Demonstration of cGMP-dependent protein kinase and cGMP-dependent phosphorylation in cell-free extracts of platelets.
Demonstration of cGMP-dependent protein kinase and cGMP-dependent phosphorylation in cell-free extracts of platelets.
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血小板无细胞提取物中 cGMP 依赖性蛋白激酶和 cGMP 依赖性磷酸化的演示。
DOI：
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发表时间：
1986
期刊：
影响因子：
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通讯作者：
U. Walter
中科院分区：
文献类型：
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作者：
R. Waldmann;S. Bauer;Claus Göbel;F. Hofmann;K. Jakobs;U. Walter
发现大鼠和人血小板的匀浆、膜和胞浆中含有cGMP依赖性蛋白激酶免疫反应性。特异性cGMP依赖性蛋白激酶免疫反应性为约1.7 pmol蛋白激酶/mg蛋白质的人血小板和0.7 pmol/mg大鼠血小板匀浆;大多数似乎与膜部分。在血小板膜中，（0.5-2 μ M）刺激表观相对分子质量Mr分别为240 000、130 000、50 000 42 000和22 000，而低浓度cGMP（0.5-2 μ M）刺激三种主要蛋白质的磷酸化，其表观Mr为130 000、50 000和46 000。制备了亲和纯化的抗cGMP依赖性蛋白激酶的抗体，其特异性地抑制cGMP依赖性蛋白激酶的活性。在人血小板膜中，这种亲和纯化的抗体抑制cGMP刺激的三种蛋白质的磷酸化，其Mr为130 000，50 000和46 000，而对cAMP依赖的和环核苷酸非依赖的蛋白质磷酸化没有影响。结果表明，血小板含有cGMP依赖性蛋白激酶和至少三种特异性底物。这些底物中的两种，表观分子Mr为130 000和50 000的蛋白质，是cAMP和cGMP依赖性蛋白激酶的底物。具有表观Mr为130 000的蛋白质似乎与血管平滑肌细胞的内在质膜蛋白密切相关，该蛋白质是膜相关cGMP依赖性蛋白激酶的底物。因此，cGMP依赖性蛋白激酶和cGMP调节的磷蛋白可能介导血小板中升高cGMP水平和抑制血小板聚集的那些激素、血管扩张剂和药物的细胞内效应。
Homogenates, membranes and cytosol of rat and human platelets were found to contain cGMP-dependent protein kinase immunoreactivity. Specific cGMP-dependent protein kinase immunoreactivity was about 1.7 pmol protein kinase/mg protein for homogenates of human platelets and 0.7 pmol/mg for homogenates of rat platelets; the majority appeared to be associated with the membrane fraction. In membranes of platelets low concentrations of cAMP (0.5-2 microM) stimulated the phosphorylation of five major proteins with apparent relative molecular masses, Mr, of 240 000, 130 000, 50 000, 42 000 and 22 000 while low concentrations of cGMP (0.5-2 microM) stimulated the phosphorylation of three major proteins with apparent Mr of 130 000, 50 000 and 46 000. An affinity-purified antibody against the cGMP-dependent protein kinase was prepared which specifically inhibited the activity of cGMP-dependent protein kinase. In membranes of human platelets this affinity-purified antibody inhibited the cGMP-stimulated phosphorylation of the three proteins with Mr of 130 000, 50 000 and 46 000 while it had no effect on the cAMP-dependent and cyclic-nucleotide-independent protein phosphorylation. The results demonstrate that platelets contain a cGMP-dependent protein kinase and at least three specific substrates for this enzyme. Two of these substrates, the proteins with apparent molecular Mr of 130 000 and 50 000, are substrates for both cAMP- and cGMP-dependent protein kinase. The protein with apparent Mr of 130 000 appears to be closely related to an intrinsic plasma membrane protein of vascular smooth muscle cells which is a substrate for a membrane-associated cGMP-dependent protein kinase. Therefore, cGMP-dependent protein kinase and cGMP-regulated phosphoproteins may mediate in platelets the intracellular effects of those hormones, vasodilators and drugs which elevate the level of cGMP and inhibit platelet aggregation.
影响因子：
20.3
作者：
B. T. Mellion;L. J. Ignarro;E. H. Ohlstein;E. Pontecorvo;A. Hyman;P. Kadowitz
通讯作者：
B. T. Mellion;L. J. Ignarro;E. H. Ohlstein;E. Pontecorvo;A. Hyman;P. Kadowitz