Omri B, Breton M F, Pavlovic-Hournac M
Eur J Biochem. 1987 May 15;165(1):83-90. doi: 10.1111/j.1432-1033.1987.tb11197.x.
Thyroid protein kinase C (PKc) from cytosols of porcine and rat thyroid glands has been characterized using histone H1 or endogenous proteins as substrates. As in many other tissues histone H1 is by far the preferred exogenous substrate of thyroid PKc. Kinetic studies with H1 showed that, compared to rat thyroids, porcine glands are particularly rich in PKc, the predominant kinase activity in this tissue. The cAMP-dependent protein kinase (PKa) level, on the contrary, is very similar in both rat and porcine thyroids. Consequently, for the same type of tissue, there may be great species differences in the PKc level and the ratios between PKc and PKa kinase activities. Chromatographic properties of thyroid PKc are similar to those described in other tissues (one major peak followed by a small shoulder) except that elution of the main peak can vary depending on the nature of the salt gradient (approximately 55 mM for NaCl and 15 mM for sodium phosphate). In the first case PKc is completely separated from the PKa activity, in the second it is coeluted with the peak of PKa type I. The one-dimensional PAGE pattern of proteins phosphorylated by porcine PKc is very similar to the pattern obtained by rat enzyme. Protein bands of 18 kDa, 22-25 kDa and 32-36 kDa are specific substrates of the thyroid PKc, after in vitro phosphorylation of cytosol proteins. A great difference in Ca2+ requirement for PKc activation was noted, depending whether histone H1 or endogenous proteins were substrates. As in other tissues, calcium was absolutely necessary for phosphorylation of histone H1 by PKc. The addition of calcium was not absolutely necessary when endogenous proteins were the substrates, either for the activation of the enzyme or for phosphorylation of the PKc-specific substrates. Almost the same rate of phosphorylation was obtained with or without calcium in the incubation medium. However the one-dimensional PAGE pattern of phosphorylated proteins was different in the presence or absence of calcium. While addition of calcium was not absolutely necessary for the phosphorylation of a great number of proteins by the PKc, its presence was indispensable for the phosphorylation of certain endogenous substrates. However, calcium alone, in the absence of phospholipids had no effect on the phosphorylation of these proteins. Endogenous proteins, phosphorylated by the PKc only when calcium was present, were resolved by the two-dimensional PAGE into several distinct spots with molecular masses of 32-35 kDa and pI range of 5-7.5.(ABSTRACT TRUNCATED AT 400 WORDS)
利用组蛋白H1或内源性蛋白质作为底物，对猪和大鼠甲状腺细胞质中的甲状腺蛋白激酶C（PKc）进行了特性研究。与许多其他组织一样，组蛋白H1是甲状腺PKc迄今为止首选的外源性底物。对H1的动力学研究表明，与大鼠甲状腺相比，猪甲状腺中PKc含量特别丰富，是该组织中的主要激酶活性。相反，环磷酸腺苷依赖性蛋白激酶（PKa）水平在大鼠和猪甲状腺中非常相似。因此，对于同一类型的组织，PKc水平以及PKc与PKa激酶活性之间的比率可能存在很大的物种差异。甲状腺PKc的色谱特性与其他组织中描述的相似（一个主峰后接一个小肩峰），只是主峰的洗脱会因盐梯度的性质而有所不同（氯化钠约为55 mM，磷酸钠约为15 mM）。在第一种情况下，PKc与PKa活性完全分离，在第二种情况下，它与I型PKa峰共洗脱。猪PKc磷酸化的蛋白质的一维聚丙烯酰胺凝胶电泳图谱与大鼠酶获得的图谱非常相似。在对细胞质蛋白进行体外磷酸化后，18 kDa、22 - 25 kDa和32 - 36 kDa的蛋白带是甲状腺PKc的特异性底物。根据组蛋白H1或内源性蛋白质是否为底物，发现PKc激活对钙离子的需求存在很大差异。与其他组织一样，钙离子对于PKc磷酸化组蛋白H1是绝对必要的。当内源性蛋白质作为底物时，无论是酶的激活还是PKc特异性底物的磷酸化，钙离子的添加都不是绝对必要的。在孵育介质中添加或不添加钙离子，磷酸化速率几乎相同。然而，存在或不存在钙离子时，磷酸化蛋白质的一维聚丙烯酰胺凝胶电泳图谱是不同的。虽然PKc对大量蛋白质的磷酸化并不绝对需要钙离子的存在，但它的存在对于某些内源性底物的磷酸化是必不可少的。然而，在没有磷脂的情况下，单独的钙离子对这些蛋白质的磷酸化没有影响。仅在有钙离子存在时被PKc磷酸化的内源性蛋白质，通过二维聚丙烯酰胺凝胶电泳解析为几个不同的斑点，分子量为32 - 35 kDa，等电点范围为5 - 7.5。（摘要截于400字）
