Carlson G M, Graves D J
J Biol Chem. 1976 Dec 10;251(23):7480-6.
Autoactivation of phosphorylase kinase in the presence of substrates has been studied to determine the cause of the hysteresis, or lag, in the phosphorylase kinase reaction. Peptide analogs corresponding to the convertible serine region of phosphorylase have been used as low molecular weight alternative substrates. Autophosphorylation of the kinase molecule was measured under conditions that favored autoactivation. Phosphorylase b and a tetradecapeptide, which was found to be a good model of phosphorylase, stimulated autoactivation by 86- and 37-fold, respectively. The tetradecapeptide also stimulated autophosphorylation of subunits A and B of the kinase molecule. This increased autophosphorylation coincided with an increased ability to convert phosphorylase. This finding supports the hypothesis that autophosphorylation is responsible for the lag in the phosphorylase kinase reaction. No evidence was obtained to suggest that the lag could be due to dissociation of the kinase. The stoichiometry of phosphate incorporation into phosphorylase kinase subunits by autophosphorylation was much greater than that reported to occur by protein kinase phosphorylation. Multiple phosphorylation sites in subunit A accounted for most of the phosphate incorporation during autophosphorylation. Saturating levels of hexa- and octapeptide analogs also caused stimulation of autophosphorylation. Possible mechanisms and experimental implications of substrate-stimulated autophosphorylation are discussed. Consideration also is given to the possible role of effectors in autophosphorylation in vivo.
为了确定磷酸化酶激酶反应中滞后现象（即延迟）的原因，研究了在底物存在下磷酸化酶激酶的自身激活。与磷酸化酶可转化丝氨酸区域对应的肽类似物已被用作低分子量替代底物。在有利于自身激活的条件下测量激酶分子的自身磷酸化。磷酸化酶b和一种十四肽（被发现是磷酸化酶的良好模型）分别将自身激活刺激了86倍和37倍。十四肽还刺激了激酶分子A和B亚基的自身磷酸化。这种自身磷酸化的增加与转化磷酸化酶能力的增强相吻合。这一发现支持了自身磷酸化是磷酸化酶激酶反应中滞后现象原因的假设。没有获得证据表明滞后可能是由于激酶的解离。通过自身磷酸化掺入磷酸化酶激酶亚基的磷酸盐化学计量比蛋白激酶磷酸化所报道的要大得多。亚基A中的多个磷酸化位点占自身磷酸化过程中大部分磷酸盐掺入。六肽和八肽类似物的饱和水平也导致自身磷酸化的刺激。讨论了底物刺激的自身磷酸化的可能机制和实验意义。还考虑了效应物在体内自身磷酸化中的可能作用。
