Evidence for an ATP-driven H⁺-pump in the plasma membrane of the bovine corneal epithelium

In a highly enriched plasma membrane fraction isolated from the bovine corneal epithelium, MgATP dependent intravesicular acidification was identified by measuring Acridine Orange quenching. The rate of acidification was increased 2·7-fold by pre-exposure of the membranes to 1% cholate which was subsequently removed by Sephadex G-50 column chromatography. However, in a lysosomal fraction whose enrichment with respect to the homogenate was 82-fold in N-acetyl-β-d-glucosaminidase, cholate pre-exposure had no significant effect on the rate of intralysosomal acidification. This difference is assumed to reflect reorientation by cholate of the H⁺-pump's normally inaccessible ATP-binding site in right-side-out vesicles of the plasma membrane-enriched fraction to a configuration in which this site becomes accessible to externally added ATP. In contrast, the ATP-binding site of the H⁺-pump in the lysosomal fraction is completely exposed to the exterior even in the absence of cholate treatment. The characteristics of the H⁺-pump in the plasma membrane fraction was subsequently determined using cholate-pretreated membrane vesicles. The rank order of nucleotide support of the H⁺-pump activity was: ATP ≫ GTP > ITP. However, UTP and CTP were totally inactive. The pump is electrogenic because the activity of the pump was enhanced in voltage-clamped membrane vesicles. Substitution of Mg²⁺ with Mn²⁺ did not change the acidification rate but Co²⁺ only partly activated whereas Ca²⁺ and Zn²⁺ were ineffective as activators. The H⁺-pump was relatively unaffected by oligomycin, azide or vanadate but completely inhibited by 10 μm NEM or NBD-Cl and 92% inhibited by 20 μm DCCD. The pH dependency of the NEM-sensitive ATPase activity showed a single optimum pH at 7·5. It is concluded that a major portion of the H⁺-pump activity in this membrane preparation is derived from the plasma membrane and that the properties of this plasma membrane H⁺-pump are similar to those of the vacuolar (V-type) H⁺-pumps described in various intracellular organelles and in the plasma membrane of other cell types.