Vanadate binding to the gastric hydrogen(1+)-transporting, potassium(1+)-stimulated ATPase and inhibition of the enzyme's catalytic and transport activities
Vanadate inhibition of the catalytic and transport activities of the [hog] gastric Mg-dependent, H+ ion transporting, and K-stimulated ATPase (EC 3.6.1.3) (H,K-ATPase) was studied. The principal experimental observations are the following: Inhibition of ATP hydrolysis is biphasic. Vanadate binding with a stoichiometry of 1.5 nmol mg-1 approximately halves K+-stimulated ATPase activity at physiological temperature. The remaining activity is inhibited by binding an additional 1.5 nmol mg-1 vanadate with lower apparent affinity. In the presence of both Mg2+ and K+, vanadate ions bind specifically to gastric vesicles with 2 affinities. Vanadate binding in the presence of nucleotide is compatible with competition for the kinetically defined high-affinity and low-affinity ATP sites. Vanadate inhibits phosphoenzyme formation and the K+-stimulated p-nitrophenyl phosphatase activity of the enzyme monophasically. A maximum of 1.5 nmol mg-1 acid-stable phosphoenzyme is formed. The half-time for vanadate dissociation from the site that inhibits p-nitrophenyl phosphate hydrolysis is 5 min. At most, 3 nmol mg-1 vanadate is required to inhibit proton transport. Vanadate probably inhibits the H,K-ATPase by binding competitively with ATP at 2 catalytic sites. Different catalytic mechanisms at the high-affinity and low-affinity sites are suggested by the different stoichiometries found for vanadate binding and phosphoenzyme formation.