The Effects of Vanadate on the Plasma Membrane ATPase of Neurospom crassa*

The plasma membrane ATPase of Neurospom cmsa, which is believed to function as an electrogenic proton pump, is extremely sensitive to inhibition by vanadium in the +5 oxidation state (vanadate). At pH 6.7 (the pH optimum of the ATPase), inhibition is half-maximal between 0.45 and 1.0 PM vanadate, depending upon the ionic composition of the reaction medium. Inhibition also depends upon pH, with the result that 1.6 PM vanadate causes an apparent shift of pH optimum to 6.0. EDTA prevents inhibition of the Neurospom ATPase by vanadate. It does not act by chelating M%‘, since other chelators (trens-1,2diaminocyclohexanetetraacetic acid (CDTA), ethyleneglycol bis(P-aminoethyl ether)N,N,N,N-tetraacetic acid (EGTA)) are ineffective and since, over a broad range of concentrations, MgClz has little effect on the degree of inhibition by vanadate. Bather, EDTA appears to complex vanadate directly. as has previously been reported in the chemical literature. We have used vanadate to explore the reaction mechanism of the Neurospom ATPase in two ways. 1) When assayed with vanadate-free ATP, the ATPase shows a sigmoid dependence upon substrate concentration, consistent with the notion that the enzyme has two or more substrate binding sites acting cooperatively. Vanadate stimulates ATPase activity at low substrate concentrations and inhibits at high concentrations, thus reducing the apparent cooperativity between sites. 2) We have previously reported (Bowman, B. J., and Slayman, C. W. (1977) J. BioL Chem. 252, 3357-3363) that Neumspom plasma membrane ATPase activity is stimulated 20 to 62% by K+ or NH+. Under these conditions, vanadate still inhibits more than 95% of the activity, and Dixon plots of the data are linear. The results can best be accounted for in terms of a single plasma membrane ATPase which is subject to nonspecific salt effects, and are difficult to reconcile with the idea that the K+-stimulated portion of ATPase activity represents a discrete enzyme involved in K+ transport.