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 μM, 30 μM and 300 μM. 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 μM). 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.