Pysiological siginificance of 240K protein of vascular smooth muscle

为了进一步阐明蛋白激酶G （PKG）磷酸化IP_3受体在PKG诱导的血管平滑肌质膜钙泵（PMCA）刺激中的作用，本研究以猪主动脉为原料制备了部分纯化的PMCA，考察了去除IP_3受体对PMCA刺激的影响，结果表明PKG激活后，PMCA活性明显受到刺激。用1型IP_3受体特异性抗体特异性免疫沉淀去除IP_3受体不影响PKG对PMCA活性的刺激，即使在没有IP_3受体的情况下仍观察到PMCA的刺激。此外，PKG以完全缺乏IP_3受体的高纯度制剂刺激PMCA，仅当PMCA浓度较低（10 ~ 20nM）时，酶已知为单体形式，而当酶浓度增加到40 nM时，酶已知为寡聚形式，并变得完全活化且对钙调素不敏感时，PKG未能刺激PMCA。热灭活PKG，不含cGMP的PKG不产生刺激。此外，发现i - α型PKG刺激PMCA，而非i - β型PKG。由此得出结论，PKG以单体形式刺激PMCA，而不磷酸化atp酶，并且在血管平滑肌中发现的两种PKG同工酶中，只有i - α PKG参与刺激。

To furhter clarify the role of phosphorylation of IP_3 receptor by protein kinase G (PKG) in the PKG induced stimulation of plasma membrane calcium pump (PMCA) of the vasucular smooth muscle, the effect of removal of IP_3 receptor on the stimulation was examed in the partially purified preparation of PMCA from porcine aorta, in which a definite stimulation of PMCA actrivity was observed upon activation of PKG.Contrary to expectation, removal of IP_3 receptor by specific immunoprecipitation with an antibody specific to type 1 IP_3 receptor did not affect the PKG stimulation of PMCA activity : the stimulation of PMCA was still observed even in the absence of IP_3 receptor. Furthermore, PKG stimulated PMCA in a highly purified preparation completely devoid of IP_3 receptor, only when the concentrations of PMCA were low (10 to 20nM), at which the enzyme is known to be in a monomeric form, while it failed to stimulate when the concentration of the enzyme was increased to 40 nM at which the enzyme is known to take an oligomeric form, and become fully actived and insensitive to calmodulin. Heat inactivated PKG,and PKG without cGMP did not produce stimulation. In addition, type Ialpha but not type Ibeta PKG was found to stimulate PMCA.It was concluded that PKG stimulates the PMCA in a monomeric form without phosphorylation of the ATPase, and that, of two PKG isozymes found in the vascular smooth muscle, only type Ialpha PKG participates in the stimulation.