Ecto-adenosinetriphosphatase in rat small intestinal brush-border membranes

Abstract

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.

Purinergic signalling in the gastrointestinal tract and related organs in health and disease

“…In situ hybridisation studies have shown that the mRNA for P2X4 receptors is localised in rat intestinal crypts [653]. The strong presence of ecto-diphosphohydrolase (apyrase) in rat small intestinal brush-border membranes has been demonstrated [611], consistent with the view that nucleotides have potent actions in mucosal epithelial cells. A study of goblet cell-like clone derived from colonic HT-29 cells led to the conclusion that ATP-stimulated increase in Cl − current does not require an increase in [Ca 2+ ] i suggesting the involvement of either another signalling pathway or direct activation of Cl − channels via purinergic receptors [305].…”

Absence of vacuolar H + -ATPases from rat small intestine brush-border membranes

“…Intact BBMV from rat small intestine also possess ATP-hydrolysing enzymes at their outer surface [5]. One of these enzymes is a levamisole-insensitive alkaline phosphatase.…”

“…Brush-border membrane vesicles (BBMV) from entire small intestine and kidneys were prepared by a Mg 2+ precipitation technique [5,7]. Vesicles were loaded with KCl buffer (150 mM KCl, 10 mM HEPES/Tris, pH 7.0), adjusted to a protein concentration of 5 mg·ml -1 , and frozen in liquid N 2 until use.…”

“…by overnight incubation at 4°C in 5 mmol·l -1 EDTA to remove the Zn 2+ necessary for enzyme function [5]. Although the renal alkaline phosphatase is readily inhibitable by levamisole, renal BBMV were pretreated in a similar fashion to small intestine vesicles to allow for comparison of the data.…”

“…Bafilomycin was used as a specific inhibitor of the vacuolar-type H + -ATPase. The reaction was stopped 15 min after ATP addition and the liberated phosphate was determined by a colorimetric assay as described earlier [5].…”