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

Abstract

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 γ‐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 × 10⁵ and 4.1–5.3 × 10⁵ 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.