Fiscus R R, Torphy T J, Mayer S E
Biochim Biophys Acta. 1984 Dec 11;805(4):382-92. doi: 10.1016/0167-4889(84)90022-3.
Methacholine (3 microM) and sodium nitroprusside (300 microM) increased cGMP-dependent protein kinase activity ratios (activity without cGMP divided by activity with 2 microM cGMP) in canine tracheal smooth muscle from a control value of 0.47 to 0.55 and 0.71, respectively. This correlates with 3-fold and 6-fold increases in cGMP concentrations in response to methacholine and sodium nitroprusside, respectively. Addition of charcoal to the homogenizing buffer prior to homogenization had no significant effect on the cGMP-dependent protein kinase response to either agent, suggesting that activation of the enzyme was not occurring as a result of cGMP release during homogenization. In order to limit cGMP dissociation from cGMP-dependent protein kinase during the assay procedure, it was necessary to perform assays at a reduced temperature (0 degree C) and with an abbreviated incubation time (2.5 min). When assayed at 30 degrees C, activated cGMP-dependent protein kinase rapidly lost activity. This inactivation occurred whether the enzyme had been activated exogenously, by exposing a supernatant fraction of canine trachealis to 0.1 microM cGMP, or endogenously, by treating intact canine trachealis with methacholine or sodium nitroprusside. By assaying instead at 0 degree C, the inactivation of cGMP-dependent protein kinase was minimized. Therefore, the activity ratio obtained by this new modified assay provided an estimate of the endogenous activation state of cGMP-dependent protein kinase. The data indicate that cGMP responses in canine trachealis to both methacholine and sodium nitroprusside are functionally linked to activation of cGMP-dependent protein kinase and are consistent with the hypothesis that cGMP, via cGMP-dependent protein kinase activation, regulates smooth muscle contractility.
乙酰甲胆碱（3微摩尔）和硝普钠（300微摩尔）可使犬气管平滑肌中依赖环鸟苷酸的蛋白激酶活性比值（无环鸟苷酸时的活性除以有2微摩尔环鸟苷酸时的活性）分别从对照值0.47增至0.55和0.71。这分别与乙酰甲胆碱和硝普钠引起的环鸟苷酸浓度增加3倍和6倍相关。在匀浆前向匀浆缓冲液中加入活性炭，对依赖环鸟苷酸的蛋白激酶对这两种试剂的反应均无显著影响，这表明该酶的激活并非匀浆过程中环鸟苷酸释放所致。为了在测定过程中限制环鸟苷酸与依赖环鸟苷酸的蛋白激酶解离，有必要在降低的温度（0摄氏度）下并缩短孵育时间（2.5分钟）进行测定。当在30摄氏度下测定时，活化的依赖环鸟苷酸的蛋白激酶迅速丧失活性。无论该酶是通过将犬气管上清液部分暴露于0.1微摩尔环鸟苷酸而外源性激活，还是通过用乙酰甲胆碱或硝普钠处理完整的犬气管而内源性激活，都会发生这种失活。改为在0摄氏度下测定时，依赖环鸟苷酸的蛋白激酶的失活被最小化。因此，通过这种新的改良测定法获得的活性比值提供了依赖环鸟苷酸的蛋白激酶内源性激活状态的估计值。数据表明，犬气管对乙酰甲胆碱和硝普钠的环鸟苷酸反应在功能上与依赖环鸟苷酸的蛋白激酶的激活相关，并且与环鸟苷酸通过依赖环鸟苷酸的蛋白激酶激活来调节平滑肌收缩性的假说一致。
