Clark M R, Azhar S, Menon K M
Biochem J. 1976 Aug 15;158(2):175-82. doi: 10.1042/bj1580175.
Choriogonadotropin and lutropin have been found to activate cyclic AMP-dependent protein kinase in ovarian cells isolated by collagenase dispersion from immature rats. The stimulatory effect of gonadotropins was dependent on both hormone concentration and incubation time. Choriogonadotropin at 1 mug/ml fully stimulated the protein kinase activity within 5 min of incubation, and this effect was specific for choriogonadotropin and lutropin-like activity. In addition, protein kinase activity has been characterized with respect to salt sensitivity, cyclic AMP binding, and its responsiveness to gonadotropins and other peptide hormones. Ovarian protein kinase was susceptible to high salt concentrations. The addition of 0.3-1.0 M-NaCl in incubation medium increased the activity ratio with a concomitant decrease in cycle AMP-dependence. The salt effect on protein kinase was observed both from hormone-treated and untreated cells. The hormone-stimulated and unstimulated protein kinase activity was completely stable in the absence of NaCl. No change in the activity ratio was observed when cellular extracts were assayed for protein kinase activity either immediately or after 2 h in the absence of added salt. Gel filtration in the absence of NaCl of cellular extracts prepared from choriogonadotropin-treated and untreated cells showned only a single peak of protein kinase activity that was sensitive to exogenously added cyclic AMP. By contrast, when 0.5 M-NaCl was included in the column buffer, the chromatography of untreated extract showed two peaks of protein kinase activity. The first peak was sensitive to added cyclic AMP, whereas the second peak was insensitive to it. Under identical experimental conditions, protein kinase from gonadotropin-treated cells showed, on gel filtration, only one peak of activity that was totally insensitive to added cyclic AMP. DEAE-cellulose column chromatography of a 20000 g supernatant fraction resulted in a peak of kinase activity that eluted in approx. 0.15 M-NaCl, similar to the similar to the elution of type II protein kinases as described by Corbin et al. (1975) (J. Biol. Chem. 250, 218-225). Choriogonadotropin stimulation produced a decrease in the capacity of protein kinase to bind exogenous cyclic [3H]AMP, with a concomitant increase in the kinase activity ratio. These results are consistent with the notion that cyclic AMP, GENERATED IN SITU Under hormonal stimulation, binds tot he regulatory subunit of protein kinase with subsequent dissociation of the active catalytic subunit from the holoenzyme.
已发现绒毛膜促性腺激素和促黄体生成素能激活通过胶原酶分散法从未成熟大鼠分离出的卵巢细胞中的环磷酸腺苷依赖性蛋白激酶。促性腺激素的刺激作用取决于激素浓度和孵育时间。1微克/毫升的绒毛膜促性腺激素在孵育5分钟内可充分刺激蛋白激酶活性，且这种作用对绒毛膜促性腺激素和促黄体生成素样活性具有特异性。此外，还对蛋白激酶活性在盐敏感性、环磷酸腺苷结合以及对促性腺激素和其他肽类激素的反应性方面进行了表征。卵巢蛋白激酶对高盐浓度敏感。在孵育培养基中添加0.3 - 1.0 M氯化钠会增加活性比，同时降低对环磷酸腺苷的依赖性。在激素处理和未处理的细胞中均观察到盐对蛋白激酶的影响。在无氯化钠的情况下，激素刺激的和未刺激的蛋白激酶活性完全稳定。在无添加盐的情况下，无论是立即测定细胞提取物的蛋白激酶活性还是在2小时后测定，活性比均无变化。对绒毛膜促性腺激素处理和未处理的细胞制备的细胞提取物在无氯化钠的情况下进行凝胶过滤，结果显示只有一个对外源添加的环磷酸腺苷敏感的蛋白激酶活性峰。相比之下，当柱缓冲液中含有0.5 M氯化钠时，未处理提取物的色谱图显示有两个蛋白激酶活性峰。第一个峰对添加的环磷酸腺苷敏感，而第二个峰对其不敏感。在相同的实验条件下，经促性腺激素处理的细胞中的蛋白激酶在凝胶过滤时仅显示一个对添加的环磷酸腺苷完全不敏感的活性峰。对20000克上清液部分进行二乙氨基乙基纤维素柱色谱分析，得到一个激酶活性峰，其在约0.15 M氯化钠中洗脱，类似于科尔宾等人（1975年）（《生物化学杂志》250卷，218 - 225页）所述的II型蛋白激酶的洗脱情况。绒毛膜促性腺激素刺激导致蛋白激酶结合外源环[3H]磷酸腺苷的能力下降，同时激酶活性比增加。这些结果与以下观点一致，即激素刺激下原位产生的环磷酸腺苷与蛋白激酶的调节亚基结合，随后活性催化亚基与全酶解离。
