Giuseppe Capozza

ATPase activity, IF1 content, and proton conductivity of ESMP from control and ischemic slow and fast heart-rate hearts.

Earlier studies by Rouslin and coworkers showed that, during myocardial ischemia in slow heart-rate species which include rabbits and all larger mammals examined including humans, there is an IF1-mediated inhibition of the mitochondrial ATPase due to an increase in the amount of IF1, bound to the ATPase (Rouslin, W., and Pullman, M.E.,J. Mol. Cell. Cardiol.19, 661–668, 1987). Earlier work by Guerrieri and colleagues demonstrated that IF1 binding to bovine heart ESMP was accompanied by parallel decreases in ATPase activity and in passive proton conduction (Guerrieri, F.,et al., FEBS Lett.213, 67–72, 1987). In the present study rabbit was used as the slow heart-rate species and rat as the fast heart-rate species. Rat is a fast heart-rate species that contains too little IF1 to down regulate the ATPase activity present. Mitochondria were prepared from control and ischemic hearts and ESMP were made from aliquots by sonication at pH 8.0 with 2 mM EDTA. Oligomycin-sensitive ATPase activity and IF1 content were measured in SMP prepared from the control and ischemic mitochondrial samples. After identical incubation procedures, oligomycin-sensitive ATPase activity, oligomycin-sensitive proton conductivity, and IF1 content were also measured in ESMP samples. The study was undertaken to corroborate further what appear to be fundamental differences in ATPase regulation between slow and fast heart-rate mammalian hearts evident during total myocardial ischemia. Thus, passive proton conductivity was used as an independent measure of these regulatory differences. The results show that, consistent with the low IF1 content of rat heart cardiac muscle mitochondria, control rat heart ESMP exhibit approximately twice as much passive proton conductivity as control rabbit heart ESMP regardless of the pH of the incubation and assay. Moreover, while total ischemia caused an increase in IF1 binding and a commensurate decrease in passive proton conductivity in rabbit heart ESMP regardless of pH, neither IF1 content nor proton conductivity changed significantly in rat heart ESMP as a result of ischemia.

Age-dependent changes in the mitochondrial F0F1 ATP synthase

The age dependence of ATP hydrolase activity and oligomycin sensitive passive proton conduction in sonicated submitochondrial particles of rat brain and rat heart has been investigated. The results show an increase of Vmax of the ATP hydrolase activity and decrease of oligomycin sensitive passive proton conduction with increase of the age of rats from 3 to 6 months. Decrease of ATPase activity and increase of oligomycin sensitive proton conduction occur with further aging to 24 months. Immunoblot analysis shows that both the F(1) and F(0) contents of mitochondria vary with the age of rats, the former exhibiting relatively larger changes.

CORRELATION BETWEEN RAT LIVER REGENERATION AND MITOCHONDRIAL ENERGY METABOLISM

The time course of changes in mitochondrial energy metabolism during liver regeneration, following partial hepatectomy, is analyzed. For 24 h after surgical operation, a lag phase in the time course of the growth of liver is observed. In this period mitochondria showed a decrease of: (1) the respiratory control index; (2) the rate of oxidative phosphorylation; (3) the amount of immunodetected beta-F1 and F01-PVP subunits of F0F1-ATP synthase. No decrease, but instead a small increase in the content of mRNA for beta-F1 was observed in this phase. After this lag phase the growth of liver started, the content of mRNA for beta F1, as well as the level of immunodetected mitochondrial beta-F1 and F01-PVP subunits, increased and oxidative phosphorylation recovered. Analysis of the relative beta F1 protein/mRNA ratio indicates a decrease of beta F1 translational efficiency which remained low up to 72 h after partial hepatectomy and reached the same ratio of control at 96 h. It is concluded that the regenerating capability of rat liver is correlated with the efficiency of oxidative phosphorylation.

The γ subunit of F1 and the PVP protein of Fo (FoI) are components of the gate of the mitochondrial FoF1 H+-ATP synthase

The γ subunit of the F1 moiety of the bovine mitochondrial H+‐ATP synthase is shown to function as a component of the gate. Addition of purified γ subunit to Fo‐liposomes inhibits transmembrane proton conduction. This inhibition can be removed by the bifunctional thiol reagent diamide. Immunoblot analysis shows that the diamide effect is likely due to disulphide bridging of the γ subunit with the PVP protein of the Fo sector.

Mitochondrial F0F1 H+-ATP synthase. Characterization of F0 components involved in H+ translocation.

The membrane F0, sector of mitochondrial ATP synthase complex was rapidly isolated by direct extraction with CHAPS from F1‐depleted submitochondrial particles. The preparation thus obtained is stable and can be reconstituted in artificial phospholipid membranes to result in oligomycin‐sensitive proton conduction, or recombined with purified F1 to give the oligomycin‐sensitive F0F1‐ATPase complex. The F0 preparation and constituent polypeptides were characterized by SDS‐polyacrylamide gel electrophoresis and immunoblot analysis. The functional role of F0 polypeptides was examined by means of trypsin digestion and reconstitution studies. It is shown that, in addition to the 8 kDa DCCD‐binding protein, the nuclear encoded protein [(1987) J. Mol. Biol. 197, 89–100], characterized as an intrinsic component of F0, (F0I, PVP protein [(1967) J. Biol. Chem. 242, 2547–2551]) is involved in H+ translocation and the sensitivity of this process to the F0 inhibitors, DCCD and oligomycin.

Age related changes of the mitochondrial energy metabolism in rat liver and heart

The influence of aging on mitochondrial energy metabolism of rat liver and rat heart has been studied by analysis of (i) respiratory rate of succinate-supplemented mitochondria in state III (coupled state in the presence of ADP + Pi); (ii) the rate of synthesis of ATP in succinate-supplemented mitochondria; (iii) the ATP hydrolase activity of sonicated submitochondrial particles. The results indicate a decrease of the F(0)F(1)-ATP synthase activity in mitochondria isolated from both organs of aged (24-month-old) as compared to young (3-month-old) rats which was accompanied by a decrease of immunodetected amount of the beta-F(1) (subunit of the catalytic F(1) sector of F(0)F(1)-ATP synthase). These effects were more evident in heart than in liver mitochondria. Analysis of the mitochondrial content of glutathione (GSH), an essential intracellular antioxidant agent, shows a decrease in mitochondria of both tissues of aged animals. Exposure of submitochondrial particles to free radicals, produced either by (60)Co or by respirtory chain (in presence of the inhibitor antimycin A) mimicked the alterations of F(0)F(1) ATP synthase obsreved in submitochondrial particles of aged rats. The possible relationship between aging process, free radical production and alteration of mitochondrial oxidative phosphorylation is discussed.

Role of the carboxyl‐terminal region of the PVP protein (F0I subunit) in the H+ conduction of F0F1 H+‐ATP synthase of bovine heart mitochondria

By means of protein sequencing, labelling with thiol reagents and reconstitution studies it is shown that the carboxyl‐terminal region of the PVP protein (F0I subunit, nuclear‐encoded protein of M r 25000) of mitochondrial F0 promotes transmembrane proton conduction by F0 and the sensitivity of this process to oligomycin.

Activation of a complex of ATPase with the natural protein inhibitor in submitochondrial particles

Almost all ATPase molecules in submitochondrial particles, isolated from beef heart mitochondria in the presence of MgATP, are in an inactive complex with the natural protein inhibitor (IF1). In de‐energized particles at high ionic strength a slow and irreversible ATPase activation is found to occur due to a dissociation of the enzyme‐inhibitor complex. The pH‐dependence of this process points out that deprotonation of IF1 molecule is an essential step in the dissociation of the complex. Zn2+ sharply accelerates ATPase activation, probably via binding with the deprotonated form of IF1. ATPase activation is completely prevented by MgATP, indicating the formation of a transient enzyme‐inhibitor complex retaining ATPase activity

F0 and F1 Subunits Involved in the Gate and Coupling Function of Mitochondrial H+ ATP Synthasea

The FoFl H+-ATP synthase of mitochondria contains five or six supernumerary subunits in addition to the eight present in prokary~tes.l-~ Two of the subunits of the mitochondrial enzyme (ATPase 6 and A6L3) are encoded by the mitochondrial genome; the others are nuclear encoded. Different from the prokaryotic enzyme, which functions as both ATP synthase and hydrolase depending on the cultural conditions, the mitochondrial complex is made to function essentially as an ATP synthase.? The mitochondrial system also seems to adapt itself to the continuously changing energy demand of tissues in various physiopathological conditions (see F. Guerrieri et al., this volume). The peripheral catalytic sector of H+-ATP synthase of coupling membranes consists universally of five subunits (a3P3y&) (FIG. 1). The H+ translocating membrane sector, Fo, as well as conserved subunits a, c, and possibly b (Escherichia coli nomenclature), has in eukaryotes a variable number of supernumerary subunits. A critical region in the H+-ATP synthase seems to be represented by a stalk connecting the F1 headpiece to Fo.1,2 This structure is likely to contribute the ion filter, the gate, and the coupling device between H+ transport and hydroanhydro catalysis. In E. coli the stalk is apparently formed by segments of y, P, and b subunits (subunit c also seems to contribute to the binding of Fo to FI4) (FIG. 1). In mitochondria a larger number of subunits are involved in the functional connection of Fo to F1 (FIG. 1). In this respect a peculiar role is played by the inhibitor protein (IF,). This binds to the complexwhen aerobic AkH+ decreases below a critical value, with consequent inhibition of ATP hydr~lysis.~ This report deals with the subunits involved in the gate and coupling function of bovine heart H+-ATP synthase. In particular, the results of the role of the FJ-PVP subunit (considered by some authors to be analogous to E. coli subunit b3), F6 and OSCP of the membrane sector, F1-y, and IFI will be presented. The following experimental approaches were used: (1) Controlled proteolytic digestion of subunits, immunodetection of products, and amino acid sequencing; (2)

Effect of cetyltrimethylammonium on ATP hydrolysis and proton translocation in the F0-F1 H+-ATP synthase of mitochondria

The amphiphylic alkyl cation cetyltrimethylammonium inhibits the catalytic activity of soluble and membrane-bound F1 in a noncompetitive fashion. In sonic submitochondrial particles the Dixon plot showed a peculiar pattern with upward deviation at cetyltrimethylammonium concentration higher than 80µM. In membrane-bound F1 the inhibition by cetyltrimethylammonium was potentiated by the F0 inhibitor ologomycin. Cetyltrimethylammonium also inhibited the oligomycin-sensitive proton conductivity in F1-containing particles but was without any effect in F1-depleted particles. Also this inhibitory effect was potentiated by oligomycin. These results indicate functional cooperative interactions between F0 and F1.