Tertiary amines as antagonists of both the luminal and cytosolic K(+)-site of gastric H,K-ATPase.

Tertiary amines like imidazole and triallylamine lower the apparent affinity of K+ in the ATP hydrolysis reaction of pig gastric H,K-ATPase in a pH and amine concentration dependent way. The mechanism and sidedness of this effect was studied by analyzing the partial reactions of the enzyme in both leaky and ion-tight vesicles. In leaky vesicles Tris and Hepes had nearly no effect on the apparent Km for K+ in the ATPase reaction, but imidazole (Ki = 13 mM) and triallylamine (Ki = 1.6 mM) markedly decreased the K+ affinity. The steady-state ATP-phosphorylation level in the absence of K+ was not or only slightly affected by these compounds. The reduction of the ATP-phosphorylation level by K+, however, again depended on both the type and concentration of tertiary amine used. A comparable K(+)-amine antagonism was observed in the dephosphorylation reaction. In tightly sealed vesicles, where no activation of K+ at the luminal side could occur, K+ reduced the affinity for ATP in the phosphorylation reaction. Triallylamine counteracted this effect. The K(+)-activated p-nitrophenylphosphatase activity in these ion-tight vesicles also showed a K(+)-triallylamine antagonism. Inhibition of H,K-ATPase activity in these vesicles by triallylamine was immediate (with nigericin present in order to allow intravesicular K+ activation), suggesting the transmembrane feature of this inhibition. These results indicate that tertiary amines decrease the affinity for K+ at both luminal and cytosolic binding sites by interaction at the cytosolic side of the membrane. This results in shifts in the equilibrium of both the E1.H<==>E1.K transition and in the dephosphorylation reaction, E2-P-->E2.K.