Divalent cations but not other activators enhance phosphorylase kinase's affinity for glycogen phosphorylase.
Divalent cations but not other activators enhance phosphorylase kinase's affinity for glycogen phosphorylase.
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二价阳离子而非其他激活剂增强磷酸化酶激酶对糖原磷酸化酶的亲和力。
DOI：
10.1021/bi9528107
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发表时间：
1996
期刊：
影响因子：
--
通讯作者：
Carlson,GM
中科院分区：
文献类型：
--
作者：
Xu,YH;Wilkinson,DA;Carlson,GM
为了更好地了解糖原磷酸化酶-b（P-b）与其唯一已知的激酶，磷酸化酶激酶（PbK）之间的物理相互作用，以及这种相互作用与PbK活化的关系，有必要进行直接结合研究。利用酶联免疫吸附法，建立了一种在多种实验条件下测定PbK与固定化p键结合的方法。一种针对PbK α亚基的单克隆抗体对PbK的p - by磷酸化或PbK与其已知效应物的相互作用没有影响，用于检测与镀P-b结合的PbK。无论PbK或p -b的浓度是否变化，都得到双曲结合曲线，并且该实验检测到由激酶的某些效应物引起的相对亲和力的变化。变构效应ADP、碱性pH和camp依赖性蛋白激酶磷酸化，这些都是PbK的激活剂，没有引起其对P-b的相对亲和力的显著变化；然而，Ca2+和Mg2+离子也刺激PbK，增加其对P-b的亲和力，其中Mg2+更有效。Mn2+抑制PbK的P-b转化活性，是其对P-b亲和力的最有效增强剂。这些结果表明，PbK的活化不一定是由其对P-b的亲和力增加引起的，尽管二价阳离子可能增强结合。在与Ca2+和Mg2+结合的实验中加入ATP类似物来模拟催化实验条件，并没有进一步影响P-b的表观亲和力，这与之前报道的P-b转化的快速平衡随机bi-bi动力学机制一致。
To better understand the physical interaction between glycogen phosphorylase-b(P-b) and its only known kinase, phosphorylase kinase (PbK), and the relationship of this interaction to the activation of PbK, direct binding studies are necessary. By utilizing an enzyme-linked immunosorbent assay, a method was developed for measuring the binding of PbK to immobilized P-bunder a variety of experimental conditions. A monoclonal antibody specific for the α subunit of PbK that had no effect on the phosphorylation of P-bby PbK or on the interaction of PbK with its known effectors was used to detect PbK bound to plated P-b. Hyperbolic binding curves were obtained regardless of whether the concentration of PbK or P-bwas varied, and the assay detected changes in relative affinity caused by certain effectors of the kinase. The allosteric effector ADP, alkaline pH, and phosphorylation by cAMP-dependent protein kinase, all activators of PbK, did not cause significant changes in its relative affinity for P-b; however, Ca2+and Mg2+ions, which also stimulate PbK, increased its affinity for P-b, with Mg2+being more effective. Mn2+, which inhibits the P-bconversion activity of PbK, was found to be the most potent enhancer of its affinity for P-b. These results indicate that activation of PbK is not necessarily caused by an increase in its affinity for P-b, although divalent cations may enhance binding. Inclusion of ATP analogs in the binding assay with Ca2+and Mg2+to simulate catalytic assay conditions did not further affect the apparent affinity for P-b, which is consistent with the previously reported rapid equilibrium random bi-bi kinetic mechanism for P-bconversion.