Vanadate binding to the gastric H,K-ATPase and inhibition of the enzyme's catalytic and transport activities.
Vanadate inhibition of the catalytic and transport activities of the gastric magnesium-dependent, hydrogen ion transporting, and potassium-stimulated adenosinetriphosphatase (EC 3.6.1.3) (H,K-ATPase) has been studied. The principal experiment observations are the following: (1) Inhibition of adenosine 5'-triphosphate (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 ions bind specifically to gastric vesicles with two affinities. Vanadate binding in the presence of nucleotide is compatible with competition for the kinetically defined high-affinity and low-affinity ATP sites. (3) 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 (4) At most, 3 nmol mg-1 vanadate is required to inhibit proton transport. The simplest interpretation of the data is that vanadate inhibits the H,K-ATPase by binding competitively with ATP at two 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.
D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors*; D000818:Animals; D017301:Ca(2+) Mg(2+)-ATPase; D017506:H(+)-K(+)-Exchanging ATPase; D007700:Kinetics; D008433:Mathematics; D008861:Microsomes / Q000201:enzymology; D010766:Phosphorylation; D011485:Protein Binding; D013270:Stomach / Q000201:enzymology*; D013552:Swine; D014638:Vanadates; D014639:Vanadium / Q000494:pharmacology*
D014639:Vanadium; D014638:Vanadates; D000251:Adenosine Triphosphatases; D017301:Ca(2+) Mg(2+)-ATPase; D017506:H(+)-K(+)-Exchanging ATPase