Ecto-adenosinetriphosphatase in rat small intestinal brush-border membranes.
Antibodies against the holo ecto-adenosinetriphosphatase (ATPase) of rat liver and antibodies against COOH-terminal peptides of the long isoform of this enzyme reacted in Western blots with a 105-kDa band from small intestinal brush-border membranes. Indirect immunofluorescence revealed reactive proteins predominantly at the apical surface of enterocytes with some staining of basolateral membranes and of vascular endothelium. Similar results were obtained with monoclonal antibodies against HA4, a protein from rat liver closely related to the ecto-ATPase. Since these results suggested the presence of an ecto-ATPase, ATP hydrolysis was studied in intact, right-side-out brush-border membrane vesicles. Nearly half of ATP hydrolysis was caused by alkaline phosphatase (AP). Besides purine and pyrimidine trinucleotides, AP also hydrolyzed ADP, AMP, pyrophosphate, and 4-nitrophenylphosphate. Inactivation of AP by cleavage of its membrane anchor and by removal of the Zn2+ necessary for its function left the ecto-ATPase that was activated by Ca2+ and Mg2+ and hydrolyzed purine and pyrimidine trinucleotides and dinucleotides, but not AMP, pyrophosphate, and 4-nitrophenylphosphate. These features are characteristic of an ATP diphosphohydrolase (EC 3.6.1.5, also called apyrase). The physiological role of the small intestinal ecto-ATPase may be the degradation of nutrient nucleotides.
Schweickhardt
C
C
Zentrum Physiologie und Pathophysiologie, Göttingen, Germany.
Sabolić
I
I
Brown
D
D
Burckhardt
G
G
D000227:Adenine Nucleotides / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000276:immunology / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000469:Alkaline Phosphatase / Q000037:antagonists & inhibitors / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D003124:Colorimetry; D004798:Enzymes / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007106:Immune Sera; D007421:Intestine, Small / Q000378:metabolism*; D008871:Microvilli / Q000378:metabolism; D017830:Octoxynol / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar
D000227:Adenine Nucleotides; D004798:Enzymes; D007106:Immune Sera; D000255:Adenosine Triphosphate; D017830:Octoxynol; D000469:Alkaline Phosphatase; D000251:Adenosine Triphosphatases; C027201:ectoATPase