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-beta-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 microM NEM or NBD-Cl and 92% inhibited by 20 microM DCCD.(ABSTRACT TRUNCATED AT 250 WORDS)