title: Dissociation between Ca2+-ATPase and alkaline phosphatase activities in plasma membranes of rat duodenum.
abstract: The presence of Ca2+-ATPase activities with high-affinity sites for Ca2+ 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 Ca2+, the substrate specificity of Ca2+-induced ATP-hydrolysis has been studied to determine whether or not alkaline phosphatase and Ca2+-ATPase are two distinct enzymes. In basolateral fragments, the rate of Ca2+-dependent ATP-hydrolysis was greater than that of ADP, AMP and p-nitrophenylphosphate at Ca2+ concentrations below 25 muM. At 0.2 mM Ca2+ the rates of ATP, ADO, AMO and p-nitrophenylphosphate hydrolysis were not significantly different. In brush border fragments the rates of ATP, ADP and AMP hydrolysis were identical at low Ca2+, but at 0.2 mM Ca2+, Ca2+-induced hydrolysis of ADO and AMO was greater than either ATP or p-nitrophenylphosphate. Alkaline phsophatase in brush border and basolateral membranes was inhibited by 75% after addition of 2.5 mM theophylline. Ca2+-stimulated ATP hydrolysis at 1 muM Ca2+ was not sensitive to theophylline in basolateral fragments while the same activity in brush border fragments was totally inhibited. At 0.2 mM Ca2+, Ca2+-induced ATP hydrolysis in both basolateral and brush border membranes was sensitive to theophylline. Oligomycin and azide had no effect on Ca2+-stimulated ATP hydrolysis, either at low or at high Ca2+ concentrations. Chlorpromazine fully inhibited Ca2+-stimulated ATP hydrolysis in basolateral fragments at 5 muM Ca2+, while it had no effect in brush border fragments. From these results we conclude that, (i) Ca2+-ATPase and alkaline phosphatase are two distinct enzymes, (ii) high-affinity Ca2+-ATPase is exclusively located in basolateral plasma membranes, (iii) alkaline phosphatase activity, present on both sides of duodenal epithelium is stimulated slightly by low Ca2+ concentrations, but this Ca2+-induced activity is inhibited by theophylline and shows no specificity with respect to ATP, ADP or AMP.
mesh_terms: D000227:Adenine Nucleotides / Q000378:metabolism; D000469:Alkaline Phosphatase / Q000378:metabolism*; D000818:Animals; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D002462:Cell Membrane / Q000201:enzymology; D004386:Duodenum / Q000201:enzymology*; D004848:Epithelium / Q000201:enzymology; D007413:Intestinal Mucosa / Q000201:enzymology*; D007700:Kinetics; D008297:Male; D008871:Microvilli / Q000201:enzymology; D010649:Phenylalanine / Q000494:pharmacology; D051381:Rats; D013379:Substrate Specificity; D013806:Theophylline / Q000494:pharmacology
chemical_list: D000227:Adenine Nucleotides; D010649:Phenylalanine; D013806:Theophylline; D000469:Alkaline Phosphatase; D000252:Calcium-Transporting ATPases