Regulatory proteins of F1F0-ATPase: Role of ATPase inhibitor

An intrinsic ATPase inhibitor inhibits the ATP-hydrolyzing activity of mitochondrial F₁F₀-ATPase and is released from its binding site on the enzyme upon energization of mitochondrial membranes to allow phosphorylation of ADP. The mitochondrial activity to synthesize ATP is not influenced by the absence of the inhibitor protein. The enzyme activity to hydrolyze ATP is induced by dissipation of the membrane potential in the absence of the inhibitor. Thus, the inhibitor is not responsible for oxidative phosphorylation, but acts only to inhibit ATP hydrolysis by F₁F₀-ATPase upon deenergization of mitochondrial membranes. The inhibitor protein forms a regulatory complex with two stabilizing factors, 9K and 15K proteins, which facilitate the binding of the inhibitor to F₁F₀-ATPase and stabilize the resultant inactivated enzyme. The 9K protein, having a sequence very similar to the inhibitor, binds directly to F₁ in a manner similar to the inhibitor. The 15K protein binds to the F₀ part and holds the inhibitor and the 9K protein on F₁F₀-ATPase even when one of them is detached from the F₁ part.     

Effect of the natural ATPase inhibitor on the binding of adenine nucleotides and inorganic phosphate to mitochondrial F1-ATPase

(1) Incubation of the beef heart mitochondrial ATPase, F₁ with Mg-ATP was required for the binding of the natural inhibitor, IF₁, to F₁ to form the inactive F₁-IF₁ complex. When F₁ was incubated in the presence of [¹⁴C]ATP and MgCl₂, about 2 mol ¹⁴C-labeled adenine nucleotides were found to bind per mol of F₁; the bound ¹⁴C-labeled nucleotides consisted of [¹⁴C]ADP arising from [¹⁴C]ATP hydrolysis and [¹⁴C]ATP. The ¹⁴C-labeled nucleotide binding was not prevented by IF₁. These data are in agreement with the idea that the formation of the F₁-IF₁ complex requires an appropriate conformation of F₁. (2) The ¹⁴C-labeled adenine nucleotides bound to F₁ following preincubation of F₁ with Mg-[¹⁴C]ATP could be exchanged with added [³H]ADP or [³H]ATP. No exchange occurred between added [³H]ADP or [³H]ATP and the ¹⁴C-labeled adenine nucleotides bound to the F₁-IF₁ complex. These data suggest that the conformation of F₁ in the isolated F₁-IF₁ complex is further modified in such a way that the bound ¹⁴C-labeled nucleotides are no longer available for exchange. (3) ³²P_i was able to bind to isolated F₁ with a stoichiometry of about 1 mol of P_i per mol of F₁ (Penefsky, H.S. (1977) J. Biol. Chem. 252, 2891–2899). There was no binding of ³²P_i to the F₁-IF₁ complex. Thus, not only the nucleotides sites, but also the P_i site, are masked from interaction with external ligands in the isolated F₁-IF₁ complex.     

Exploration of nucleotide binding sites in the mitochondrial membrane by 2-azido-[α-P]ADP

The ADP/ATP carrier of beef heart mitochondria is able to bind 2-azido-[α-³²P]ADP in the dark with a K_d value of 8 μM. 2-Azido ADP is not transported and it inhibits ADP transport and ADP binding. Photoirradiation of beef heart mitochondria with 2-azido-[α-³²P]ADP results mainly in photolabeling of the ADP/ATP carrier protein; photolabeling is prevented by carboxyatractyloside, a specific inhibitor of ADP/ATP transport. Upon photoirradiation of inside-out submitochondrial particles with 2-azido-[α-³²P]ADP, both the ADP/ATP carrier and the β subunit of the membrane-bound F₁-ATPase are covalently labeled. The binding specificity of 2-azido-[α-³²P]ADP for the β subunit of F₁-ATPase is ascertained by prevention of photolabeling of isolated F₁ by preincubation with an excess of ADP.     

The extent of mitochondrial F1-ATPase and adenine nucleotide carrier activity with ϵ-ATP

1. The use of 1,N⁶-ethenoadenosine 5′-triphosphate (?-ATP), a synthetic, fluorescent analog of ATP, by whole rat liver mitochondria and by submitochondrial particles produced via sonication has been studied.2. Direct [³H]adenine nucleotide uptake studies with isolated mitochondria, indicate the ?-[³H]ATP is not transported through the inner membrane by the adenine nucleotide carrier and is therefore not utilized by the 2,4-dinitrophenol-sensitive F₁-ATPase (EC 3.6.1.3) that functions in oxidative phosphorylation. However, ?-ATP is hydrolyzed by a Mg²⁺-dependent, 2,4-dinitrophenol-insensitive ATPase that is characteristic of damaged mitochondria.3. ?-ATP can be utilized quite well by the exposed F₁-ATPase of sonic submitochondrial particles. This ?-ATP hydrolysis activity is inhibited by oligomycin and stimulated by 2,4-dinitrophenol. The particle F₁-ATPase displays similar K_m values for both ATP and ?-ATP; however, the V with ATP is approximately six times greater than with ?-ATP.4. Since ?-ATP is a capable substrate for the submitochondrial particle F₁-ATPase, it is proposed that the fluorescent properties of this ATP analog might be employed to study the submitochondrial particle F₁-ATPase complex, and its response to various modifiers of oxidative phosphorylation.     

Stereospecific bioactions of 5-hydroxyicosatetraenoate

Interaction of mitochondrial F₁-ATPase with the isolated natural inhibitor protein resulting in the inhibition of multi-site ATP hydrolysis is accompanied by the loss of activity at low ATP concentrations when single-site hydrolysis should occur. Catalytic site occupancy by [¹⁴C]nucleotides in F₁-ATPase during steady-state [¹⁴C]ATP hydrolysis, which is saturated in parallel with single-site catalysis, is prevented after blocking the enzyme with the inhibitor protein.     

Effect of ε subunit on the rotation of thermophilic Bacillus F1-ATPase

F₁-ATPase is an ATP-driven motor in which γε rotates in the α₃β₃-cylinder. It is attenuated by MgADP inhibition and by the ε subunit in an inhibitory form. The non-inhibitory form of ε subunit of thermophilic Bacillus PS3 F₁-ATPase is stabilized by ATP-binding with micromolar K_d at 25 °C. Here, we show that at [ATP] > 2 μM, ε does not affect rotation of PS3 F₁-ATPase but, at 200 nM ATP, ε prolongs the pause of rotation caused by MgADP inhibition while the frequency of the pause is unchanged. It appears that ε undergoes reversible transition to the inhibitory form at [ATP] below K_d.     

Inactivation and the site of labeling of F1-ATPase from Mycobacterium phlei by dicyclohexylcarbodiimide, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole and quinacrine mustard

The F₁-ATPase from Mycobacterium phlei is inactivated by dicyclohexylcarbodiimide (DCCD), 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) and quinacrine mustard. The inactivation is both time-and concentration-dependent and in the case of DCCD being more pronounced at acidic pH. The minimum inactivation half-time ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$) for DCCD, NBD-Cl and quinacrine mustard was observed to be 14, 6 and 7 min, respectively. Inactivation of F₁-ATPase resulted in the incorporation of [¹⁴C]DCCD as well as [¹⁴C]NBD-Cl into α and γ subunits. The incorporation of label into α and γ subunits, utilizing [¹⁴C]NBD-Cl, was reversible by dithiothreitol. Complete inactivation, by linear extrapolation to zero activity, revealed that 4 mol [¹⁴C]DCCD and 4 mol [¹⁴C]NBD-Cl bind per mol F₁-ATPase. Kinetic and binding studies show that these probes bind to site(s) distinct from ATP-binding site in F₁-ATPase from M. phlei.     

Activation of F1 -ATPase isolated from potato tuber mitochondria

The ATP-hydrolyzing activity of F₁-ATPase purified from potato tubers mitochondria was stimulated 2- and 3.5-fold by anions, chloride and bicarbonate, respectively, and 5.5- and 6.5-fold by detergents, octyl glucoside and lauryl dimethylamine oxide (LDAO), respectively. The maximal specific activity of the activated F₁, 129 μmol/min per mg protein is the highest activity of plant mitochondrial F₁ hitherto reported and exceeds several-fold values reported earlier. In the absence of activators F₁-catalyzed ATP hydrolysis exhibits non-linear double-reciprocal plots of [ATP]⁻¹ vs v⁻¹ indicative of negative cooperativity, while in the presence of activators, linear plots are observed. It is suggested that the activators reduce the cooperativity originating from the interaction between different subunits of the enzyme.     

Acceleration of the ATP-binding rate of F1-ATPase by forcible forward rotation

F₁-ATPase (F₁) is a reversible ATP-driven rotary motor protein. When its rotary shaft is reversely rotated, F₁ produces ATP against the chemical potential of ATP hydrolysis, suggesting that F₁ modulates the rate constants and equilibriums of catalytic reaction steps depending on the rotary angle of the shaft. Although the chemomechanical coupling scheme of F₁ has been determined, it is unclear how individual catalytic reaction steps depend on its rotary angle. Here, we report direct evidence that the ATP-binding rate of F₁ increases upon the forward rotation of the rotor, and its binding affinity to ATP is enhanced by rotation.     

Overexpression, Purification, and Characterization of Human and Bovine Mitochondrial ATPase Inhibitors: Comparison of the Properties of Mammalian and Yeast ATPase Inhibitors

Mitochondrial ATP synthase (F₁F₀-ATPase) is regulated by an intrinsic ATPase inhibitor protein. In this study, we overexpressed and purified human and bovine ATPase inhibitors and their properties were compared with those of a yeast inhibitor. The human and bovine inhibitors inhibited bovine ATPase in a similar way. The yeast inhibitor also inhibited bovine F₁F₀-ATPase, although the activity was about three times lower than the mammalian inhibitors. All three inhibitors inhibited yeast F₁F₀-ATPase in a similar way. The activities of all inhibitors decreased at higher pH, but the magnitude of the decrease was different for each combination of inhibitor and ATPase. The results obtained in this study show that the inhibitory mechanism of the inhibitors was basically shared in yeast and mammals, but that mammalian inhibitors require unique residues, which are lacking in the yeast inhibitor, for their maximum inhibitory activity. Common inhibitory sites of mammalian and yeast inhibitors are suggested.     

Purification and functional properties of the DCCD-reactive proteolipid subunit of the H+-translocating ATPase from Mycobacterium phlei

Interaction of N,N′-dicyclohexylcarbodiimide (DCCD) with ATPase of Mycobacterium phlei membranes results in inactivation of ATPase activity. The rate of inactivation of ATPase was pseudo-first order for the initial 30–65% inactivation over a concentration range of 5–50 μM DCCD. The second-order rate constant of the DCCD-ATPase interaction was k = 8.5·10⁵ M^?1·min^?1. The correlation between the initial binding of [¹⁴C]DCCD and 100% inactivation of ATPase activity shows 1.57 nmol DCCD bound per mg membrane protein. The proteolipid subunit of the F₀F₁-ATPase complex in membranes of M. phlei with which DCCD covalently reacts to inhibit ATPase was isolated by labeling with [¹⁴C]DCCD. The proteolipid was purified from the membrane in free and DCCD-modified form by extraction with chloroform/methanol and subsequent chromatography on Sephadex LH-20. The polypeptide was homogeneous on SDS-acrylamide gel electrophoresis and has an apparent molecular weight of 8000. The purified proteolipid contains phosphatidylinositol (67%), phosphatidylethanolamine (18%) and cardiolipin (8%). Amino acid analysis indicates that glycine, alanine and leucine were present in elevated amounts, resulting in a polarity of 27%. Cysteine and tryptophan were lacking. Butanol-extracted proteolipid mediated the translocation of protons across the bilayer, in K⁺-loaded reconstituted liposomes, in response to a membrane potential difference induced by valinomycin. The proton translocation was inhibited by DCCD, as measured by the quenching of fluorescence of 9-aminoacridine. Studies show that vanadate inhibits the proton gradient driven by ATP hydrolysis in membrane vesicles of M. phlei by interacting with the proteolipid subunit sector of the F₀F₁-ATPase complex.     

猪心线粒体Fo的纯化、重建及其质子转运功能

比较了猪心线粒体F_oF₁-ATPase膜部分F_o的四种纯化方法.结果表明,用NaBr从亚线粒体除去F_oF₁-ATPase的水溶性部分F₁-ATPase后,再以CHAPS增溶,并经蔗糖梯度离心,可获得高纯度的F_o.SDS-聚丙烯酰胺凝胶电泳鉴定表明,纯化的F_o含有b、OSCP（寡霉素敏感授予蛋白）、d、a、e、F₆、IF₁、A6L和c等9种亚基.用去污剂稀释法将纯化的F_o在脂质体上重建后,重建F_o表现较高的被动转运质子活性.这为在体外深入研究F_o的活性、构象与膜脂的关系,以及F_o与F₁-ATPase的组装等提供了很好的实验模型.     

ADP and ATP binding to noncatalytic sites of thiol-modulated chloroplast ATP synthase

A modified ‘cold chase’ technique was used to study tight [¹⁴C]ADP and [¹⁴C]ATP binding to noncatalytic sites of chloroplast ATP synthase (CF₀F₁). The binding was very low in the dark and sharply increased with light intensity. Dissociation of labeled nucleotides incorporated into noncatalytic sites of CF₀F₁ or CF₁ reconstituted with EDTA-treated thylakoid membranes was also found to be light-dependent. Time dependence of nucleotide dissociation is described by the first order equation with a k _d of about 5 min⁻¹. The exposure of thylakoid membranes to 0.7–24.8 μM nucleotides leads to filling of up to two noncatalytic sites of CF₀F₁. The sites differ in their specificity: one preferentially binds ADP, whereas the other – ATP. A much higher ATP/ADP ratio of nucleotides bound at noncatalytic sites of isolated CF₁ dramatically decreases upon its reconstitution with EDTA-treated thylakoid membranes. It is suggested that the decrease is caused by conformational changes in one of the α subunits induced by its interaction with the δ subunit and/or subunit I–II when CF₁ becomes bound to a thylakoid membrane.     

Interaction of beef-heart mitochondrial F1-ATPase with immobilized ATP in the presence of dimethylsulfoxide

Dimethylsulfoxide [Me₂SO, 30% (v/v)] promotes the formation of ATP from ADP and phosphate catalyzed by soluble mitochondrial F₁-ATPase. The effects of this solvent on the interaction of beef-heart mitochondrial F₁ with the immobilized ATP of Agarose-hexane-ATP were studied. In the presence of Me₂SO, F₁ bound less readily to the immobilized ATP, but once bound was more difficult to elute with exogenous ATP. This suggests that not only was the binding affinity for adenine nucleotide at the first binding site affected but that adenine nucleotide binding affinity at the second and/or third sites, which interact cooperatively with the first site to release bound nucleotide, was also affected. A reduction in the binding of [³H]ADP to these sites was shown. A change in the conformation of F₁ in 30% (v/v) Me₂SO was demonstrated by crosslinking and by the increased resistance of the enzyme to cold denaturation.     

Regulation of the mitochondrial ATPasein situ in cardiac muscle: Role of the inhibitor subunit

The mitochondrial F₁-ATPase inhibitor protein, IF₁, binds to subunits of the F₁-ATPase bothin vitro andin situ under nonenergizing conditions, i.e., under conditions that allow a net hydrolysis of ATP by the mitochondrial ATPase to take place. This reversible IF₁ binding occurs in a wide variety of cell types including (anaerobic) baker's yeast cells and (ischemic) mammalian cardiomyocytes under conditions that limit oxidative phosphorylation. The binding of inhibitor results in a marked slowing of ATP hydrolysis by the undriven mitochondrial ATP synthase. An apparent main function of this reversible IF₁ binding, at least in cells that undergo aerobic-anaerobic switching, is the mitigation of a wasteful hydrolysis of ATP produced by glycolysis during anoxic or ischemic intervals, by the mitochondrial ATPase. While this apparent main function is probably of considerable importance in cells that normally either can or must undergo aerobic-anaerobic switching such as baker's yeast cells and skeletal myocytes, one wonders why a full complement of IF₁ has been retained in certain cells that normally do not undergo such aerobic-anaerobic switching, cells such as adult mammalian cardiomyocytes of many species. While some mammalian species have, indeed, not retained a functional complement of IF₁ in their cardiomyocytes, those that have can benefit significantly from its presence during intervals of myocardial ischemia.This mini-review is dedicated to the memory of Professor Efraim Racker.     

Covalent modification of the non-catalytic sites of the H+-ATPase from chloroplasts with 2-azido-[α-32P]ATP and its effect on ATP synthesis and ATP hydrolysis

Incubation of the isolated H⁺-ATPase from chloroplasts, CF₀F₁, with 2-azido-[α-³²P]ATP leads to the binding of this nucleotide to different sites. These sites were identified after removal of free nucleotides, UV-irradiation and trypsin treatment by separation of the tryptic peptides by ion exchange chromatography. The nitreno-AMP, nitreno-ADP and nitreno-ATP peptides were further separated on a reversed phase column, the main fractions were subjected to amino acid sequence analysis and the derivatized tyrosines were used to distinguish between catalytic (β-Tyr362) and non-catalytic (β-Tyr385) sites. Several incubation procedures were developed which allow a selective occupation of each of the three non-catalytic sites. The non-catalytic site with the highest dissociation constant (site 6) becomes half maximally filled at 50 μM 2-azido-[α-³²P]ATP, that with the intermediate dissociation constant (site 5) at 2 μM. The ATP at the site with the lowest dissociation constant had to be hydrolyzed first to ADP before a replacement by 2-azido-[α-³²P]ATP was possible. CF₀F₁ with non-covalently bound 2-azido-[α-³²P]ATP and after covalent derivatization was reconstituted into liposomes and the rates of ATP synthesis as well as ATP hydrolysis were measured after energization of the proteoliposomes by ΔpH/Δϕ. Non-covalent binding of 2-azido-ATP to any of the three non-catalytic sites does not influence ATP synthesis and ATP hydrolysis, whereas covalent derivatization of any of the three sites inhibits both, the degree being proportional to the degree of derivatization. Extrapolation to complete inhibition indicates that derivatization of one site (either 4 or 5 or 6) is sufficient to block completely multi-site catalysis. The rates of ATP synthesis and ATP hydrolysis were measured as a function of the ADP and ATP concentration from uni-site to multi-site conditions with covalently derivatized and non-derivatized CF₀F₁. Uni-site ATP synthesis and ATP hydrolysis were not inhibited by covalent derivatization of any of the non-catalytic sites, whereas multi-site catalysis is inhibited. These results indicate that multi-site catalysis requires some flexibility between β- and α-subunits which is abolished by covalent derivatization of β-Tyr385 with a 2-nitreno-adenine nucleotide. Conformational changes connected with energy transduction between the F₀-part and the F₁-part are either not required for uni-site ATP synthesis or they are not impaired by the derivatization of any of the three β-Tyr385.     

Unisite Hydrolysis of [γ32P]ATP by Soluble Mitochondrial F1ATPase and Its Release by Excess ADP and ATP. Effect of Trifluoperazine

Some of the characteristics of unisite hydrolysis of [³²P]ATP as well as the changes that occur on the transition to multisite catalysis were further studied. It was found that a fraction of [³²P]ATP bound at the catalytic sites of F₁ under unisite conditions undergoes both hydrolysis and release induced by medium nucleotides upon addition of millimolar concentrations of ADP or ATP. The fraction of [³²P]ATP that undergoes release is similar to the fraction that undergoes hydrolytic cleavage, indicating that the rates of the release and hydrolytic reactions of bound [³²P]ATP are in the same range. As part of studies on the mechanisms through which trifluoperazine inhibits ATP hydrolysis, its effect on unisite hydrolysis of [³²P]ATP was also studied. Trifluoperazine diminishes the rate of unisite hydrolysis by 30–40%. The inhibition is accompanied by a nearly tenfold increase in the ratio of [³²P]ATP/³²Pi bound at the catalytic site and a 50% diminution in the rate of ³²Pi release from the enzyme into the media. Trifluoperazine also induces heterogeneity of the three catalytic sites of F₁ in the sense that in a fraction of F₁ molecules, the high-affinity catalytic site has a turnover rate lower than the other two. Trifluoperazine does not modify the release of previously bound [³²P]ATP induced by medium nucleotides. The latter indicates that hindrances in the release of Pi do not necesarily accompany alterations in the release of ATP even though both species lie in the same site.     

High-Pressure Effects on Vacuolar H+-ATPase from Etiolated Mung Bean Seedlings

A high-hydrostatic-pressure technique was employed to study the structure-function relationship of plant vacuolar H⁺-ATPase from etiolated mung bean seedlings (Vigna radiata L.). When isolated vacuolar H⁺-ATPase was subjected to hydrostatic pressure, the activity of ATP hydrolysis was markedly inhibited in a time-, protein concentration- and pressure-dependent manner. The pressure treatment decreased both V _max and K _m of solubilized vacuolar H⁺-ATPase, implying an increase in ATP binding affinity, but a decrease in the ATP hydrolysis activity. Physiological substrate, Mg²⁺-ATP, augmented the loss of enzymatic activity upon pressure treatment. However, ADP, AMP, and Pi exerted substantial protective effects against pressurization. Steady-state ATP hydrolysis was more sensitive to pressurization than single-site ATPase activity. The inactivation of solubilized vacuolar H⁺-ATPase by pressure may result from changes in protein–protein interaction. The conformational change of solubilized vacuolar H⁺-ATPase induced by hydrostatic pressure was further determined by spectroscopic techniques. The inhibition of vacuolar H⁺-ATPase under pressurization involved at least two steps. Taken together, our work indicates that subunit–subunit interaction is crucial for the integrity and the function of plant vacuolar H⁺-ATPase. It is also suggested that the assembly of the vacuolar H⁺-ATPase complex is probably not random, but follows a sequestered pathway.     

Purification and properties of the latent F1-ATPase of Micrococcus lysodeikticus

The latent coupling factor (F₁)-ATPase of Micrococcus lysodeikticus has been purified to homogeneity as determined by a number of criteria including, non-denaturing polyacrylamide gel electrophoresis, crossed immunoelectrophoresis and analytical ultracentrifugation. By inclusion of 1 mM phenylmethyl sulfonyl fluoride, a serine protease inhibitor, in the shock-wash step of release of F₁ from the membranes, the spontaneous activation of both crude and purified ATPase by endogenous membrane protease(s) can be prevented, thereby yielding a highly latent ATPase preparation. Equilibrium ultracentrifugation of the latent ATPase gave a molecular weight of 400 000. The ATPase contained five different subunits α, β, γ, δ, and ? and their molecular weights determined by SDS-polyacrylamide gel electrophoresis were 60 000, 54 000, 37 000, 27 000 and 9000, respectively. The subunit composition was determined with ¹⁴C-labelled, F₁-ATPase prepared from cells grown on medium containing [U-¹⁴C]-labelled algal protein hydrolysate. Within the limitations of this method the results tentatively suggest a subunit composition of 3 : 3 : 1 : 1 : 3.     

Myocardial ischemic preconditioning and mitochondrial F1F0-ATPase activity

A short period of ischemia followed by reperfusion (ischemic preconditioning) is known to trigger mechanisms that contribute to the prevention of ATP depletion. In ischemic conditions, most of the ATP hydrolysis can be attributed to mitochondrial F₁F₀-ATPase (ATP synthase). The purpose of the present study was to examine the effect of myocardial ischemic preconditioning on the kinetics of ATP hydrolysis by F₁F₀-ATPase. Preconditioning was accomplished by three 3-min periods of global ischemia separated by 3 min of reperfusion. Steady state ATP hydrolysis rates in both control and preconditioned mitochondria were not significantly different. This suggests that a large influence of the enzyme on the preconditioning mechanism may be excluded. However, the time required by the reaction to reach the steady state rate was increased in the preconditioned group before sustained ischemia, and it was even more enhanced in the first 5 min of reperfusion (101 ± 3.0 sec in preconditioned vs. 83.4 ± 4.4 sec in controls, p 0.05). These results suggest that this transient increase in activation time may contribute to the cardioprotection by slowing the ATP depletion in the very critical early phase of post-ischemic reperfusion.