Steady-state kinetic properties of FoF1-ATPase: the pH effect.
1. The kinetic properties of FoF1-ATPase from submitochondrial particles isolated from rat heart were studied, with emphasis to the pH effect. The velocity data were treated according to the Hill equation, and the results were discussed on the basis of the knowledge on the soluble F1-ATPase properties. 2. Three kinetic phases were observed in the range of pH 6.0-8.5, with apparent dissociation constant values (K0.5) of 0.001, 0.04 and 1.5 mM (respectively sites I, II and III) at pH 7.0. Their contribution to the total activity of the enzyme were pH-dependent on the range of 6.0-7.0, but not from 7.0 to 8.5, where the maximal velocity (V) for site III was some 4-fold larger than for site II, and the total V of sites II and III was some 40-fold larger than V assumed for site I. Therefore, two catalytic sites seem to participate significantly in the catalysis at steady-state condition. 3. Azide increased the sites II and III K0.5 values as well as decreased the site III V. In the presence of bicarbonate these two sites were not distinguishable, and the kinetic parameters at pH 7.0 were similar to those for sites II and III combined. Both azide and bicarbonate did not have a significant effect on site I, and this behavior was not pH-dependent. 4. The studies on the effect of pH on the kinetic parameters showed the following results: (1) the optimum pH for V was around 8.5; (2) decrease in the K0.5 values at pH below 7.0 for site II, and increase at pH over 7.0 for sites II and III; (3) in the pH range of 6.0-8.5 the Hill coefficient increased for site II, decreased for site III, and an intermediary effect was observed for the sites II and III combined, with a Michaelis-Menten behavior in the highest affinity pH, which was found in the physiological range.
Uyemura
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Departamento de Física e Química, Faculdade de Ciências Farmacêuticas-USP, Ribeirão Preto, Brazil.
Curti
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D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001386:Azides / Q000494:pharmacology; D001639:Bicarbonates / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008297:Male; D008929:Mitochondria, Heart / Q000201:enzymology; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar
D001386:Azides; D001639:Bicarbonates; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases