Ghijsen W E, de Jong M D, van Os C H
Biochim Biophys Acta. 1980 Jul;599(2):538-51. doi: 10.1016/0005-2736(80)90198-4.
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.
先前已有报道称，在大鼠十二指肠上皮细胞的刷状缘以及基底外侧质膜中存在对Ca2+具有高亲和力位点的Ca2+-ATP酶活性（吉森，W.E.J.M.和范奥斯，C.H.（1979年）《自然》279卷，802 - 803页）。由于这两种质膜都含有碱性磷酸酶（EC 3.1.3.1），而碱性磷酸酶也可被Ca2+激活，因此研究了Ca2+诱导的ATP水解的底物特异性，以确定碱性磷酸酶和Ca2+-ATP酶是否为两种不同的酶。在基底外侧膜片段中，当Ca2+浓度低于25μM时，Ca2+依赖性ATP水解速率大于ADP、AMP和对硝基苯磷酸酯的水解速率。在0.2 mM Ca2+时，ATP、ADP、AMP和对硝基苯磷酸酯的水解速率无显著差异。在刷状缘膜片段中，低Ca2+浓度下ATP、ADP和AMP的水解速率相同，但在0.2 mM Ca2+时，Ca2+诱导的ADP和AMP水解大于ATP或对硝基苯磷酸酯的水解。加入2.5 mM茶碱后，刷状缘和基底外侧膜中的碱性磷酸酶活性被抑制75%。在基底外侧膜片段中，1μM Ca2+时Ca2+刺激的ATP水解对茶碱不敏感，而刷状缘膜片段中的相同活性则被完全抑制。在0.2 mM Ca2+时，基底外侧膜和刷状缘膜中Ca2+诱导的ATP水解对茶碱敏感。寡霉素和叠氮化物在低Ca2+或高Ca2+浓度下对Ca2+刺激的ATP水解均无影响。氯丙嗪在5μM Ca2+时完全抑制基底外侧膜片段中Ca2+刺激的ATP水解，而对刷状缘膜片段无影响。从这些结果我们得出结论：（i）Ca2+-ATP酶和碱性磷酸酶是两种不同的酶；（ii）高亲和力Ca2+-ATP酶仅位于基底外侧质膜；（iii）十二指肠上皮两侧存在的碱性磷酸酶活性在低Ca2+浓度下受到轻微刺激，但这种Ca2+诱导的活性被茶碱抑制，且对ATP、ADP或AMP无特异性。
