小鼠受精卵中蛋白激酶C底物的鉴定

为研究蛋白激酶C（protein kinase C，PKC）在小鼠早期发育中的调节作用，运用超排卵和体外受精技术，采用体外磷酸化和放射自显影的方法，鉴定小鼠1-细胞期受精卵中PKC的底物。经特殊的反复冻融处理，消除卵中内源性蛋白激酶活性。55个受精卵的样品中加入部分纯化的PKC，结合应用较强的PKC抑制剂H-7和星形孢菌素以及促分裂原活化蛋白激酶抑制剂PD098059作为对照，观察到12条PKC底物蛋白的放射自显影带，根据标准蛋白质对值绘制的标准曲线计算，这些磷酸化蛋白的相对分子量分别约为120kDa、100kDa、79kDa、63kDa、59kDa、47kDa、40kDa、34kDa、32kDa、26kDa、24kDa和22kDa。实验结果表明，PKC可通过底物蛋白活性的调节，在小鼠早期发育中发挥重要作用。

Identification of Substrates of Protein Kinase C at 1-Cell Fertilized Eggs in Mouse

To study regulatory effects of protein kinase C (PKC) in early development of mouse, identifi- cation of the PKC substrates at fertilized eggs was carried out with super-ovulation, in vitro fertilization, in vitro phosphorylation and autoradiography. The endogenous protein kinase in fertilized egg lysates was deactivated through multiply freezing and thawing cycles. The sample without purified PKC did not show any autoradiography band, so it served as blank control in this study. The sample of 55 fertilized eggs with partially purified PKC showed 12 bands in autoradiography. While less autoradiography bands were observed in samples containing both purified PKC and more potent PKC inhibitor (H-7 or staurosporin). According to Rf value of proteins, the relative molecular weights of these phosphorylated proteins were determined as 120 kDa, 100 kDa, 79 kDa, 63 kDa, 59 kDa, 47 kDa, 40 kDa, 34 kDa, 32 kDa, 26 kDa, 24 kDa and 22 kDa, respectively. These results suggested that PKC played an important role in early development of mouse.