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

Archives of Biochemistry and Biophysics
Dimas C BelisarioJesús R del Castillo

Abstract

Distal colon absorbs K+ through a Na+-independent, ouabain-sensitive H+/K+-exchange, associated to an apical ouabain-sensitive H+/K+-ATPase. Expression of HKalpha2, 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 Mg2+-dependent and activated by K+, Cs+ and NH4+ 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]-gamma-ATP, forming an acyl-phosphate bond, in an Mg2+-dependent, vanadate-sensitive process. K+ inhibited phosphorylation, effect blocked by ouabain. H+/K+-ATPase is an alpha/beta-heterodimer, whose subunits, identified by Tandem-mass spectrometry, seems to correspond to HKalpha2 and Na+/K+-ATPase beta1-subunit, respectively. Thus, colonic ouabain-sensitive H+/K+-ATPase is a distinctive P-type ATPase.

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Citations

Jan 30, 2015·Molecular Neurobiology·Rocío Retamales-OrtegaNibaldo C Inestrosa
Jan 17, 2014·American Journal of Physiology. Renal Physiology·Gilles Crambert
Apr 5, 2014·American Journal of Physiology. Gastrointestinal and Liver Physiology·Christopher WelshJaques Belik
Dec 29, 2020·OncoTargets and Therapy·Yang-Yang BaoWei-Wei Yong

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