Functional molecular masses of vacuolar membrane H⁺‐ATPase from Saccharomyces cerevisiae as studied by radiation inactivation analysis

The functional molecular masses of the vacuolar membrane H+-ATPase in Saccharomyces cerevisiae under two kinetic conditions for ATP hydrolysis were measured by radiation inactivation. When vacuolar membrane vesicles were exposed to gamma-rays from 60Co, the activities catalyzing a single-cycle and multi-cycles of ATP hydrolysis both decreased as single-exponential functions of the radiation dosage. By applying the target theory, the functional molecular masses for single- and multi-cycle hydrolyses of ATP were determined to be approx. 0.9-1.1 X 10(5) and 4.1-5.3 X 10(5) Da, respectively. N,N'-Dicyclohexylcarbodiimide (DCCD) did not inhibit the former reaction but strongly inhibited the latter. It is suggested that the ATPase with a minimal composite of subunits a and b, in which subunit c is not necessarily involved operationally, can catalyze single-cycle hydrolysis of ATP, whereas for multi-cycle hydrolysis of ATP, the ATPase requires a properly organized oligomeric structure with subunits a-c, which may direct a positive cooperative mechanism of ATP hydrolysis and coupled H+ translocation in a DCCD-sensitive manner.