Purification and characterization of the ouabain-sensitive H/K-ATPase from guinea-pig distal colon |
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Authors: | Dimas C Belisario |
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Institution: | Laboratorio de Fisiología Molecular, Centro de Biofísica y Bioquímica, Instituto Venezolano de Investigaciones Científicas (IVIC), Apartado 21827, Caracas 1020-A, Venezuela |
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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 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 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]-γ-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 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. |
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Keywords: | Mammalian distal colon Potassium absorption HKα2 gene Na+/K+-ATPase β1-subunit |
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