Nucleotide Sequence of the F(1)-ATPase alpha Subunit Gene from Maize Mitochondria

The α subunit of the F₁-ATPase complex of maize is a mitochondrial translational product, presumably encoded by the mitochondrial genome. Based on nucleotide and amino acid homology, we have identified a mitochondrial gene, designated atpα, that appears to code for the F₁-ATPase α subunit of Zea mays. The atpα gene is present as a single copy in the maize. Texas cytoplasm and is actively transcribed. The maize α polypeptide has a predicted length of 508 amino acids and a molecular mass of 55,187 daltons. Amino acid homologies between the maize mitochondrial α subunit and the tobacco chloroplast CF₁ and Escherichia coli α subunits are 54 and 51%, respectively. The origin of the atpα gene is discussed.     

Purification and Partial Characterization of Two Soluble NAD(P)H Dehydrogenases from Arum maculatum Mitochondria

Two enzyme systems carrying out the oxidation of NAD(P)H in the presence of various electron acceptors have been isolated and partially characterized from the supernatant of frozen-thawed mitochondria from Arum maculatum spadices. The two systems contain flavoproteins and differ by their ability to oxidize NADH or NADPH, optimum pH and pI values, sensitivity to Ca²⁺ and EGTA, denaturation by 4 molar urea, molecular mass, and number of subunits. These properties, together with methodological considerations, are compatible with the location of these enzyme activities on the outer surface of the inner mitochondrial membrane, and support the hypothesis of the existence of two separate dehydrogenases responsible for the mitochondrial oxidation of cytosolic NADH and NADPH.     

Isolation and Antigenic Characterization of Corn Mitochondrial F(1)-ATPase

Corn mitochondrial F₁-ATPase was purified from submitochondrial particles by chloroform extraction. Enzyme stored in ammonium sulfate at 4°C was substantially activated by ATP, while enzyme stored at −70°C in 25% glycerol was not. Enzyme in glycerol remained fully active (8-9 micromoles P_i released per minute per milligram), while the ammonium sulfate preparations steadily lost activity over a 2-month storage period. The enzyme was cold labile, and inactived by 4 minutes at 60°C. Treatment with octylglucoside resulted in complete loss of activity, while vanadate had no effect on activity. The apparent subunit molecular weights of corn mitochondrial F₁-ATPase were determined by SDS-polyacrylamide gel electrophoresis to be 58,000 (α), 55,000 (β), 35,000 (γ), 22,000 (δ), and 12,000 (ε). Monoclonal and polyclonal antibodies used in competitive binding assays demonstrated that corn mitochondrial F₁-ATPase was antigenically distinct from the chloroplastic CF₁-ATPases of corn and spinach. Monoclonal antibodies against antigenic sites on spinach CF₁-ATPase β and γ subunits were used to demonstrate that those sites were either changed substantially or totally absent from the mitochondrial F₁-ATPase.     

Epsilon subunit, an endogenous inhibitor of bacterial F(1)-ATPase, also inhibits F(0)F(1)-ATPase

Since the report by Sternweis and Smith (Sternweis, P. C., and Smith, J. B. (1980) Biochemistry 19, 526-531), the epsilon subunit, an endogenous inhibitor of bacterial F(1)-ATPase, has long been thought not to inhibit activity of the holo-enzyme, F(0)F(1)-ATPase. However, we report here that the epsilon subunit is exerting inhibition in F(0)F(1)-ATPase. We prepared a C-terminal half-truncated epsilon subunit (epsilon(DeltaC)) of the thermophilic Bacillus PS3 F(0)F(1)-ATPase and reconstituted F(1)- and F(0)F(1)-ATPase containing epsilon(DeltaC). Compared with F(1)- and F(0)F(1)-ATPase containing intact epsilon, those containing epsilon(DeltaC) showed uninhibited activity; severalfold higher rate of ATP hydrolysis at low ATP concentration and the start of ATP hydrolysis without an initial lag at high ATP concentration. The F(0)F(1)-ATPase containing epsilon(DeltaC) was capable of ATP-driven H(+) pumping. The time-course of pumping at low ATP concentration was faster than that by the F(0)F(1)-ATPase containing intact epsilon. Thus, the comparison with noninhibitory epsilon(DeltaC) mutant shed light on the inhibitory role of the intact epsilon subunit in F(0)F(1)-ATPase.     

The IF(1) inhibitor protein of the mitochondrial F(1)F(0)-ATPase

Recent studies on the IF(1) inhibitor protein of the mitochondrial F(1)F(0)-ATPase from molecular biochemistry to possible pathophysiological roles are reviewed. The apparent mechanism of IF(1) inhibition of F(1)F(0)-ATPase activity and the biophysical conditions that influence IF(1) activity are summarized. The amino acid sequences of human, bovine, rat and murine IF(1) are compared and domains and residues implicated in IF(1) function examined. Defining the minimal inhibitory sequence of IF(1) and the role of conserved histidines and conformational changes using peptides or recombinant IF(1) is reviewed. Luft's disease, a mitochondrial myopathy where IF(1) is absent, is described with respect to IF(1) relevance to mitochondrial bioenergetics and clinical observations. The possible pathophysiological role of IF(1) in conserving ATP under conditions where cells experience oxygen deprivation (tumor growth, myocardial ischemia) is evaluated. Finally, studies attempting to correlate IF(1) activity to ATP conservation in myocardial ischemic preconditioning are compared.     

豌豆根胞质V1—ATPase的鉴定

利用免疫印迹,免疫电镜和ATP水解活性的测定对豌豆（Pisum sativum L.)根细胞胞质中V1-ATPase复合物的存在进行鉴定。用兔抗绿豆V-typeH^ -ATPase的A,B亚基的抗体进行的immuno-blotting和胶体金电镜结果都表明,胞质中存在有A,B亚基。活性测定结果进一步表明胞质具有ATP水解活性,这些结果说明豌豆根胞质具有活性的V1-ATPase复合物。这是首次直接证明植物中有胞质V1-ATPase的存在。     

豌豆根胞质V1-ATPase的鉴定

利用免疫印迹、免疫电镜和ATP水解活性的测定对豌豆(Pisum sativum L.)根细胞胞质中V1-ATPase复合物的存在进行鉴定.用兔抗绿豆V-type H+-ATPase 的A、B亚基的抗体进行的immuno-blotting和胶体金电镜结果都表明,胞质中存在有A、B亚基.活性测定结果进一步表明胞质具有ATP水解活性.这些结果说明豌豆根胞质具有有活性的V1-ATPase复合物.这是首次直接证明植物中有胞质V1-ATPase的存在.     

Purification and characterization of the F1-ATPase from Clostridium thermoaceticum.

The F1 portion of the H+-ATPase from Clostridium thermoaceticum was purified to homogeneity by solubilization at low ionic strength, ion-exchange chromatography, and gel filtration. The last indicated the Mr to be 370,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the pure enzyme revealed four bands with Mr corresponding to 60,000, 55,000, 37,000, and 17,000 in an apparent molar ratio of 3:3:1:1. The purified enzyme would bind to stripped membranes to reconstitute dicyclohexylcarbodiimide-sensitive ATPase activity. Phosphohydrolase activity, measured at 58 degrees C, was optimal at pH 8.5. In the presence of a 1 mM excess of Mg2+ over the concentration of ATP, the Km for ATP was 0.4 mM, and the Vmax was 6.7 mumol min-1 mg-1. Unlike the membrane-bound F1F0 complex, the F1-ATPase was relatively insensitive to the inhibitors dicyclohexylcarbodiimide and tributyltin chloride. Both the complex and the F1-ATPase were inhibited by quercetin, azide, 7-chloro-4-nitro-benz-2-oxa-1,3-diazole, and free magnesium, and both were stimulated by primary alcohols and sulfite. In whole cells, the F1F0-ATPase catalyzed the synthesis of ATP in response to a pH gradient.     

Purification and Characterization of a Glycerol-Resistant CF(0)-CF(1) and CF(1)-ATPase from the Halotolerant Alga Dunaliella bardawil

The isolation of the chloroplast ATP synthase complex (CF₀-CF₁) and of CF₁ from Dunaliella bardawil is described. The subunit structure of the D. bardawil ATPase differs from that of the spinach in that the D. bardawil α subunit migrates ahead of the β subunit and ε-migrates ahead of subunit II of CF₀ when separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The CF₁ isolated from D. bardawil resembles the CF₁ isolated from Chladmydomonas reinhardi in that a reversible, Mg²⁺-dependent ATPase is induced by selected organic solvents. Glycerol stimulates cyclic photophosphorylation catalyzed by D. bardawil thylakoid membranes but inhibits photophosphorylation catalyzed by spinach thylakoid membranes. Glycerol (20%) also stimulates the rate of ATP-P_i exchange catalyzed by D. bardawil CF₀-CF₁ proteoliposomes but inhibits the activity with the spinach enzyme. The ethanol-activated, Mg²⁺-ATPase of the D. bardawil CF₁ is more resistant to glycerol inhibition than the octylglucoside-activated, Mg²⁺-ATPase of spinach CF₁ or the ethanol-activated, Mg²⁺-dependent ATPase of the C. reinhardi CF₁. Both cyclic photophosphorylation and ATP-P_i exchange catalyzed by D. bardawil CF₀-CF₁ are more sensitive to high concentrations of NaCl than is the spinach complex.     

Binding of the inhibitor protein IF(1) to bovine F(1)-ATPase

In the structure of bovine F₁-ATPase inhibited with residues 1-60 of the bovine inhibitor protein IF₁, the α-helical inhibitor interacts with five of the nine subunits of F₁-ATPase. In order to understand the contributions of individual amino acid residues to this complex binding mode, N-terminal deletions and point mutations have been introduced, and the binding properties of each mutant inhibitor protein have been examined. The N-terminal region of IF₁ destabilizes the interaction of the inhibitor with F₁-ATPase and may assist in removing the inhibitor from its binding site when F₁F_o-ATPase is making ATP. Binding energy is provided by hydrophobic interactions between residues in the long α-helix of IF₁ and the C-terminal domains of the β_DP-subunit and β_TP-subunit and a salt bridge between residue E30 in the inhibitor and residue R408 in the C-terminal domain of the β_DP-subunit. Several conserved charged amino acids in the long α-helix of IF₁ are also required for establishing inhibitory activity, but in the final inhibited state, they are not in contact with F₁-ATPase and occupy aqueous cavities in F₁-ATPase. They probably participate in the pathway from the initial interaction of the inhibitor and the enzyme to the final inhibited complex observed in the structure, in which two molecules of ATP are hydrolysed and the rotor of the enzyme turns through two 120° steps. These findings contribute to the fundamental understanding of how the inhibitor functions and to the design of new inhibitors for the systematic analysis of the catalytic cycle of the enzyme.     

Kinetic Analysis of Corn Mitochondrial F(1)-ATPase

The activation and catalytic mechanism of corn mitochondrial F₁ were examined for the two distinct forms of the enzyme which appear upon storage in ammonium sulfate or glycerol. Apparently irreversible differences in the stability of the two active forms were found. Nucleosidetriphosphate induced activation of the enzyme was found to produce lasting effects on subsequent catalysis. These effects varied with both the nucleotide used for activation, and the hydrolyzed species. The substrate and metal specificity were examined with the ATP activated enzyme. Mg²⁺ and Ca²⁺ were found to be the most effective at promoting ATP hydrolysis. The substrates were hydrolyzed in the order GTP > ITP > ATP regardless of which nucleotide was used for activation. While ATP and GTP hydrolysis exhibited kinetics typical of other ATPases, ITP showed a transition from negative to positive cooperativity at low substrate concentrations. Bicarbonate was found to affect primarily the kinetics of ATP hydrolysis. AMP-PNP proved to be a potent inhibitor with respect to ATP hydrolysis. The results are discussed in terms of possible catalytic mechanisms and the similarities of the corn mitochondrial F₁ to other ATPases.     

Rotation of Escherichia coli F(1)-ATPase.

By applying the same method used for F(1)-ATPase (TF(1)) from thermophilic Bacillus PS3 (Noji, H., Yasuda, R., Yoshida, M., and Kinosita, K., Jr. (1997) Nature 386, 299-302), we observed ATP-driven rotation of a fluorescent actin filament attached to the gamma subunit in Escherichia coli F(1)-ATPase. The torque value and the direction of the rotation were the same as those observed for TF(1). F(1)-ATPases seem to share common properties of rotation irrespective of the sources.     

Rotary catalysis of the stator ring of F(1)-ATPase

F(1)-ATPase is a rotary motor protein in which 3 catalytic β-subunits in a stator α(3)β(3) ring undergo unidirectional and cooperative conformational changes to rotate the rotor γ-subunit upon adenosine triphosphate hydrolysis. The prevailing view of the mechanism behind this rotary catalysis elevated the γ-subunit as a "dictator" completely controlling the chemical and conformational states of the 3 catalytic β-subunits. However, our recent observations using high-speed atomic force microscopy clearly revealed that the 3 β-subunits undergo cyclic conformational changes even in the absence of the rotor γ-subunit, thus dethroning it from its dictatorial position. Here, we introduce our results in detail and discuss the possible operating principle behind the F(1)-ATPase, along with structurally related hexameric ATPases, also mentioning the possibility of generating hybrid nanomotors. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).     

The stochastic chemomechanics of the F(1)-ATPase molecular motor

We report a theoretical study of the F(1)-ATPase molecular rotary motor experimentally studied by R. Yasuda, H. Noji, M. Yoshida, K. Kinosita Jr., H. Itoh [Nature 410 (2001) 898]. The motor is modeled as a stochastic process for the angle of its shaft and the chemical state of its catalytic sites. The stochastic process is ruled by six coupled Fokker-Planck equations for the biased diffusion of the angle and the random jumps between the chemical states. The model reproduces the experimental observations that the motor proceeds by substeps and the rotation rate saturates at high concentrations of adenosine triphosphate or at low values of the friction coefficient. Moreover, predictions are made about the dependence of the rotation rate on temperature, and about the behavior of the F(1) motor under the effect of an external torque, especially, in the regime of synthesis of adenosine triphosphate.     

The coupled chemomechanics of the F(1)-ATPase molecular motor

The enzyme F(1)-ATPase is a rotary nanomotor in which the central gamma subunit rotates inside the cavity made of alpha(3)beta(3) subunits. The experiments showed that the rotation proceeds in steps of 120 degrees and each 120 degrees step consists of 80 degrees and 40 degrees substeps. Here the Author proposes a stochastic wave mechanics of the F(1)-ATPase motor and combines it with the structure-based kinetics of the F(1)-ATPase to form a chemomechanic coupled model. The model can reproduce quantitatively and explain the experimental observations about the F(1) motor. Using the model, several rate-limited situations about gamma subunit rotation are proposed, the effects of the friction and the load on the substeps are investigated and the chemomechanic coupled time during ATP hydrolysis cycle is determined.     

Reversible ischemic inhibition of F(1)F(0)-ATPase in rat and human myocardium

The physiological role of F(1)F(0)-ATPase inhibition in ischemia may be to retard ATP depletion although views of the significance of IF(1) are at variance. We corroborate here a method for measuring the ex vivo activity of F(1)F(0)-ATPase in perfused rat heart and show that observation of ischemic F(1)F(0)-ATPase inhibition in rat heart is critically dependent on the sample preparation and assay conditions, and that the methods can be applied to assay the ischemic and reperfused human heart during coronary by-pass surgery. A 5-min period of ischemia inhibited F(1)F(0)-ATPase by 20% in both rat and human myocardium. After a 15-min reperfusion a subsequent 5-min period of ischemia doubled the inhibition in the rat heart but this potentiation was lost after 120 min of reperfusion. Experiments with isolated rat heart mitochondria showed that ATP hydrolysis is required for effective inhibition by uncoupling. The concentration of oligomycin for 50% inhibition (I(50)) for oxygen consumption was five times higher than its I(50) for F(1)F(0)-ATPase. Because of the different control strengths of F(1)F(0)-ATPase in oxidative phosphorylation and ATP hydrolysis an inhibition of the F(1)F(0)-ATPase activity in ischemia with the resultant ATP-sparing has an advantage even in an ischemia/reperfusion situation.     

Osmolytes protect mitochondrial F(0)F(1)-ATPase complex against pressure inactivation

We have previously reported that carbohydrates and polyols protect different enzymes against thermal inactivation and deleterious effects promoted by guanidinium chloride and urea. Here, we show that these osmolytes (carbohydrates, polyols and methylamines) protect mitochondrial F(0)F(1)-ATPase against pressure inactivation. Pressure stability of mitochondrial F(0)F(1)-ATPase complex by osmolytes was studied using preparations of membrane-bound submitochondrial particles depleted or containing inhibitor protein (IP). Hydrostatic pressure in the range from 0.5 to 2.0 kbar causes inactivation of submitochondrial particles depleted of IP (AS particles). However, the osmolytes prevent pressure inactivation of the complex in a dose-dependent manner, remaining up to 80% of hydrolytic activity at the highest osmolyte concentration. Submitochondrial particles containing IP (MgATP-SMP) exhibit low ATPase activity and dissociation of IP increases the hydrolytic activity of the enzyme. MgATP-SMP subjected to pressure (2.2 kbar, for 1 h) and then preincubated at 42 degrees C to undergo activation did not have an increase in activity. However, particles pressurized in the presence of 1.5 M of sucrose or 3.0 M of glucose were protected and after preincubation at 42 degrees C, showed an activation very similarly to those kept at 1 bar. In accordance with the preferential hydration theory, we believe that osmolytes reduce to a minimum the surface of the macromolecule to be hydrated and oppose pressure-induced alterations of the native fold that are driven by hydration forces.     

Purification and Identification of the Fusicoccin Binding Protein from Oat Root Plasma Membrane

Fusicoccin (FC), a fungal phytotoxin, stimulates the H⁺-ATPase located in the plasma membrane (PM) of higher plants. The first event in the reaction chain leading to enhanced H⁺-efflux seems to be the binding of FC to a FC-binding protein (FCBP) in the PM. We solubilized 90% of the FCBP from oat (Avena sativa L. cv Victory) root PM in an active form with 1% octyl-glucoside. The FCBP was stabilized by the presence of protease inhibitors. The FCBP was purified by affinity chromatography using FC-linked adipic acid dihydrazide agarose (FC-AADA). Upon elution with 8 molar urea, two major protein bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with molecular weights of 29,700 and 31,000 were obtained. Successive chromatography on DEAE Bio-Gel A, hexyl agarose, and FC-AADA resulted in the same two bands when the FC-AADA was eluted with sodium dodecyl sulfate. A direct correlation was made between ³H-FC-binding activity and the presence of the two protein bands. The stoichiometry of the 29,700 and 31,000 molecular weight bands was 1:2. This suggests that the FCBP occurs in the native form as a heterotrimer with an apparent molecular weight of approximately 92,000.