Isolation and localization of H, K-ATPase in colonic epithelial cell.

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1)Solubilization of H,K-ATPase: Approximately 30% of the ouabain-sensitive H,K-ATPase activity in crude membrane fraction of epithelial cells from guinea pig distal colon has been solubilized with 0.05% n-Dodecyl-b-D-maltoside. 2)Histochemistry: Cerium-based cytochemical method failed to reveal the localization of the ouabainsensitive H,K-ATPase because the reaction product of ATPase activity was not blocked by ouabain. 3)cDNA cloning: cDNAs encoding the colonic H,K-ATPase were isolated and its nucleotide sequence was determined. 155 b Hpall fragment of the rat Na,K-ATPase a1 cDNA was used as a probe to screen a cDNA library constructed in l_<zap>ll from poly(A)+ RNA isolated from the mucosa of the guinea pig distal colon. The cDNA is 4242 nucleotides in length and contains 144 bp of 5'-untranslated region, 3102 bp of an open reading frame and 996 bp of 3'-untranslated region. The deduced amino acid sequence predicts a protein of 1033 residues which belongs to the P-type family of membrane-transporting ATPases and exhibits 67% amino acid identity to the rat Na,K-ATPase a1, and 64% identity to the rat gastric H,K-ATPase a subunit. In immunoblotting, a polyclonal antibody raised against a synthetic peptide (residues 10-24) identified a membrane protein of 94 kd in the distal, but not in the proximal colon. These findings suggest that the cloned cDNA represents the putative H,K-ATPase in the distal colon.