Identification of a vanadate-sensitive potassium-dependent proton pump from rabbit colon

Abstract

Membrane vesicles prepared from rabbit distal colon were used to study colonic transport mechanisms. Differential and sucrose-Ficoll density gradient centrifugation of the mucosal homogenate yielded fractions which supported ATP-dependent proton transport, as measured with the fluorescent weak base acridine orange. Quenching of acridine orange absorbance in light microsomes and microsome-derived density gradient fractions was MgATP-dependent and was reversed with nigericin; these characteristics suggest the presence of one or more ATP-driven proton pumps. Proton transport in the microsomal fraction was inhibited by N-ethylmaleimide more than by orthovanadate, and was dependent on extravesicular chloride. Vesicles in a microsome-derived gradient fraction were inhibited by orthovanadate more than by N-ethylmaleimide. N-ethylmaleimide pretreatment of this gradient fraction uncovered a vesicle population with characteristics similar to the gastric H+,K+ATPase: proton transport was abolished by orthovanadate and the experimental anti-ulcer drug SCH 28080, was enhanced by potassium, and was not affected by chloride. ATP-generated proton gradients in this fraction were not dissipated by the proton ionophore 3,3',4',5-tetrachlorosalicylanilide. We conclude that two ATP-driven proton pumps are present in mucosa from distal rabbit colon; one with characteristics of N-ethylmaleimide-sensitive organelle associated proton pumps, and one similar to the gastric proton-potassium exchanger.