Regulation mechanisms of plasma membrane Ca^<2+>-pumping ATPase of vascular smooth muscle

(1)在猪主动脉质膜富集区(IC50=106um)发现对腺苷抑制高度敏感的2型磷脂酰肌醇激酶(PI-K)，用它可以特异性地检测质膜Ca~(2+)泵送TAPase的活性，以响应cGMP或cGMP依赖的蛋白激酶(G-Kinase)的刺激作用。(2)PI激酶活性可被cGMP或G-K所刺激，但腺苷不能抑制质膜Ca~(2+)&Gt；-泵送ATPase的活性。用Kosk-Kosicka等人的钙调素亲和层析法从猪主动脉中部分纯化出2+&gt；-泵ATPase。(J.Biol.，261,3333-3338,1986)被G-Kinase以浓度依赖的方式刺激。然而，腺苷不能抑制激活，也不需要PI、PIP或PIP_&lt；2+&gt；存在。此外，在部分…中未检测到任何PI磷酸化活性(4)在相同的条件下，部分纯化的制剂对Ca~(2+)泵ATPase的活性有剂量依赖性的刺激作用，还原条件下，用SDS-PAGE分析，G-激酶磷酸化了两个相对分子质量分别为240-和138-kDa的蛋白。只有240-kDa蛋白的磷酸化依赖于G-激酶的浓度，而138-kDa蛋白的浓度不变。(5)用传统的钙调素亲和层析方法获得的高纯度的钙泵ATPase缺乏138-和240-kDa的G-激酶底物蛋白，对G-激酶没有反应。(6)部分纯化的Ca~(2+)&GT；用一种新的钙调素亲和层析方法将G-K与质膜Ca~(2+)~(2+)~(2+)和G~(2+)~(2+)-PATPase共孵育后，用一种新的亲和层析方法将240和138 kDa的G-KK底物蛋白从两种分子量分别为135和145 kDa的Ca~(2+)~(2+)和G~(2+)泵ATPase中完全分离出来。(7)这些结果表明，PI激酶不参与G-K对质膜Ca~(2+)~(2+)和GT~(2+)泵的刺激，240-kDa蛋白的磷酸化是G-K诱导的刺激作用的原因。质膜Ca~(2+)~(2+)~(2+)泵不受G-激酶的刺激而发生直接磷酸化。较少

(1) Type 2 phosphatidylinositol kinase (PI kinase) with high sensitivity to inhibition by adenosine (IC50=106 muM) was found in a plasma membrane-enriched fraction of porcine aorta, with which the plasma membrane Ca^<2+>-pumping TAPase activity, responsive to stimulatory action of cGMP or cGMP-dependent protein kinase (G-kinase), could be specifically measured.(2) The PI kinase activity was stimulated by cGMP or G-kinase, but adenosine did not inhibit the G-kinase stimulation of Ca^<2+>-pumping ATPase in the membrane fraction.(3) The plasma membrane Ca^<2+>-pumping ATPase that was partially purified from porcine aorta by the calmodulin affinity chromatographic method of Kosk-Kosicka et al. (J. Biol. Chem., 261, 3333-3338, 1986) was found to be stimulated in a concentration-dependent manner by G-Kinase. However, the activation was not inhibited by adenosine and did not require the presence of PI, PIP or PIP_<2+>. Furthermore any PI phosphorylating activity was not detected in the partia … More lly purified Ca^<2+>-pumping ATPase preparation.(4) Under identical conditions under which a dose-dependent stimulation of Ca^<2+>-pumping ATPase activity of the partially purified preparation was observed, G-kinase phosphorylated two proteins with molecular masses of 240- and 138-kDa as assessed by SDS-Page under reducing condition. Only the phosphorylation of 240-kDa protein was dependent on the concentration of G-kinase, that of 138-kDa protein being unchanged.(5) A highly purified Ca^<2+>-pumping ATPase preparation that was obtained by a conventional calmodulin affinity chromatographic method lacked the 138- and 240-kDa G-kinase substrate proteins and did not respond to G-kinase.(6) Fractionation of the partially purified Ca^<2+>-pumping ATPase after the incubation with G-kinase by a newly developed calmodulin affinity chromatographic method resulted in complete separation of the 240- and 138-kDa G-kinase substrate proteins from two isoforms of Ca^<2+>-pumping ATPase with molecular masses of 135- and 145-kDa.(7) From these results, it was concluded that PI kinase is not involved in the stimulation of plasma membrane Ca^<2+>-pumping ATPase by G-kinase and that the phosphorylation of 240-kDa protein is responsible for the G-kinase induced stimulation. Direct phosphorylation of the Ca^<2+>-pump dose not occur in association with the G-kinase stimulation of plasma membrane Ca^<2+>-pumping ATPase. Less