiols were probably located in a hydrophobic microenvironment since alkylation of this set of sulfhydryl groups was sensitive to the hydrophobic character of the thiol reagent. The reactivity of the other class of sulfhydryl groups was not influenced by the nature of the substituent. When the enzyme was deactivated by oxaloacetate, the two classes of sulfhydryl groups became unreactive with the alkylating agents. Masking of these groups may reflect a conformational change of the enzyme.

[Reactivity and specificity of alpha-dicarbonyl compounds towards essential aminoacyl residues of D-beta-hydroxybutyrate dehydrogenase from the inner mitochondrial membrane]. / Réactivité et spécificité de composés alpha-dicarbonylés vis-à-vis de résidus aminoacyl essentiels à l'activité de la D-beta-hydroxybutyrate déshydrogénase de la membrane interne mitochondriale.

The effects of a alpha-dicarbonyl chromophoric reagent: 4-hydroxy-3-nitrophenylglyoxal on the D-beta-hydroxybutyrate dehydrogenase have been compared to those of phenylglyoxal, a specific arginyl reagent in proteins. Both reagents inactivate irreversibly the enzyme. Kinetic experiments show that only one molecule of these reagents per molecule of enzyme is sufficient to inactivate the enzyme. The second order inactivation rate constant is more than 500 times higher with the chromophoric reagent than with phenylglyoxal. A pseudosubstrate (methylmalonate) in presence of coenzyme (NAD) strongly protects enzyme against inactivation by both reagents. Coenzyme alone has no effect on inactivation by phenylglyoxal while it protects whether inhibitor is the chromophoric reagent or N-ethylmaleimide: a thiol specific reagent. These results indicate: 1. That one arginyl residue is essential for D-beta-hydroxybutyrate dehydrogenase activity (experiments with phenylglyoxal). 2. That the presence of a nitro group on position 3 and a hydroxyl-group on position 4 strongly increase the reactivity of the alpha-dicarbonyl groups, but the specificity of the chemical reaction with arginyl residues seems to be lost for the benefit of cysteyl residues.

Influence of the energetic state of mitochondria on the inhibition of oxidative phosphorylation by N-ethylmaleimide.

N-Ethylmaleimide inhibitory effect on oxidative phosphorylation, adenylic nucleotide translocation, succinate dehydrogenase and succinoxidase activities was studied as a function of the energetic state of mitochondria. 1. Using a reversible thiol reagent (mersalyl), in order to protect the phosphate carrier against irreversible action of N-ethylmaleimide, it was found that: (a) when mersalyl-pretreated mitochondria were in a 'non-energized' state, i.e. preincubated without a substrate and in the presence of rotenone, only a slight inhibition of succinate oxidation coupled to ATP synthesis by N-ethylmaleimide was observed. (b) when mersalyl-pretreated mitochondria were in an 'energized' state, i.e. preincubated in the presence of an oxidizable substrate, N-ethylmaleimide strongly inhibited the coupled oxidation of succinate. 2. Mitochondrial energization was also shown to enhavce the inhibitory effect of N-ethylmaleimide on adenylic nucleotide translocation and succinoxidase activity. However, other sulphydrul groups seem to be involved in the inhibition mechanism, but their function is unknown. 3. As N-ethylmaleimide inhibitory effect increased, an enhancement of N-[14C]ethylmaleimide binding to mitochondrial sulphydryl groups was obtained.

Variations of D (-)-beta-hydroxybutyrate dehydrogenase activity of rat liver mitochondria during post-natal development.

1. D(-)-beta-hydroxybutyrate dehydrogenase specific activity of rat liver mitochondria changes during ontogenesis: at birth, the activity is low, then increases to a maximum at 12 days, decreases until 50 days to keep constant thereafter. At the same time, mitochondrial protein amount increases regularly while succinatecytochrome c reductase specific activity slightly increases after birth to keep constant afterwards. 2. The observed changes in activity of D(-)-beta-hydroxybutyrate dehydrogenase are not related to possible interactions between the enzyme and phospholids since addition of lecithin to mitochondria does not change the activity. 3. Electrophoresis of mitochondrial proteins isolated from rats at different development stages demonstrates the presence of a protein band characterized by the same electrophoretic mobility as beta-hydroxybutyrate dehydrogenase and by significative changes of its proportion during maturation: the relative amount of this protein increases from the new-born to the 10-12 days old rat, to decrease afterwards. 4. These findings may signify that the increased activity of the enzyme with a maximum at 10-12 days followed by a decrease is related to the rate of the enzymes biosynthesis.

Influence of the energetic state of rat liver mitochondria on the sensitivity of the phosphate carrier towards SH reagents.

Phosphate transport into rat liver mitochondria was measured by the swelling technique in 0.1 M ammonium phosphate. Energized or non-energized mitochondria were preincubated with different thiol reagents and evidence is given that with a slow-reacting thiol reagent, ethacrynate, the inactivation of the phosphate carrier is obtained when mitochondria are energized, while poor or no inactivation occurs when mitochondria are non-energized or preincubated with Pi. Moreover, the inactivation depends on the presence of Mg2+ and on the nature of the substrate. Some comparative essays were done using N-ethylmaleimide as a thiol reagent, but no energy-linked variation of N-ethylmaleimide inhibition on phosphate transport was obtained. Taking into account the fact that both thiol-reagents incorporation into rat liver mitochondria is sitmulated by the presence of substrate, the different behaviour of these two thiol-reagents towards Pi transport is discussed on the basis of their different reactivity with SH groups.

[Demonstration of the variations in accessibility or reactivity of mitochondrial SH groups according to the energy status of the mitochondria]. / Mise en évidence des variations d'accessibilité ou de réactivité des groupes SH mitochondriaux suivant l'état énergétique des mitochondries.

Evidence for energy dependent variations of mitochondrial SH groups accessibility or reactivity was obtained using alkylating agents. These variations are thought to be consecutive to conformational changes affecting some proteins among them the phosphate carrier and the adenylic nucleotide translocator.

Thiols related to mitochondrial ATPase and transports: unmasking upon conformational changes supported by the comparative effects of ethacrynate and dihydroethacrynate.

Comparison between the effects on various rat liver mitochondrial functions of ethacrynate, a thiol reagent inhibitor of oxidative phosphorylations [3, 4] and those of dihydroethacrynate its saturated derivative which is not a thiol reagent, has been performed. Both, ethacrynate and dihydroethacrynate increase oxygen consumption by mitochondria in state 4 (succinate as substrate) in a concentration dependent way (from 1 to 5 X 10(-4) M EA or DHEA). This activation is followed, only with ethacrynate, by an inhibition appearing sooner with higher concentrations. After preincubation or mitochondria with ethacrynate (1 to 5 X 10(-4) M), the stimulation of respiration by (ADP + Pi) is completely inhibited whereas it is only weakly affected by dihydroethacrynate at the same concentrations. Ethacrynate and dihydroethacrynate provoke variations of intramitochondrial Mg2+ and K+ levels which need energy from the respiratory chain. These are affected by Pi or (Pi + ADP) in a different way with ethacrynate and with dihydroethacrynate. After preincubation with mitochondria, ethacrynate and to a smaller extent dihydroethacrynate, inhibit partially ADP translocation; ADP increases the inhibitory effect of EA on translocation and not that of dihydroethacrynate. Ethacrynate increases the oligomycin sensitive ATPase activity and dihydroethacrynate still more. After a ten minutes preincubation with mitochondria, ethacrynate and dihydroethacrynate hardly affect the 2.4 DNP stimulated ATPase activity. Preincubation with succinate or ADP strongly increases the ethacrynate inhibition whereas it decreases dihydroethacrynate inhibition. Ethacrynate and dihydroethacrynate do not affect the efflux of Pi produced by ATP hydrolysis but ethacrynate enforces the inhibitory effect of mersalyl (Mg2+ containing medium). After ten minutes of preincubation with mitochondria, ethacrynate binds 25 nmoles of -SH/mg protein (DTNB titration) and dihydroethacrynate has no effect. These results show an effect of ethacrynate on two types of thiols linked with energy conservation mechanisms and ADP translocation. These thiols could be unmasked or made accessible by conformational modifications of the inner membrane upon energization or addition of ADP.