ECTO-ADENOSINE-TRIPHOSPHATASE IN RAT SMALL-INTESTINAL BRUSH-BORDER MEMBRANES

Antibodies against the hole ecto-adenosmetriphosphatase (ATPase) of ra t liver and antibodies against COOH-terminal peptides of the long isof orm of this enzyme reacted in Western blots with a 105-kDa band from s mall intestinal brush-border membranes. Indirect immunofluorescence re vealed reactive proteins predominantly at the apical surface of entero cytes with some staining of basolateral membranes and of vascular endo thelium. Similar results were obtained with monoclonal antibodies agai nst HA4, a protein from rat liver closely related to the ecto-ATPase. Since these results suggested the presence of an ecto-ATPase, ATP hydr olysis was studied in intact, right-side-out brush-border membrane ves icles. Nearly half of ATP hydrolysis was caused by alkaline phosphatas e (AP). Besides purine and pyrimidine trinucleotides, AP also hydrolyz ed ADP, AMP, pyrophosphate, and 4-nitrophenylphosphate. Inactivation o f AP by cleavage of its membrane anchor and by removal of the Zn2+ nec essary for its function left the ecto-ATPase that was activated by Ca2 + and Mg2+ and hydrolyzed purine and pyrimidine trinucleotides and din ucleotides, but not AMP, pyrophosphate, and 4-nitrophenylphosphate. Th ese features are characteristic of an ATP diphosphohydrolase (EC 3.6.1 .5, also called apyrase). The physiological role of the small intestin al ecto-ATPase may be the degradation of nutrient nucleotides.