Abstract

The presence of Ca 2+-ATPase activities with high-affinity sites for Ca 2+ in brush border as well as basolateral plasma membranes of rat duodenal epithelium has been reported previously (Ghijsen, W.E.J.M. and van Os, C.H. (1979) Nature 279, 802–803). Since both plasma membranes contain alkaline phosphatase (EC 3.1.3.1), which also can be stimulated by Ca 2+, the substrate specificity of Ca 2+-induced ATP-hydrolysis has been studied to determine whether or not alkaline phosphatase and Ca 2+-ATPase are two distinct enzymes. In basolateral fragments, the rate of Ca 2+-dependent ATP-hydrolysis was greater than that of ADP, AMP and p- nitrophenylphosphate at Ca 2+ concentrations below 25 μM. At 0.2 mM Ca 2+ the rates of ATP, ADP, AMP and p- nitrophenylphosphate hydrolysis were not significantly different. In brush border fragments the rates of ATP, ADP and AMP hydrolysis were identical at low Ca 2+, but at 0.2 mM Ca 2+, Ca 2+-induced hydrolysis of ADP and AMP was greater than either ATP or p- nitrophenylphosphate . Alkaline phosphatase in brush border and basolateral membranes was inhibited by 75% after addition of 2.5 mM theophylline. Ca 2+-stimulated ATP hydrolysis at 1 μM Ca 2+ was not sensitive to theophylline in basolateral fragments while the same activity in brush border fragments was totally inhibited. At 0.2 mM Ca 2+, Ca 2+-induced ATP hydrolysis in both basolateral and brush border membranes was sensitive to theophylline. Oligomycin and azide had no effect on Ca 2+-stimulated ATP hydrolysis, either at low or at high Ca 2+ concentrations. Chlorpromazine fully inhibited Ca 2+-stimulated ATP hydrolysis in basolateral fragments at 5 μM Ca 2+, while it had no effect in brush border fragments. From these results we conclude that, (i) Ca 2+-ATPase and alkaline phosphatase are two distinct enzymes, (ii) high-affinity Ca 2+-ATPase is exclusively located in basolateral plasma membranes, (iii) alkaline phosphatase activity, present on both sides of duodenal epithelium, is stimulated slightly by low Ca 2+ concentrations, but this Ca 2+-induced activity is inhibited by theophylline and shows no specificity with respect to ATP, ADP or AMP.

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