The equilibrium between the mitochondrial ATPase (F1) and its natural inhibitor in submitochondrial particles

1. The reversible equilibrium between the mitochondrial ATPase (F₁) and its naturally occurring inhibitor in Mg-ATP submitochondrial particles has been studied under different conditions.2. High ionic strength favours dissociation of the ATPase inhibitor as tested by ATPase and ATP-driven transhydrogenase activities.3. Dissociation of the ATPase inhibitor results in an increased maximal velocity of the ATPase activity measured in the presence of uncoupler and an increased affinity for adenine nucleotides, in particular for ATP.4. Association of the ATPase inhibitor with inhibitor-depleted Mg-ATP particles causes a slowing of the initial rate of succinate oxidation.5. The antibiotic aurovertin stimulates the ATPase activity of Mg-ATP particles preinculbated in the presence of a supply of oxidative energy. Bound aurovertin impedes the association of inhibitor-deficient particles with ATPase inhibitor.6. The fluorescence of aurovertin bound to inhibitor-containing particles is much less than that of aurovertin bound to inhibitor-depleted particles.7. The oligomycin-sensitivity-conferring protein, added either alone or in the presence or absence of membranous components of the ATPase complex, has little or no effect on the fluorescence of the F₁-aurovertin complex.8. It is suggested that the ATPase inhibitor brings F₁ in a conformation denoted 1F₁ that binds aurovertin with a low quantum yield, a decreased affinity and an increased binding capacity.     

Effects of quercetin and F1 inhibitor on mitochondrial ATPase and energy-linked reactions in submitochondrial particles

Quercetin (3,3′,4′,5,7-pentahydroxyflavone) shares certain properties with the mitochondrial ATPase inhibitor protein. At low concentrations it inhibits both soluble and particulate mitochondrial ATPase and has no effect on oxidative phosphorylation in submitochondrial particles. Unlike the mitochondrial inhibitor protein quercetin inhibits the ATP-dependent reduction of NAD⁺ by succinate in fully reconstituted submitochondrial particles. A comparison of various flavones indicates that the hydroxyl groups at the 3′ and perhaps 3 position are important for the inhibition of ATPase activity.     

The interaction between the mitochondrial ATPase (F1) and the ATPase inhibitor

1. The naturally occurring mitochondrial ATPase inhibitor inhibits the mitochondrial ATPase (F₁) non-competitively.2. The interaction between inhibitor and inhibitor-depleted F₁ or submitochondrial particles is diminished when the ratio of ATP/ADP is low or when energy is generated by substrate oxidation.3. The dissociation of the inhibitor from coupled Mg-ATP particles is promoted when substrates are being oxidized. This results in the appearance of a large uncoupler-stimulated ATPase activity. Activation of the uncoupler-stimulated ATPase activity is also achieved by incubation of the particles with ADP.4. The ATPase activity of Mg-ATP particles is determined by the turnover capacity of F₁. When endogenous inhibitor is removed, energy dissipation becomes the rate-limiting step. This energy dissipation can be activated by an uncoupler.5. Evidence is presented for the existence of a non-inhibited intermediate F₁-inhibitor complex.     

Interaction of aurovertin with submitochondrial particles,deficient in ATPase inhibitor

1. Binding of aurovertin to submitochondrial particles deficient in ATPase inhibitor is accompanied by an enhancement of the fluorescence by at least 100-fold.2. This change in fluorescence proceeds in three phases. The slowest change may be due to a conformational change in F₁, induced by the antibiotic bound during the rapid phases, giving rise to an increase in the quantum yield of the bound fluorochrome.3. Phosphate and ATP quench the fluorescence of the particle-aurovertin complex and ADP enhances it; the rate and extent of these changes are dependent on the availability of free Mg²⁺.4. There is at least one binding site on the submitochondrial particles, where ATP, ADP and phosphate can bind reversibly and for which these ligands compete. These interactions are dependent on the availability of free Mg²⁺ and are partly sensitive to oligomycin.5. Binding studies reveal two binding sites for aurovertin on inhibitor-free particles, one with high affinity and one with a lower affinity. Ligands such as phosphate and ATP decrease both the quantum yield and the affinity of the particles for aurovertin. They also increase the total concentration of binding sites, and affect the relative contribution of weak and strong binding sites.6. A model is presented in which changes of the aurovertin fluorescence reflect conformational changes of the ATPase induced by its ligands.     

Purification and properties of ATPase inhibitor from rat liver mitochondria

(1) The ATPase inhibitor protein has been isolated from rat liver mitochondria in purified form. The molecular weight determined by sodium dodecyl sulfate gel electrophoresis is approximately 9500, and the isoelectric point is 8.9.

(2) The protein inhibits both the soluble ATPase and the particle-bound ATPase from rat liver mitochondria. It also inhibits ATPase activities of soluble F₁, and inhibitor-depleted submitochondrial particles derived from bovine heart mitochondria.

(3) On particle-bound ATPase the inhibitor has its maximal effect if incubated in the presence of Mg²⁺. ATP at slightly acidic pH.

(4) The inhibitor has a minimal effect on P_i-ATP exchange activity in sonicated submitochondrial particles. However, unexpectedly the inhibitor greatly stimulates P_i-ATP exchange activity in whole mitochondria while the low ATPase activity of the mitochondria is not affected. The possible mechanism of action of the inhibitor on intact mitochondria is offered.     

Spontaneous aggregation of the mitochondrial natural ATPase inhibitor in salt solutions as demonstrated by gel filtration and neutron scattering. Application to the concomitant purification of the ATPase inhibitor and F1-ATPase

(1) The natural ATPase inhibitor (IF₁) from beef heart mitochondria has a tendency to form aggregates in aqueous solutions. The extent of aggregation and the structure of the aggregates were assessed by gel filtration and small-angle neutron scattering. IF₁ polymerization was found to depend on the salt concentrations, pH of the medium and concentration of IF₁. The higher the salt concentration, the lower the aggregation state. Aggregation of IF₁ was decreased at slightly acidic pH. It increased with the concentration of IF₁ as expected from the law of mass action. (2) Neutron scattering showed the aggregation of IF₁ in 2 M ammonium sulfate solutions. The predominant species is the dimer which has a somewhat elongated shape. (3) The Sephadex G-50 chromatography that is supposed to deprive beef heart submitochondrial particles of loosely bound IF₁ (Racker, E. and Horstman, L.L. (1967) J. Biol. Chem. 242, 2547–2551) was shown to have a limited effectiveness as a trap for IF₁. The reason was that IF₁ released from the particles formed high molecular weight aggregates that were not separated from the membrane vesicles by Sephadex G-50 chromatography. (4) The above observations provide the basis for a simple method of purification of beef heart IF₁ which combines the recovery of the supernatant from submitochondrial particles with the last three steps of the IF₁ preparation described by Horstman and Racker (J. Biol. Chem. (1970) 265, 1336–1344). The particles recovered in the sediment were deprived of IF₁ and could therefore be used for preparation of F₁-ATPase. The advantage of this method is that both IF₁ and F₁-ATPase can be prepared from the same batch of mitochondria.     

Titration of the binding sites for the oligomycin-sensitivity conferring protein in beef heart submitochondrial particles

The binding parameters of the oligomycin-sensitivity conferring protein (OSCP) in inside-out particles from beef heart mitochondria have been tested by means of two assays, the oligomycin-sensitive ATP-Pi exchange, and the oligomycin-sensitive ATP hydrolysis. The total number of OSCP binding sites in A particles was equal to 220 pmol/mg particle protein. Each mole of ATPase active site was able to bind 1.1 +/- 0.5 mol OSCP with Kd 1.7 nM.