Effect of physiological electron donors and acceptors on F1-ATPase.

The hydrolytic activity of F1-ATPase isolated from rat liver was enhanced in the presence of NADH, FADH2, QH2 or reduced cyt c. The extent of this activation depended largely on substrate concentration. F1-ATPase sensitivity to bicarbonate or dinitrophenol activators decreased in the presence of any of those electron donors, which originated as well a slight sensitivity to oligomycin and a sensitivity increase to the inhibitory anion OCN-. In the presence of oxidized carriers the sensitivity to bicarbonate, dinitrophenol, or OCN- was not modified, and the enzyme remained oligomycin insensitive.

Molecular species of phosphatidylcholine and phosphatidylethanolamine in subcellular membranes from rat liver.

Four subfractions of phosphatidycholine and phosphyatidylethanolamine according to the degree of unsaturation of their fatty acids have been separated from lipid extracts of microsomes, and inner and outer mitochondrial membranes. The predominant species found in the three membranes contained one saturated and one unsaturated fatty acid. In microsomes completely saturated species of both phosphatidylcholine and phosphatideylethanolamine were practically nonexistent. In outer mitochondrial membranes species with two unsaturated fatty acids were absent. In the inner mitochondrial membranes, however, disaturated species and those with two unsaturated fatty acids were found.

Effect of bicarbonate and other anions on the oxidized and reduced forms of F1-ATPase.

The effect of activating anions on the hydrolysis of ATP catalyzed by mitochondrial ATPase was higher on the oxidized than on the reduced form of the enzyme. On the contrary the effect of inhibitory anions on this reaction was more manifest on the reduced form of the enzyme. Kinetic data show that both activating and inhibitory anions compete for the same sites of the ATPase. A unifying mechanism of action is suggested according to which the anions could establish coordination bonds with the suggested iron atoms of the catalytic site. The preferential displacement of electrons of such bonds towards the ligand, or towards the metal atom, would lead respectively to an inhibition or to an activation of the enzyme.

Catalytic sites of mitochondrial ATPase with different properties. Effect of citrate, free ATP and ADP in the presence of dithionite.

The extent of stimulation of the hydrolytic activity of mitochondrial ATPase by the reducing agent dithionite has been found to depend on substrate concentration both for the membrane bound enzyme and for the isolated and purified F1ATPase. The results suggest the existence of three catalytic sites differing in their standard reduction potential. The activating effect of free ATP on the hydrolytic activity of rat liver F1-ATPase has been found to be more pronounced on the reduced form of the enzyme. On the contrary, the inhibitory effect of ADP was higher on the oxidized form of F1-ATPase. Citrate has also been found to be an inhibitor of F1-ATPase; its effect was more pronounced on the reduced form of the enzyme, and exhibited a competitive pattern of inhibition with respect to free ATP. The results obtained have been interpreted in the sense that free ATP and ADP may be modifying the standard reduction potential of the enzyme, and suggest the existence of three independent redox cycles in ATPase governed by the exchange of ADP and Pi for the newly synthesized ATP.

Effect of uncouplers and inhibitors of oxidative phosphorylation on the reduced and oxidized forms of mitochondiral ATPase.

A series of uncouplers and inhibitors of oxidative phosphorylation have been studied with regard to their effect on the hydrolytic activity of the reduced and oxidized forms of isolated or membrane-bound mitochondrial ATPase. Uncouplers (2,4-dinitrophenol, dicoumarol), which are also activators of the hydrolytic activity of ATPase, were more potent activators on the oxidized form of the enzyme. Inhibitors of oxidative phosphorylation (oligomycin, azide and amytal) had a more potent inhibitory effect on the hydrolytic activity of ATPase in its reduced form. Purified F1-ATPase, oligomycin insensitive in the oxidized form of the enzyme, became sensitive to oligomycin in the reduced form. An interpretation of the results suggests the presence of a mechanism that unifies the action of these different compounds on the synthesis and hydrolysis of ATP catalyzed by mitochondrial ATPase.

Reversible modification of rat liver F1-ATPase by a redox reaction.

F1-ATPase from rat liver mitochondria exhibited a change in properties when reduced by dithionite: an increase in activity together with a disappearance of its sensitivity to bicarbonate stimulation was observed. A complete reversion to the original properties was achieved with the oxidizing agent 2,6-dichlorophenolindophenol.

Rat liver mitochondrial F1-ATPase, an FAD containing ferroprotein.

F1-ATPase isolated from rat liver mitochondria has been found to contain approximately 1 mole of FAD and 6 g atoms of nonheme iron per mole of enzyme.

Effect of pH on the sensitivity of mitochondrial ATPase to free ATP, ADP and anions.

The pH activity curves of the hydrolyzing activity of ATPase obtained with rat liver mitochondria or with the isolated F1-ATPase at three different substrate concentrations exhibited different optimum pH values. At 0.06 mM ATPMg2+ maximal activity was reached at pH 7; at 0.67 mM ATPMg2+ the optimum value was around 8; and at 3 mM ATPMg2+ between pH 8.2 and 9. These results suggest the presence of different catalytic sites in the enzyme with different affinities for the substrate and different optimum pH values. The sensitivity to the activating anions dinitrophenol and bicarbonate decreased with increasing pH values; the decrease in the activating effect was sharper when approaching the optimum pH value at any of the three substrate concentrations tested. These results might indicate that either OH-, dinitrophenol, or bicarbonate could compete for a regulatory site or sites in ATPase. The activating effect of free ATP on the hydrolyzing activity of isolated F1-ATPase was found to be dependent on the pH of the medium. The activating effect was more pronounced above the optimum corresponding to each of the three ATPMg2+ concentrations used, whereas the inhibitory effect of ADP was more manifest at pH values below that optimum point.