Interaction of Mg2+ with F0.F1 mitochondrial ATPase as related to its slow active/inactive transition.
Bovine heart submitochondrial particles incubated with a low concentration of ADP in the presence of Mg2+ and passed through a Sephadex column equilibrated with EDTA exhibit sensitivity of their initial ATPase activity to preincubation with Mg2+. By using particles thus prepared, several characteristics of a Mg(2+)-specific inhibitory site on F0.F1 ATPase were studied. The inhibition was shown to be both time- and Mg(2+)-concentration-dependent, with an equilibrium constant (at infinite time) of 2 x 10(-6) M (25 degrees C, pH 7.5). The dependence of the pseudo-first-order rate constant for the inhibition process on Mg2+ concentration suggests the presence of a single Mg(2+)-binding site with K8 = 1.1 x 10(-4) M. The data obtained are consistent with a two-step mechanism of Mg(2+)-F0.F1 interaction which results in a loss of the ATPase activity; it includes rapid pH-dependent binding of Mg2+ at the site with K8 = 1.1 x 10(-4) M, followed by a slow interconversion of the Mg(2+)-F1 complex into inactive ATPase (kin. = 0.65 min-1, kact. = 0.01 min-1). The Mg(2+)-inhibited ATPase is very slowly (t1/2 approximately 90 min) re-activated in the presence of EDTA. The rate of EDTA-induced re-activation is pH-independent and can be dramatically increased by added ATP, Pi and sulphite. The dissociation constants for free ATP and P1 (5 x 10(-7) M and 1 x 10(-3) M respectively) and the maximal activation rates were determined by measuring the hyperbolic dependencies of the EDTA-induced re-activation of Mg(2+)-de-activated ATPase on the concentrations of the accelerating ligands. Taken together, the data obtained show two functionally detectable free nucleotide-specific binding sites, one site for Pi and one Mg(2+)-specific ATPase-inhibitory site on the F0.F1 mitochondrial ATP synthase complex.
Bulygin
V V
VV
Department of Biochemistry, School of Biology, Moscow State University, U.S.S.R.
Vinogradov
A D
AD
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000818:Animals; D002417:Cattle; D004492:Edetic Acid / Q000494:pharmacology; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology*; D008929:Mitochondria, Heart / Q000201:enzymology*; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D013367:Submitochondrial Particles / Q000201:enzymology*; D013997:Time Factors
D000244:Adenosine Diphosphate; D004492:Edetic Acid; D006180:Proton-Translocating ATPases; D008274:Magnesium