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.     

Interaction of ATPase from submitochondrial fragments and a natural inhibitor protein during delta-mu-H+ generation on a membrane

An addition of the inhibitor protein (IF1) to submitochondrial particles (SMP) essentially free of endogenous IF1 (AS-SMP) results in a synchroneous inhibition of ATP hydrolysis and ATP-dependent reduction of NAD+ by succinate without any effect on the oxidative phosphorylation rate. The binding of IF1 to the membrane-bound ATPase leads to the loss of the inhibitor protein sensitivity to trypsin despite the delta mu H+ generation. The data obtained are consistent with a model according to which there exist the hydrolase and synthetase forms of F1 and contradict the generally accepted concepts on the delta mu H+-dependent dissociation of the F1-IF1 complex.     

The ATPase inhibitor protein in oxidative phosphorylation. The rate-limiting factor to phosphorylation in submitochondrial particles.

1. Purified luciferase and luciferin were used to study the time course of phosphorylation in submitochondrial particles. The light emitted was detected by a single-photon counter, using a multichannel analyser, and the results were analysed by an 'on-line' digital computer. 2. Using NADH as substrate, phosphorylation showed, in general, four phases. These were (i) a period of increasing rate ('lag'); (ii) a period of constant (positive) rate; (iii) a period of zero net rate (plateau), when the phosphorylation potential was maintained at its equilibrium value, and (iv) a period of negative rate (atp hydrolysis) after all the oxygen has been consumed. 3. The lag phase, several seconds in length, was a function of the inhibitor protein content of the particles. It was decreased in particles treated to remove the inhibitor protein, either by prior energisation of the particles with NADH, or by addition of aurovertin, which competes with the inhibitor protein for the ATPase. It was concluded that the ATPase inhibitor inhibits both ATP synthesis and hydrolysis by the ATPase. 4. The rate constant for the release of the inhibitor protein from the energised membrane was determined from the time course of ATP production during the lag phase. The activation energy of this process was measured from the temperature dependence of the lag, and was shown to be 13.3 kcal/mol, lower than the activation energy of ATP synthesis or NADH oxidation. 5. The rate constant for inhibitor release was dependent on 'energisation' of the membrane, being lower in the presence of uncouplers. However, it was possible to decrease the rate constant considerably with agents that collapsed the membrane potential without uncoupling the membrane. It was concluded that the inhibitor protein responded to the membrane potential component of the energisation. 6. A kinetic model for energy-dependent dissociation of the ATPase-inhibitor complex is proposed.     

Effect of the protonmotive force on ATP-linked processes and mobilization of the bound natural ATPase inhibitor in beef heart submitochondrial particles

In an attempt to determine whether the natural ATPase inhibitor (IF₁) plays a role in oxidative phosphorylation, the time course of ATP synthesis and ATP hydrolysis in inside-out submitochondrial particles from beef heart mitochondria either possessing IF₁ (Mg-ATP particles) or devoid of IF₁ (AS particles) was investigated and compared to movements of IF₁, as assessed by an isotopic assay. The responses of the above reactions to preincubation of the particles in aerobiosis with NADH or succinate were as follows: (1) The few seconds lag that preceded the steady-rate phase of ATP synthesis was shortened and even abolished both in Mg-ATP particles and AS particles. The rate of ATP synthesis in the steady state was independent of the length of the lag. (2) ATPase was slowly activated, maximal activation being obtained after a 50-min preincubation; there was no direct link between the development of the protonmotive force (maximal within 1 sec) and ATPase activation. (3) Bound IF₁ was slowly released; the release of bound IF₁ as a function of the preincubation period was parallel to the enhancement of ATPase activity; the maximal amount of IF₁ released was a small fraction of the total IF₁ bound to the particles (less than 20%). (4) The double reciprocal plots of the rates of ATP and ITP hydrolysis vs. substrate concentrations that were curvilinear in the absence of preincubation with a respiratory substrate became linear after aerobic preincubation with the substrate. The data conclusively show that only ATPase activity in submitochondrial particles is correlated with the release of IF₁, and that the total extent of IF₁ release induced by respiration is limited. On the other hand, the kinetics of ATPase in control and activated particles are consistent with the existence of two conformations of the membrane-bound F₁-ATPase, directed to ATP synthesis or ATP hydrolysis and distinguishable by their affinity for IF₁.     

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.     

Radiation modification of the ATPase properties of submitochondrial particles

The appreciable changes in hydrophobic properties of ATPase of rat liver submitochondrial particles were discovered 3 days after whole-body X-irradiation with a dose of 7 Gy. The sensitivity of ATPase activity to stimulating anions decreased. The radiation damages to ATPase were detected and the ratio of concentrations of magnesium and ATP exceeded a unity.     

ATP synthesis and hydrolysis in submitochondrial particles subjected to an acid—base transition: Effects of the ATPase inhibitor protein

ATP hydrolysis or succinate oxidation by inhibitor-rich submitochondrial particles leads to a 3-fold increase in ATPase activity, with concomitant loss of about 30% of bound inhibitor protein. An acid—base transition causes similar, but smaller, effects (a 30% ATPase increase, and a loss of 8% of the inhibitor). Omitting the electrical component of the gradient completely abolished these effects. The inhibitor protein inhibits ADP phosphorylation induced by an acid—base transition but not by NADH oxidation. This is suggested to reflect the slow movement of the inhibitor protein and the brief period of acid—base jump phosphorylation.     

Effect of anions on the ATPase activity of submitochondrial particles

The effects of anions on the ATPase activity of submitochondrial particles from mouse liver cells were investigated. Thiocyanite decreased the ATP hydrolysis, acting as a competitive inhibitor with respect to sulfite. All the anions tested changed the ATPase activity noncompetitively towards Mg-ATP. The hydrolysis of CTP, GTP, ITP and UTP was insensitive to sulfite and thiocyanate. In the presence of Mn2+, Ca2+, Co2+, Zn2+ and Ba2+ an anion-dependent hydrolysis of ATP took place. It was assumed that the anions control the rate of the limiting step of the ATPase reaction, since sulfite and thiocyanate change the activation energy of ATP hydrolysis. The data obtained are discussed in terms of a previously proposed mechanism of the anions effect on the activity of mitochondrial ATPase.     

Simultaneous synthesis and hydrolysis of ATP regulated by the inhibitor protein in submitochondrial particles

Coupled submitochondrial particles from bovine heart with ATP synthases devoid of control by the inhibitor protein of Pullman and Monroy [J. Biol. Chem. 238, 3762-3769 (1963)] can be prepared by incubation of Mg-ATP particles in 50 mM phosphate, 250 mM sucrose, and greater than 95% D2O (pD 7.8) at 38 degrees C. As monitored with oxonol, the respiring particles build up and maintain a delta psi about 5-10% lower than that of the starting preparation. With oligomycin delta psi of the two preparations is the same. In the presence of an ATP trap (hexokinase and glucose), the two types of particles carry out oxidative phosphorylation at comparable rates. Low concentrations of oligomycin induce a small enhancement of the rate of ATP synthesis in non-controlled particles. In the absence of an ATP trap, net accumulation of ATP, as driven by electron transport in particles without control by the inhibitor protein, is low. Apparently this is due to lack of control by the inhibitor protein of ATP hydrolysis that occurs during oxidative phosphorylation.     

Kinetics of the interaction of ATPase of submitochondrial fragments and a natural protein-inhibitor

Conditions were selected which enable a quantitative assay of the ATPase inhibitor protein in submitochondrial particles. It was found that the isolated soluble inhibitor exhibits a marked pH-dependent hysteretic behaviour, i. e., an instant jump of pH for the inhibitor solution from 4.8 to 8.2 induced a slow alteration of its activity as measured by the inhibition of ATP hydrolysis by submitochondrial particles. In acid media (pH less than 6.8), the inhibitor is in the active, whereas in alkaline media (pH greater than 6.8) in the inactive state; the apparent pKa value for the cooperative active/inactive transition is 6.8. Treatment of the inhibitor protein with diethylpyrocarbonate, a specific reagent for histidine, completely abolishes its inhibitory activity. Two types of the inhibitor protein--ATPase interaction were revealed, i.e., reversible (ATP-independent) and irreversible (ATP-dependent) ones. Both reactions, i.e., ATP hydrolysis and ATP inhibition by the inhibitor in the presence of Mg2+ are characterized by a hyperbolic dependence of the reaction rate on ATP concentration; however, for both reactions the apparent KmATP values (50 and 5 microM, respectively) differ significantly (pH 8.0). Thus, the inhibitor--ATPase interaction shows that there exists a specific site for ATP in the ATPase which is different from the catalytic one. A model for the inhibitor protein interaction with ATPase which takes account of a slow pH-dependent conformational transformation of the inhibitor protein is proposed.     

The use of aurovertin to determine the F1 content of submitochondrial particles and the ATPase complex.

(1) The concentration of aurovertin-binding sites calculated from fluorimetric titrations of submitochondrial particles is equal to the F1 concentration, calculated from the concentration of F1-binding sites in stripped particles. (2) Direct binding experiments show that the fluorescence enhancement of aurovertin bound to submitochondrial particles and the isolated ATPase complex is less (or absent) at higher concentrations than at lower concentrations. The binding data can be described by 'specific' and 'non-specific' binding. The concentration of the 'specific' sites is twice that derived from fluorimetric titrations. (3) After dissociation of the bound F1 with LiCl, fluorimetric titrations with aurovertin yield linear Scatchard plots. The fluorescence enhancement and KD are equal to those of the beta-subunit-aurovertin complex. The concentration of beta-subunits is double the concentration of F1. (4) It is concluded that both for submitochondrial particles and the isolated ATPase complex the most reliable and simple way to determine the F1 content is to dissociate the F1 with LiCl, spin down the insoluble material and titrate the supernatant (containing free beta-subunit) with aurovertin.     

The interaction of mitochondrial F1-ATPase with the natural ATPase inhibitor protein

The interaction of soluble mitochondrial ATPase from beef heart with the natural ATPase inhibitor was studied. It was found that the phosphorylation of small amounts of ADP by phosphoenolpyruvate and pyruvate kinase, and an ensuing catalytic cycle supports the binding of the inhibitor to the enzyme. The association of the inhibitor with F1-ATPase does not increase the content of ATP in the F1-ATPase-inhibitor complex. The inhibitor of catalytic activity bathophenanthroline-Fe2+ chelate prevents the interaction, while the association of the inhibitor with F1-ATPase is delayed if the reaction is carried out in 2H2O. The date indicate that a transient state involved in the catalytic cycle is the form of the enzyme that interacts with the inhibitor. The proton-motive force-induced dissociation of the inhibitor from particulate ATPase is prevented by bathophenanthroline-Fe2+ chelate and nitrobenzofurazan chloride, which indicates that a functional catalytic (beta) subunit is required for the proton-motive force-induced release of the inhibitor. The data suggest a direct involvement of catalytic (beta) subunit in the mechanism by which the F1-ATPase senses the proton-motive force.     

Reaction mechanism of the membrane-bound ATPase of submitochondrial particles from beef heart

Submitochondrial particles from beef heart, washed with dilute solutions of KCl so as to activate the latent, membrane-bound ATPase, F1, may be used to study single site catalysis by the enzyme. [gamma-32P]ATP, incubated with a molar excess of catalytic sites, a condition which favors binding of substrate in only a single catalytic site on the enzyme, is hydrolyzed via a four-step reaction mechanism. The mechanism includes binding in a high affinity catalytic site, Ka = 10(12)M-1, a hydrolytic step for which the equilibrium constant is near unity, and two product release steps in which Pi dissociates from catalytic sites about 10 times more rapidly than ADP. Catalysis by the membrane-bound ATPase also is characterized by a 10(6)-fold acceleration in the rate of net hydrolysis of [gamma-32P]ATP, bound in the high affinity catalytic site, that occurs when substrate is made available to additional catalytic sites on the enzyme. These aspects of the reaction mechanism of the ATPase of submitochondrial particles closely parallel the reaction mechanism determined for solubilized, homogeneous F1 (Grubmeyer, C., Cross, R. L., and Penefsky, H. S. (1982) J. Biol. Chem. 257, 12092-12100). The finding that removal of the enzyme from the membrane does not significantly alter the properties of single site catalysis lends support to models of ATP synthesis in oxidative phosphorylation, catalyzed by membrane-bound F1, that have been based on the study of the soluble enzyme.