EFFECTS OF W-7, W-5, VERAPAMIL AND DILTIAZEM ON VACUOLAR PROTON TRANSPORT - COMPARISON OF VACUOLAR H+-ATPASE AND H+-PPASE FROM ROOTS OF ZEA-MAYS

The vacuolar membrane of plant cells is characterized by two proton pumps: the vacuorar H+-ATPase (V-ATPase; EC 3.6.1.3) and the vacuolar H+-PPase (V-PPase; EC 3.6.1.1). Recently, Du Pont and Morrissey reported that Ca2+ stimulates hydrolytic activity of purified V-ATPase (Arch. Biochim. Biophys., 1992, 294: 341-346). Since this effect may be due to degradation during purification further investigation of Ca2+ regulation of native V-ATPase was done. However, native tonoplast membranes contain a Ca2+/H+ antiport activity, which interferes with effects of calcium ions on proton transport activity of vacuolar ATPase. Therefore, the effects of anti-calmodulin drugs (W-7, W-5, calmidazolium), and calcium channel antagonists (Verapamil, Diltiazem) on proton transport activities of the vacuolar-type H+-ATPase and H+-PPase in tonoplast enriched membrane vesicle preparations from roots of Zea mays L. were studied. The concentrations for half maximal inhibition of vacuolar H+-ATPase (H+-PPase) were: 71 (191) mu M W-7,470 (> 800) mu M W-5, 26 (24) mu M calmidazolium (= compound R 24571), 398 (700) mu M Verapamil, and 500 (1330) mu M Diltiazem. Estimation of Hill coefficients (n(H)) for the inhibition by Verapamil showed a further difference between the two vacuolar proton pumps (H+-ATPase, n(H)=2.02; H+-PPase, n(H)=0.96). The data indicate that the vacuolar H+-ATPase itself is affected by these chemicals. It is suggested that some biological activities of W-7, W-5, Verapamil, and Diltiazem are due to their effects on proton translocation by the vacuolar-type H+-ATPase.