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 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.
Belisario
Dimas C
DC
Laboratorio de Fisiología Molecular, Centro de Biofísica y Bioquímica, Instituto Venezolano de Investigaciones Científicas (IVIC), Apartado 21827, Caracas 1020-A, Venezuela.
Rocafull
Miguel A
MA
del Castillo
Jesús R
JR
D000595:Amino Acid Sequence; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D016764:Cell Polarity; D003106:Colon / Q000166:cytology / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D015971:Gene Expression Regulation, Enzymologic; D006168:Guinea Pigs; D017506:H(+)-K(+)-Exchanging ATPase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D008297:Male; D008969:Molecular Sequence Data; D009092:Mucous Membrane / Q000166:cytology; D010042:Ouabain / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012964:Sodium / Q000378:metabolism; D053719:Tandem Mass Spectrometry
D007527:Isoenzymes; D010750:Phosphoproteins; D010042:Ouabain; D012964:Sodium; D017506:H(+)-K(+)-Exchanging ATPase