The H + ‐ATPase of the plasma membrane from yeast

The H + ‐ATPase of the plasma membrane from Saccharmyces cerevisiae has been isolated, purified and reconstituted into asolectin liposomes. The kinetics of ATP hydrolysis have been compared for the H + ‐ATPase in the plasma membrane, in a protein/lipid/detergent micelle (isolated enzyme) and in asolectin proteoliposomes (reconstituted enzyme). In all three cases the kinetics of ATP hydrolysis can be described by Michaelis‐Menten kinetics with K m = 0.2 mM MgATP (plasma membranes), K m = 2.4 mM MgATP (isolated enzyme) and K m = 0.2 mM MgATP (reconstituted enzyme). However, the maximal turnover decreases only by a factor of two during isolation of the enzyme and does not change during reconstition; the activation of the H + ‐ATPase by free Mg 2+ is also only slightly influenced by the detergent. The dissociation constant of the enzyme‐Mg 2+ complex K a , does not alter during isolation and the dissociation constant of the enzyme‐substrate complex, K s , increases from K s =30 μM (plasma membranes) to K s = 90 μM (isolated enzyme). ATP binding to the H + ‐ATPase (‘single turnover’ conditions) for the isolated and the reconstituted enzyme resulted in both cases in a second‐order rate constant k 1 = 2.6 · 10 4 M −1 · s −1 . From these observations it is concluded that the detergent used (Zwittergent TM 3‐14) interacts reversibly with the H + ‐ATPase and that practically all H + ‐ATPase molecules are reconstituted into the liposomes with the ATP‐binding site being directed to the outside of the vesicle.