Hathaway G M, Zoller M J, Traugh J A
J Biol Chem. 1981 Nov 25;256(22):11442-6.
Casein kinase II, purified from reticulocytes, was covalently labeled with the ATP affinity analog, 5'-p-fluorosulfonylbenzoyl adenosine. The reaction was monitored by the decrease in enzyme activity and showed saturation kinetics with respect to the sulfonyl compound. This suggested a rapid equilibrium was established between the enzyme and affinity reagent prior to a slower, rate-determining step in the overall inactivation process. The enzyme was protected from the covalent modification by ATP and a series of ATP analogs. Their effectiveness in preventing inactivation by the affinity labeling reagent paralleled their ability to function as inhibitors of the phosphotransferase reaction. When radioactive p-fluorosulfonyl [14C]benzoyl adenosine was used to inactivate the enzyme, the alpha subunit was labeled and incorporation of radioactivity into the alpha subunit was blocked when ADP was included in the reaction mixture. Thus the ATP binding site of casein kinase II was shown to be contained within the domain of the alpha subunit of the alpha 2 beta 2 complex.
从网织红细胞中纯化得到的酪蛋白激酶II，用ATP亲和类似物5'-对氟磺酰苯甲酰腺苷进行共价标记。通过酶活性的降低来监测反应，并且该反应相对于磺酰化合物呈现出饱和动力学。这表明在整个失活过程中较慢的限速步骤之前，酶与亲和试剂之间迅速建立了平衡。ATP和一系列ATP类似物可保护该酶免受共价修饰。它们在防止亲和标记试剂导致失活方面的有效性与其作为磷酸转移酶反应抑制剂的能力相当。当使用放射性对氟磺酰[14C]苯甲酰腺苷使该酶失活时，α亚基被标记，并且当反应混合物中包含ADP时，放射性掺入α亚基的过程被阻断。因此，酪蛋白激酶II的ATP结合位点被证明位于α2β2复合物的α亚基结构域内。
