Mosbaugh D W, Kunkel T A, Stalker D M, Tcheng J E, Meyer R R
Nucleic Acids Res. 1976 Sep;3(9):2341-52. doi: 10.1093/nar/3.9.2341.
Unlike other beta-class eukaryotic DNA polymerases, the enzyme purified from the Novikoff hepatoma is inhibited by both sulfhydryl blocking agents N-ethylmaleimide (NEM) and p-hydroxymercuribenzoate (pHMB). The degree of sensitivity varies depending on the enzyme purity, pH of the reaction, and the presence of sulfhydryl reducing agents. Novikoff beta-polymerase activity is unaffected by the presence of 2-mercaptoethanol (2-Me) or dithiothreitol (DTT); however, the combination of 2-mercaptoethanol and NEM or pHMB acts to reverse the inhibition of the sulfhydryl blocking agent. The reversal of inhibition involves more than just a titration of NEM with 2-mercaptoethanol since a) the combination of these two reagents actually stimulates the DNA polymerase, and b) dithiothreitol did not reverse the inhibition. Binding of the polymerase to DNA did not affect the enzyme sensitivity to NEM.
与其他β类真核生物DNA聚合酶不同,从诺维科夫肝癌中纯化出的这种酶会受到巯基封闭剂N - 乙基马来酰亚胺(NEM)和对羟基汞苯甲酸(pHMB)的抑制。其敏感程度因酶的纯度、反应的pH值以及巯基还原剂的存在而有所不同。诺维科夫β聚合酶的活性不受2 - 巯基乙醇(2 - Me)或二硫苏糖醇(DTT)的影响;然而,2 - 巯基乙醇与NEM或pHMB的组合可逆转巯基封闭剂的抑制作用。抑制作用的逆转不仅仅是用2 - 巯基乙醇滴定NEM,因为:a)这两种试剂的组合实际上会刺激DNA聚合酶;b)二硫苏糖醇不能逆转这种抑制作用。聚合酶与DNA的结合并不影响该酶对NEM的敏感性。