Role of protein kinase in cell cycle regulation

1)为了阐明蛋白激酶C在（C激酶）在细胞周期调节中的作用，使用非洲爪蟾卵母细胞提取物调查了该酶的直接靶标。2）分子量约为25，000用C激酶和酪蛋白激酶II作为催化剂，在非洲爪蟾卵母细胞的胞浆组分中检测到25 kDa蛋白3）用热处理和阴离子交换柱层析法将25 KDa的蛋白质纯化至接近均一的纯度，收率达60%以上，动力学分析表明，该蛋白质的Km值为0.5- 1.0。1.0 μ M，表明25 kDa蛋白可能是这两种丝氨酸/苏氨酸激酶的天然替代物。5）通过广泛的磷酸化，无论使用何种蛋白激酶，每摩尔该蛋白都掺入约2摩尔磷酸，C激酶磷酸化位点被鉴定为位于该蛋白氨基端区域的两个丝氨酸残基。该蛋白的氨基酸序列分析表明，该蛋白的氨基酸组成与卵黄蛋白相似，但不完全相同; 9）该蛋白主要位于约克组分中，其氨基酸组成与卵黄蛋白相似，但不完全相同。但这种活性主要是在用含Triton X-100的缓冲液提取约克蛋白后检测到的。该蛋白在其他亚细胞组分中也被检测到，并且根据胞质、微粒体和质膜组分的顺序，其量减少。10）该25 kDa蛋白的功能以及该蛋白被C激酶和酪蛋白激酶II磷酸化的生理意义仍有待于研究。

1) In an attempt to elucidate the role of protein kinase C (C kinase) in cell cycle regulation, the direct target of this enzyme was surveyed using the extract of Xenopus laevis oocytes.2) An excellent phosphate acceptor protein with molecular mass of about 25,000 (25kDa protein) was detected in cytosolic fraction of Xenopus oocytes employing C kinase and casein Kinase II as catalyst3) This 25KDa protin was purified to near homogeneity with high yield of more than 60% using heat-treatment and anion-exchange column chromatography.The kinetic analyzes showed that Km values for this protein were caluculaed to be 0.5-1.0muM with either enzyme and suggested that the 25 kDa protein might be a candidate of natural subst5rate for these two serine/threonine kinases.5) By extensive phosphorylation, about two moles of phosphate were incorporated per mole of this protein irrespective of the protein kinase employed.The phosphorylated sites by C kinase were identified as the two serine residues locating on amino-terminal region of this protein. However, the phosphorylated sites by casein kinase II have not been conclusively identified.The data base analysis of the animo acid sequence of amino-terminal region of this 25kDa protein suggested to be a newly identified rotein.8) Amino acid comosition of this 25kDa protein indicated that this protein was similar to phosvitin, but not identical.9) The 25kDa protein was located mainly in york fraction, but this activity was detected mostly after extraction of york proteins with the buffer containing Triton X-100. This protein was also detected in other subcellular fractions and the amount was decreased according to the order of cvtosolic, microsomal and pasma membrane fractions.10) The function of this 25 kDa protein and physiological significance of the phosphorylation of this protein by C kinase and casein kinase II have been remained to be studied.