| Budget Amount *help |
¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1991: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 1990: ¥800,000 (Direct Cost: ¥800,000)
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| Research Abstract |
(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
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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
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