D.C. Gautheron

Vesicular preparation of a highly coupled ATPase-ATP synthase complex from pig heart mitochondria.

1. A method is described to prepare an ATPase-ATP synthase complex from pig heart mitochondria exhibiting a very high ATP-32Pi exchange activity (1.6 mumol/min per mag protein in optimal conditions). 2. The preparation is virtually devoid of nucleoside diphosphokinase and adenylate kinase activities. 3. Freeze-fracture studies show that the ATPase-ATP synthase complex is integrated in lipid vesicles of 400-600 A in diameter. 4. It contains the endogenous natural proteic inhibitor which seems to behave as a coupling factor. 5. The rate of ATP hydrolysis catalyzed by the ATPase-ATP synthase complex is competitively inhibited by ADP, while the presence of ADP increases the initial rate of 32Pi incorporation into ATP. 6. The 32Pi incorporation into ATP can occur at a rate almost equal to that of nucleoside triphosphate (NTP) hydrolysis provided that the rate of NTP hydrolysis is kept low and that the ADP concentration is high enough. In these conditions, a very high coupling between NTP hydrolysis and ATP synthesis can be demonstrated.

Heart mitochondrial creatine kinase solubilization: Effect of mitochondrial swelling and SH group reagents

Abstract The influence of mitochondrial swelling on the binding of creatine kinase to the inner membrane of pig heart mitochondria has been studied. Creatine kinase is not solubilized in isotonic mediums such as 250 m m sucrose or 125 m m KCl which do not induce mitochondrial swelling; however, it is solubilized when mitochondria swell in 100 m m potassium phosphate. This latter effect is suppressed when swelling is prevented by respiratory inhibitors or mersalyl. In hypotonic mediums creatine kinase is strongly released only when permeant anions are present; despite a high degree of swelling creatine kinase dissociation is minimal in 25 m m sucrose or KCl. Solubilization may also be obtained when swelling is induced by P j , accumulation inside mitochondria or when, after an osmotic shock in distilled water, they are resuspended in potassium phosphate or chloride but not in sucrose. Creatine kinase may be strongly released in isotonic KCl but not in sucrose when mitochondria are treated with parahydroxymercuribenzoic acid which induces swelling in the former medium (by allowing Cl − entry). A similar effect was obtained with other organic mercurials (parachloromercuriphenyl sulfonic acid, mersalyl) but not with disulfides (5,5′-dithiobis(2-nitrobenzoic acid), 6,6′-dithiodinicotinic acid, 2,2′-dithiodipyridine). Mercurials are able to solubilize creatine kinase from mitochondria suspended in distilled water so it is apparent that they have a direct effect on the association of the enzyme with the mitochondrial inner membrane. These results show that mitochondrial swelling is a prerequisite but not a sufficient condition for creatine kinase dissociation: the presence of ions or mercurials seems necessary for an efficient creatine kinase Solubilization.

Rabbit heart mitochondrial hexokinase: Solubilization and general properties

In rabbit heart, results show that two isoenzymes of hexokinase (HK) are present. The enzymatic activity associated with mitochondria consists of only one isoenzyme; according to its electrophoretic mobility and its apparent Km for glucose (0.065 mM), it has been identified as type I isoenzyme. The bound HK I exhibits a lower apparent Km for ATPMg than the solubilized enzyme, whereas the apparent Km for glucose is the same for bound and solubilized HK. Detailed studies have been performed to investigate the interactions which take place between the enzyme and the mitochondrial membrane. Neutral salts efficiently solubilize the bound enzyme. Digitonin induces only a partial release of the enzyme bound to mitochondria; this result could be explained by the existence of contacts between the outer and the inner mitochondrial membranes [C. R. Hackenbrock (1968) Proc. Natl. Acad. Sci. USA 61, 598-605]. Furthermore, low concentrations (0.1 mM) of glucose 6-phosphate (G6P) or ATP4- specifically solubilize hexokinase. The solubilizing effect of G6P and ATP4-, which are potent inhibitors of the enzyme, can be prevented by incubation of mitochondria with Pi or Mg2+. In addition, enzyme solubilization by G6P can be reversed by Mg2+ only when the proteolytic treatment of the heart homogenate is omitted during the course of the isolation of mitochondria. These results concerning the interaction of rabbit heart hexokinase with the outer mitochondrial membrane agree with the schematic model proposed by Wilson [(1982) Biophys. J. 37, 18-19] for the brain enzyme. This model involves the existence of two kinds of interactions between HK and mitochondria; a very specific one with the hexokinase-binding protein of the outer mitochondrial membrane, which is suppressed by glucose 6-phosphate, and a less specific, cation-mediated one.

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.

Dissociation and reassociation of creatine kinase with heart mitochondria; pH and phosphate dependence.

Abstract Experimental evidence is given of mitochondrial creatine kinase ability to dissociate from or reassociate with mitochondrial membrane as compared to the behaviour of adenylate kinase. CK release occurs for P i concentrations higher than 5 mM and is strongly pH-dependant. Solubilized CK is able to reassociate with mitochondrial inner membrane when either P i concentration or pH are decreased. The possible physiological effects of events, such as ischemia, which modify the intracellular pH or P i concentration are discussed, in view of the special role which has been attributed to mitochondrial CK in the transfer of energy in heart cells.

Competition between ADP and nucleotide analogues to occupy regulatory site(s) related to hysteretic inhibition of mitochondrial F1-ATPase

Abstract The regulatory site(s) responsible for ADP-induced hysteretic inhibition of pig heart mitochondrial F 1 -ATPase appeared to be specific of adenine nucleotides. The site(s) cannot be readily occupied by guanosine analogues although GTP is hydrolyzed at the catalytic sites. The length of the phosphate chain must be that of a nucleoside-diphosphate. Adenosine β,γ-imidotriphosphate, dialdehyde derivative of ADP also bind to the site(s) while ribosering opened analogues do not. It is also demonstrated that saturation of only one site, specifically by ADP, might be sufficient to induce hysteretic inhibition. However it cannot be excluded that other site(s), less specific, must also be saturated by nucleotides to permit ADP-inhibitory effects.

On the amino acids involved in the ATPase site of mitochondrial F1 and the implication of the subunit C

Abstract The data on the pH dependence of the K m for Mg-ATP and the V m of the ATPase of pig heart mitochondrial F 1 indicate the presence of two groups of different p K s which modify the enzyme activity. The first p K at pH 9.6 ± 0.2 may be related to the possible presence of arginine and/or tyrosine residues in the ATPase site; the second p K at pH 7.2 ± 0.2 could be due to the presence of a histidine residue in the ATPase site or to the involvement of amino groups in the ATPase site. The inhibition induced by photooxidation in the presence of Rose Bengal is not pH dependent in the pH range corresponding to the p K of histidine. The inhibition induced by diethylpyrocarbonate cannot be reversed by hydroxylamine and the characteristics of this inhibition rather correspond to the reaction of the inhibitor with amino groups. Pyridoxal phosphate also inhibits the ATPase activity of F 1 by reaction with amino groups. The presence of ATP or phosphate partially protects against the inhibition induced by diethylpyrocarbonate or pyridoxal phosphate, which indicates that amino groups may be directly or indirectly involved in the binding of nucleotide and phosphate to F 1 . Glutaraldehyde also inhibits the enzyme by reacting with amino groups and inducing a crosslinking of the subunits. The disappearance of subunit C is well correlated with the decrease of ATPase activity, indicating that subunit C is essential in the ATPase activity.

Effects of SH group reagents on creatine kinase interaction with the mitochondrial membrane

Solubilization of the specific mitochondrial isoenzyme of creatine kinase (CKm) from rabbit heart mitochondria by treatment with SH group reagents has been studied. From the various compounds tested only the negatively charged organomercurials are able to induce an extensive solubilization of the enzyme. This effect is fully reversible since the solubilized enzyme readily reassociates with the membrane when the bound organomercurial is removed by treatment of the homogenate by an excess of dithiothreitol. Solubilization by negatively charged organomercurials can be partly prevented by pretreatment of mitochondria with either disulfide or uncharged organomercurials. No clear-cut relationship has been pointed out when the amount of SH titrated by various reagents has been compared with the extent of CKm solubilization. More detailed studies with para-chloromercuribenzoate (pCMB) show that extensive CKm solubilization (about 75%) occurs for pCMB concentration as low as 25 microM, whereas pronounced inhibition of the enzyme is observed only for concentrations greater than 200 microM. By cross-reassociation of enzyme solubilized either by para-hydroxymercuribenzoate (pHMB) or by 20 mM sodium phosphate (NaPi) with mitochondria depleted of CKm by pHMB or by NaPi treatment, SH groups whose titration impedes CKm reassociation with the mitochondrial membrane have been tentatively located on the enzyme. Thus, negatively charged organomercurials, could induce a reversible conformational modification of the enzyme which is no longer able to bind on the inner mitochondrial membrane. Furthermore, our results show that the binding of an excess of mitochondrial CK, which has been previously reported, could reflect unspecific binding since it occurs only on mitoplasts incubated in very hypotonic medium, but not in isotonic medium.

Masking of co-operativity of nucleotide sites in pig heart mitochondrial ATPase (F1) by heating

Although the soluble mitochondrial ATPase (F,) [l] is an enzyme complex constituted of 5 polypeptides with different stoichiometry, most studies report linear Michaelis and Menten kinetics [2-41 for ATP hydrolysis and its inhibition by ADP. Kinetic studies did not reveal several nucleotide sites in purified F,-ATPases** except in the case of rat liver ATPase by Ebel and Lardy [S] who observed a concave curvature of reciprocal plots of initial velocity with MgATP as the variable substrate, in the absence of other anions. In a previous paper [6], we applied the Senior and Brooks procedure [7] to purify pig heart mitochondrial ATPase (Fi). The resulting preparation behaved mainly as that of beef heart in other laboratories (cf reviews from Senior [8], Pedersen [9] and Penefsky [lOI 1. The present work shows that with soluble pig heart mitochondrial ATPase (F,), prepared without the heating step, interaction between ATP and ADP sites can be demonstrated by kinetic studies: reciprocal plots of initial velocity with ATP as the variable substrate, become curved with increasing ADP concentrations (Hill coefficient from 1 to 2.1). This

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.