The H+‐ATPase of the plasma membrane from yeast

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Abstract
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 Km= 0.2 mM MgATP (plasma membranes), Km= 2.4 mM MgATP (isolated enzyme) and Km= 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 Mg2+ is also only slightly influenced by the detergent. The dissociation constant of the enzyme‐Mg2+ complex Ka, does not alter during isolation and the dissociation constant of the enzyme‐substrate complex, Ks, increases from Ks=30 μM (plasma membranes) to Ks= 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 k1= 2.6 · 104 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.