Kinetic studies of chromaffin granule H+-ATPase and effects of bafilomycin A1.

Vacuolar type H+-ATPase purified from bovine chromaffin granules did not show simple Michaelis-Menten type kinetics, and had apparent Km values of 5 microM, 30 microM and 300 microM. These three Km values suggested the presence of catalytic cooperativity during steady-state hydrolysis. The single turnover rate was 10(-3)-fold the maximal velocity of the enzyme and similar to the rate estimated from the velocity of steady-state hydrolysis with the smallest Km value (5 microM). The H(+)-ATPase was inhibited by the stoichiometric binding of bafilomycin A1, a specific inhibitor of vacuolar type H(+)-ATPase. This inhibitor not only lowered the rate of ATP hydrolysis at the single catalytic site, but also affected the catalytic cooperativity of the enzyme.