Purification and characterization of the ouabain-sensitive H +/K +-ATPase from guinea-pig distal colon

Distal colon absorbs K + through a Na +-independent, ouabain-sensitive H +/K +-exchange, associated to an apical ouabain-sensitive H +/K +-ATPase. Expression of HKα2, gene associated with this ATPase, induces K +-transport mechanisms, whose ouabain susceptibility is inconsistent. Both ouabain-sensitive and ouabain-insensitive K +-ATPase activities have been described in colonocytes. However, native H +/K +-ATPases have not been identified as unique biochemical entities. Herein, a procedure to purify ouabain-sensitive H +/K +-ATPase from guinea-pig distal colon is described. H +/K +-ATPase is Mg 2+-dependent and activated by K +, Cs + and NH 4 + but not by Na + or Li +, independently of K +-accompanying anion. H +/K +-ATPase was inhibited by ouabain and vanadate but insensitive to SCH-28080 and bafilomycin-A. Enzyme was phosphorylated from [ 32P]-γ-ATP, forming an acyl-phosphate bond, in an Mg 2+-dependent, vanadate-sensitive process. K + inhibited phosphorylation, effect blocked by ouabain. H +/K +-ATPase is an α/β-heterodimer, whose subunits, identified by Tandem-mass spectrometry, seems to correspond to HKα2 and Na +/K +-ATPase β1-subunit, respectively. Thus, colonic ouabain-sensitive H +/K +-ATPase is a distinctive P-type ATPase.