Vanadate-sensitive ATPase from chromaffin granule membranes formed a phosphoenzyme intermediate and was activated by phosphatidylserine

Vanadate-sensitive ATPase (115 kDa molecular weight) in adrenal chromaffin granules is an intrinsic membrane enzyme with its catalytic site located at the outer surface of the granules. Upon incubation with [γ-³²P]ATP, the purified ATPase formed an alkaline-labile phosphoenzyme intermediate, which was inhibited by vanadate but not by Na⁺ or K⁺. Ratio of ATPase or phosphatase activity and formation of phosphoenzyme intermediate was constant during purification after the first glycerol density gradient centrifugation. Phosphatidyl-serine specifically activated the enzyme about three-fold by increasing the V_max value without changing the K_m for ATP. Other phospholipids, including phosphatidyl-glycerol, phosphatidylcholine, phosphatidylinositol, and phosphatidylethanolamine, as well as lysophospholipids and detergents, had no effect. These results indicated that the vanadate-sensitive ATPase belongs to the P-type ATPases, which differ from known cation-translocating P-type ATPases.