The Effects of Magnesium on Nucleoside Phosphatase Activity in Frog Skin

Abstract

Histochemical tests, employing the Wachstein-Meisel medium, indicate that nucleoside triphosphatase activity is found predominantly in two areas of the frog skin epidermis: (1) in mitochondria, where activity is enhanced by dinitrophenol, Mg ~ dependent, but inhibited by fixation; and (2) apparently associated with cell membranes of the middle and outer portions of the epidermis, where activity is inhibited by Mg 2+, unaffected by dinitrophenol, and only slightly reduced by fixation. Spectrophotometric analysis shows that Mg ~+ in the medium does not increase spontaneous hydrolysis of ATP, thus obviating the possible explanation that changes in substrate concentrations in the medium lead to alterations in the "staining" distributions. It is postulated that perhaps the two enzymes differ in their requirements for substrate--one requiring the polyphosphate to be in complexed form with Mg ~-, the other uncomplexed. Concentrations of Mg ~ required to inhibit cell membrane nucleoside triphosphatase activity also inhibit the electrical potential difference and shortcircuit current of the fl'og skin. Although these observations might be taken as presumptive evidence of the cell membrane enzyme as a component of the ion pump system, because of certain dissimilarities with respect to the biochemists' "transport ATPase" and for other reasons discussed in the paper, any definite conclusions in this regard are premature.

The Role of Microfilaments in Frog Skin Ion Transport

“…The variable effect of calcium on the electrical potential differences and sodium transport rate have been reported by other workers (2,3,20) and were also observed in the present study. This variability is probably a complex function of season, hormonal levels, and inherent calcium levels.…”

“…We chose this area because filaments are more prominent than in basal layers and because histoehemical tests show that N a -K ATPase activity is greater in this region (3,5).…”

8‐BuS‐ATP derivatives as specific NTPDase1 inhibitors

“…Fixed slices were pre‐incubated for 30 min at room temperature (RT) in 50 mM Tris‐maleate buffer, pH 7.4, containing 2 mM CaCl 2 , 250 mM sucrose and 2.5 mM levamisole as an inhibitor of alkaline phosphatases. Enzymatic reaction was performed for 1 h at 37°C in the same buffer supplemented with 5 mM MnCl 2 to inhibit intracellular staining (Dahl and Pratley, ), 2 mM Pb(NO 3 ) 2 , 3% Dextran T‐250 and in the presence of 200–500 μM ATP with or without 8‐BuS derivatives. For control experiments, substrate was either omitted or added in the absence of divalent cations, which are essential for NTPDase activity.…”

Ticlopidine in Its Prodrug Form Is a Selective Inhibitor of Human NTPDase1

“…Fixed slices were preincubated for 30 min at RT in 50 mM Tris-maleate buffer, pH 7.4, containing 2 mM CaCl 2 , 250 mM sucrose, and 2.5 mM levamisole as an inhibitor of alkaline phosphatases. Enzymatic reaction was performed for 1 h at 37°C in the same buffer supplemented with 5 mM MnCl 2 to inhibit intracellular staining [ 48 ], 2 mM Pb(NO 3 ) 2 , 3% Dextran T-250 and in the presence of 200 μ M ATP with or without 100 μ M ticlopidine. For the control experiment, substrate was either omitted or added in the absence of divalent cations, which are essential for NTPDases' activity.…”