Structural rearrangements in soluble mitochondrial ATPase

Treatment of isolated factor F1 by 1% dimethylsuberimidate in the presence of 50 mM (NH4)2SO4 leads to the formation of four different types of cross-linked dimers of the subunits, on average one dimer per molecule of the enzyme. This treatment results in 60-70% inactivation of factor F1. Factor F1 treated with dimethylsuberimidate does not show a change in the sedimentation coefficient and is not inactivated in the cold; it is not inactivated in the presence of Mg2+ either, nor is it activated by anions. Incubation of the cross-linked factor F1 with ADP does not lead to inactivation, although the ability to tightly bind ADP is retained. The total quantity of tightly bound ADP reaches 5 mol per mol of the cross-linked factor F1. Cross-linking of factor F1 also prevents the slow inactivation of the enzyme coupled with the hydrolysis of Mg-ATP and Mg-GTP. The dependence of the inactivation rate constant on the concentration of Mg-ATP and Mg-GTP at substrate concentrations of 0.05-2 mM is characterized by the same values of Km,app as those of the ATPase and GTPase activities of factor F1. The probability of the inactivation of factor F1 per turnover remains constant for all the concentrations of the substrates studied and is 2 . 10(-6) per turnover for the ATPase reaction and 2 . 10(-5) per turnover for the GTPase reaction. Moderate hydrostatic pressure (up to 150 atmospheres) greatly accelerates ATP-induced inactivation of factor F1. The activation volume (delta V*) of the inactivation process is equal to 5.1 . 10(-4) cm3/g, which is evidence of considerable changes in the extent of protein hydration during inactivation. Inactivation of the enzyme under pressure is accompanied by dissociation into subunits. Dimethyladipimidate, which does not cause intersubunit cross-linking in the molecule of factor F1, does not alter the properties of the native enzyme. It is suggested that the formation of one intersubunit cross-link in the molecule of factor F1 by dimethylsuberimidate affects the ability of the enzyme to undergo co-operative rearrangements of the quaternary structure under the influence of Mg2+, ADP, ATP, anions, and low temperature. The rate constants of ATP binding to the active site of factor F2 (k+1) = 2 . 10(8) M-1 . min-1), of ATP release from the active site (k-1 = 2 . 10(-2) min-1), and of ADP and Pi release from the active site (k2 = 5 . 10(3) min-1) have been determined. The results obtained confirm the correctness of Boyer's idea, according to which ATP is formed in the active site of mitochondrial ATPase without any external source of energy. Energy is used at the stage of the release of synthesized ATP from the active site of ATPase in the solution.     

Functional role of soluble mitochondrial ATPase subunits

A preparation of soluble mitochondrial ATPase (coupling factor F₁) containing no and minor subunits has been isolated. The minor-subunits-deficient F₁ was found to be competent in ATP hydrolysis. However, it did not demonstrate a coupling effect in EDTA-submitochondrial particles. A portion of the ATPase activity of EDTA particles, stimulated by the minor-subunits-deficient F₁, was insensitive to oligomycin. ATPase activity of Na⁺-particles was changed only slightly by this F₁. It is suggested that and subunits are necessary to form specific contacts between the F₁ molecule and components of the mitochrondrial membrane.Abbreviations SMP submitochondrial particles - F₁ coupling factor (soluble mitochondrial ATPase) - PCB^– phenyl dicarbaundecaborane anions     

Substrate specificity of soluble mitochondrial ATPase]

The parameters of the hydrolysis of ATP and several analogs by soluble mitochondrial ATPase were determined. Vmax of the reaction decreases within the range: 2'-desoxy-ATP greater than ATP greater than etheno-ATP greater than GTP greater than 3'-O-methylATP greater than UTP. ATP, 2'-desoxypATP, 3'O-methyl-ATP, GTP, and etheno-ATP are hydrolysed by soluble mitochondrial ATPase with close Km(app) values. CTP is not hydrolysed by the enzyme and does not inhibit the ATPase reaction at a concentration of 10(-2) M. Nucleoside triphosphate derivatives with an "open" ribose cycle 9-[1',5'-dihydroxy-4-(S)-hydroxymethyl-3'-oxapent-2' (R)-yl]adenyl-5'-triphosphate, and 1-[1',5'-dihydroxy-4'-(S)-hydroxymethyl-3'-oxapent-2'(R)-yl[cytosine-5'-triphosphate are effective inhibitors of ATPase (Ki approximately 5.10(-5)M). Mitochondrial ATPase binds the ATP analogs that have hydrocarbon radicals-(CH2)2-, -(CH2)3-, and (CH2)4- instead of the ribose residues: 9-(2'hydroxyethyl)adenyl-2'-triphosphate, 9-(3'-hydroxypropyl)-adenine-3'-triphosphate, and 9-(4'-hydroxybutyl)adenine-4'-triphosphyl)adenine-4'-triphosphate were not hydrolysed by the enzyme, although they inbibit the ATPase reaction (Ki 2.10(-4)M). 9-(2'-hydroxyethyl)adenine-2'-triphosphate is hydrolysed by ATPase eight times more slowly than ATP. It is suggested that the hydrolysis of the substrates of mitochondrial ATPase is- preceded by the binding of the substrates in a tense conformation in the active site of the enzyme.     

Inhibition of muscle actomyosin ATPase activity by mitochondrial ATPase inhibitor.

Ascites hepatoma cell line AH-130 was tested for the ability to transport various amino acids and glutathione before and after γ-glutamyl transpeptidase of the cells was affinity-labeled and inactivated by 6-diazo-5-oxo-L-norleucine, a glutamine analog. The rate of uptake of alanine, glycine, leucine and glutamine by the cells remained unchanged after γ-glutamyl transpeptidase was inactivated by this affinity label. This indicated that γ-glutamyl transpeptidase of the cell was not involved in the transport process of these amino acids tested. The uptake of glutathione was also tested before and after affinity labeling the enzyme. The total amount of the radioactivity incorporated into the cells was not significantly affected by the enzyme inactivation. However, the relative amount of incorporated intact glutathione was found to be slightly but significantly increased after membraneous γ-glutamyl transpeptidase was inactivated by the affinity label, while that of component amino acid, glycine, was found to decrease. This indicated that glutathione was taken up by the cell in its intact form as well as in degraded forms into its component amino acids, and γ-glutamyl transpeptidase in the ascites tumor cell AH-130 seemed to be involved in the metabolic process via the latter system.     

Functional role of soluble mitochondrial ATPase subunits.

A preparation of soluble mitochondrial ATPase (coupling factor F1) containing no gamma and delta minor subunits has been isolated. The minor-subunits-deficient F1 was found to be competent in ATP hydrolysis. However, it did not demonstrate a "coupling" effect in EDTA-submitochondrial particles. A portion of the ATPase activity of EDTA particles, stimulated by the minor-subunits-deficient F1, was insensitive to oligomycin. ATPase activity of Na+-particles was changed only slightly by this F1. It is suggested that gamma and delta subunits are necessary to form specific contacts between the F1 molecule and components of the mitochrondrial membrane.     

Conformational changes in soluble mitochondrial ATPase by the spin probe method]

Modification of soluble mitochondrial ATPase (factor F1) by spin-labelled iodoacetamide and spin-labelled methyleneketone does not cause and change in the catalytic properties of the enzyme. The temperature dependence of tau corr. of labels bound to factor F1 testifies to conformational changes in the enzyme at temperatures of 18--20 degrees C and 34--37 degrees C. At these temperature intervals, breaks are observed in the temperature dependence of the ATPase reaction rate in the Arrenius plot. The results obtained indicate that the thermally induced conformational changes in factor F1 affect large areas of the protein molecule. The interaction of factor F1 with the hydrophobic spin probes, namely fatty acid derivatives, was studied. It was shown that the interaction of foctor F1 with Mg2+, Mg-ATP, Mg-ADP and ADP, results in an increase in the ability of the enzyme to adsorb spin probes.     

Action of alkyl cations and the natural ATPase inhibitor from mitochondria on soluble mitochondrial ATPase

The effect of the natural ATPase inhibitor and octylguanidine on the ATPase activity of soluble oligomycin-insensitive mitochondrial F₁ were compared. Both compounds induced a maximal inhibition of 60–80% in various preparations of F₁ studied. The inhibition was of the uncompetitive type with respect to MgATP, and the action of the compounds was partially additive. The data suggest that octylguanidine reproduces the action of the natural ATPase inhibitor. Alkylammonium salts also affect the ATPase activity in a similar form. F₁ bound to Sepharose-hexylammonium is largely inactive, whilst free hexylammonium at higher concentrations induces only a partial inhibition of the activity. This suggests that the degree of immobilization of F₁ is related to the magnitude of inhibition of ATPase activity induced by alkyl cations. The binding of F₁ to Sepharose-hexylammonium is prevented by high concentrations of Na⁺ or K⁺.     

Binding of ADP to beef-heart mitochondrial ATPase (F1)

1. ADP binding to beef-heart mitochondrial ATPase (F1), in the absence of Mg2+, has been determined by separating the free ligand by ultrafiltration and determining it in the filtrate by a specially modified isotachophoretic procedure. 2. Since during the binding experiments the 'tightly' bound ADP (but not the ATP) dissociates, it is necessary to take this into account in calculating the binding parameters. 3. The binding data show that only one tight binding site (Kd about 0.5 microM) for ADP is present. 4. It is not possible to calculate from the binding data alone the number of or the dissociation constants for the weak binding sites. It can be concluded, however, that the latter is not less than about 50 microM.     

Influence of aurovertin on mitochondrial ATPase activity.

Investigations have been made of the kinetic effects of the antibiotic aurovertin on the ATPase and ITPase activity of isolated rat liver mitochondrial ATPase. Unusual patterns of inhibition, decreasing slope, and increasing y-intercept values of double reciprocal plots, were observed with Mg-ATP as the substrate under various conditions. Under specified conditions, aurovertin stimulated hydrolysis of MgATP. The inhibition of MgITP hydrolysis was uncompetitive. Aurovertin eliminated the HCO3-minus stimulation of MgATP hydrolysis. The implications of these findings for the mechanism of mitochondrial ATPase are briefly discussed.     

Conformational changes of soluble mitochondrial ATPase as controlled by hydrophobic interactions within the enzyme.

At 30° C soluble mitochondrial ATPase from baker's yeast shows non-linear kinetics with respect to Mg-ATP; the apparent Km values for Mg-ATP are 0.6 and 2.0 mM. At lower temperatures, 5° C and 12° C, the kinetics of the enzyme are linear with a Km for Mg-ATP of approximately 0.6 mM. Octylguanidine induces non-linear kinetics at 12° C. As octylguanidine and increases in temperature augment hydrophobic interactions within the enzyme, it is concluded that the strength of hydrophobic bonding within the protein regulates its conformational changes. Methanol activates the enzyme only at relatively high temperature which further indicates that the protein may exist in two active conformations.     

Action of alkyl cations and the natural ATPase inhibitor from mitochondria on soluble mitochondrial ATPase.

The effect of the natural ATPase inhibitor and octylguanidine on the ATPase activity of soluble oligomycin-insensitive mitochondrial F1 were compared. Both compounds induced a maximal inhibition of 60-80% in various preparation of F1 studied. The inhibition was of the uncompetitive type with respect to MgATP, and the action of the compounds was partially additive. The data suggest that octylguanidine reproduces the action of the natural ATPase inhibitor. Alkylammonium salts also affect the ATPase activity in a similar form. F1 bound to Sepharose-hexylammonium is largely inactive, whilst free hexylammonium at higher concentrations induces only a partial inhibition of the activity. This suggests that the degree of immobilization of F1 is related to the magnitude of inhibition of ATPase activity induced by alkyl cations. The binding of F1 to Sepharose-hexylammonium is prevented by high concentrations of Na+ or K+.     

A simple method of purification of a soluble oligomycin-insensitive mitochondrial ATPase.

A simple method for the purification of the soluble oligomycin-insensitive mitochondrial ATPase from heart is described. It consists of adsorption of the of the enzyme to Sepharose hexylammonium followed by elution with KCl and a precipitation step with (NH₄)₂SO₄. In sodium dodec'yl sulfate gels, the enzyme shows the A, B, C, D, and E subunits; however, the D and E subunits appear only when the gels are loaded with a high concentration of protein. The K_m for Mg-ATP is approximately 0.6 mm and is inhibited by ADP.     

Modulation of oat mitochondrial ATPase activity by CA2+ and phytochrome

The activity of a Mg(2+)-dependent ATPase present in highly purified preparations of Avena mitochondria was photoreversibly modulated by red/far-red light treatments. These results were obtained either with mitochondria isolated from plants irradiated with white light prior to the extraction or with mitochondria isolated from unirradiated plants only when purified phytochrome was exogenously added to the reaction mixture. Red light, which converts phytochrome to the far red-absorbing form (Pfr) depressed the ATPase activity, and far-red light reversed this effect. Addition of exogenous CaCl2 also depressed the ATPase activity, and the kinetics of inhibition were similar to the kinetics of the Pfr effects on the ATPase. The calcium chelator, ethyleneglycol-bis(beta-amino-ethyl ether)-N,N' -tetraacetic acid, blocked the effects of both CaCl2 and Pfr on the ATPase. These results are consistent with the interpretation that Pfr promotes a release of Ca2+ from the mitochondrial matrix, thereby inducing an increase in the concentration of intermembranal and extramitochondrial Ca2+.     

Regulation by alcohols and anions of soluble ATPase activity in chloroplasts and mitochondria

The alcohols and anions present at the medium before the beginning of ATPase reaction increased or decreased the lag-time of ATPase hydrolysis by chloroplast coupled factor according to the extent of their inhibitory or stimulating action. The effect of alcohols and anions on the ATPase activity of coupled factor of chloroplasts and mitochondria develops less than 2 s in case of their introduction during the ATP hydrolysis.