Phosphatase Activities of Rat Intestinal Enterocytes and Their Relation to Diverse Luminal pH, with Special References to the Possible Localization of Phytase along the Brush Border Membrane

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Phosphatase activities associated with the intestinal brush border membrane (BBM) of the rat were examined histochemically in relation to the characteristic environment of the intestine, where luminal pH fiuctuates drastically between alkaline and acid pH ranges. Special attention was given to intestinal alkaline phosphatase (IALP) and phytase on the BBM. Whole body fresh-frozen sections of young rats and their rapidly frozen and freeze-substituted small intestines, embedded in Technovit 7100, were processed for the histochemical demonstration of phosphatase activity at three different pH values (9.2, 7.3, and 5.2), representing the deviation of luminal pH in vivo. Either an azo-dye method or lead-salt method was employed using naphthol AS-MX phosphate and ATP as substrate, respectively. With the azo-dye method, intense phosphatase reactions were demonstrated along the BBM at all three pH ranges. Phosphatase reactions of the BBM at pH 9.2 and 7.3 were abolished by L(+)-phenylalanine, heat pre-treatment, and EDTA chela-tion although some reaction remained at pH 7.3 after the treatment with EDTA or L(+)-phenylalanine. Phosphatase reactions of the BBM at pH 5.2 were resistant to L(+)-phenylalaniue, L(+)-tartrate, PCMB and EDTA chelation, implying that the characteristics of the enzyme responsible for phosphohydrolysis at acid pH values differed from those at higher pH values. The lead-salt method in which ATP was used as substrate revealed intense reactions - which were dependent on Mg⁺⁺ and stimulated by Ca⁺⁺ and resistant to L(+)-phenylalanine - to be localized along the BBM at alkaline and neutral pH values, but not at acid pH values. In vitro experiments showed progressive hydrolysis of naphthol AS-MX phosphate by purified phytase at pH 5.2, in a dose-dependent manner, and suggested the possible involvement of phytase in the phosphatase reactions of the BBM at acid pH.
These data indicate that the phosphatase reactions at alkaline and neutral pH values, associated with the BBM of the rat intestine, represent IALP and Mg⁺⁺/Ca⁺⁺-ATPase, while those at acid pH appear to correspond to phytase activity, something which has not been demonstrated by histochemical methods despite the availability of extensive data based on biochemical analyses.