Leung W C, Dubbs D R, Trkula D, Kit S
J Virol. 1975 Sep;16(3):486-97. doi: 10.1128/JVI.16.3.486-497.1975.
To characterize and compare the thymidine (TdR) and deoxycytidine (CdR) kinase isozymes of uninfected and herpesvirus-infected cells: (i) the subcellular distribution of the isozymes has been studied; (ii) a specific assay for CdR kinase has been devised; (iii) the TdR kinase isozymes have been partially purified; and (iv) the purified enzymes have been analyzed by disc polyacrylamide gel electrophoresis, isoelectric focusing, and glycerol gradient centrifugation and by substrate competition and dCTP inhibition studies. The results indicate that there are interesting individual differences with respect to nucleoside acceptor specificity between the cytosol and mitochondrial pyrimidine deoxyribonucleoside kinases of uninfected cells and between the enzymes induced by different herpesviruses. In the cytosol of uninfected mouse, chicken, and owl monkey kidney cells, two different proteins, TdR kinase F and CdR kinase 2, catalyze the phosphorylations of TdR and CdR, respectively. TdR kinase F does not phosphorylate CdR, nor does CdR kinase 2 phosphorylate TdR. A second TdR kinase isozyme present in HeLa(BU25) mitochondria (TdR kinase B) also lacks CdR phosphorylating activity. In contrast, a genetically distinctive deoxypyrimidine kinase (TdR kinase A) of mouse, human, and chick mitochondria catalyzes the phosphorylation of both TdR and CdT. Three herpesviruses, marmoset herpesvirus and herpes simplex virus types 1 and 2, induce in the cytosol fraction of LM(TK-) mouse cells isozymes which share common properties with mitochondrial TdR kinase A, including the ability to catalyze the phosphorylation of both TdR and CdR. However, the herpesvirus-induced deoxypyrimidine kinases differ from mitochondrial TdR kinase A with respect to sedimentation coefficient, sensitivity to dCTP inhibition, and antigenic determinants. The herpesvirus-specific and the mitochondrial deoxypyrimidine kinases exhibit a preference for TdR over CdR as nucleoside acceptor. Pseudorabies virus and herpesvirus of turkeys induce cytosol TdR kinases resembling the other herpesvirus-induced TdR kinases in several properties, but like cellular TdR kinase F, the pseudorabies virus and herpesvirus of turkeys TdR kinases lack detectable CdR phosphorylating activities. Finally, a marmoset herpesvirus nutant resistant to bromodeoxyuridine, equine herpesvirus type 1, and Herpesvirus aotus induces neither TdR nor CdR phosphorylating enzymes during productive infections.
为了鉴定和比较未感染细胞及疱疹病毒感染细胞中的胸苷（TdR）和脱氧胞苷（CdR）激酶同工酶：（i）研究了同工酶的亚细胞分布；（ii）设计了一种CdR激酶的特异性检测方法；（iii）对TdR激酶同工酶进行了部分纯化；（iv）通过圆盘聚丙烯酰胺凝胶电泳、等电聚焦、甘油梯度离心以及底物竞争和dCTP抑制研究对纯化的酶进行了分析。结果表明，未感染细胞的胞质溶胶和线粒体嘧啶脱氧核糖核苷激酶之间以及不同疱疹病毒诱导的酶之间，在核苷受体特异性方面存在有趣的个体差异。在未感染的小鼠、鸡和猫头鹰猴肾细胞的胞质溶胶中，两种不同的蛋白质，即TdR激酶F和CdR激酶2，分别催化TdR和CdR的磷酸化。TdR激酶F不磷酸化CdR，CdR激酶2也不磷酸化TdR。HeLa（BU25）线粒体中存在的第二种TdR激酶同工酶（TdR激酶B）也缺乏CdR磷酸化活性。相反，小鼠、人和鸡线粒体中一种遗传上独特的脱氧嘧啶激酶（TdR激酶A）催化TdR和CdT的磷酸化。三种疱疹病毒，即狨猴疱疹病毒、单纯疱疹病毒1型和2型，在LM（TK-）小鼠细胞的胞质溶胶部分诱导出与线粒体TdR激酶A具有共同特性的同工酶，包括催化TdR和CdR磷酸化的能力。然而，疱疹病毒诱导的脱氧嘧啶激酶在沉降系数、对dCTP抑制的敏感性和抗原决定簇方面与线粒体TdR激酶A不同。疱疹病毒特异性和线粒体脱氧嘧啶激酶表现出对TdR作为核苷受体的偏好超过CdR。伪狂犬病病毒和火鸡疱疹病毒诱导的胞质溶胶TdR激酶在几个特性上类似于其他疱疹病毒诱导的TdR激酶，但与细胞TdR激酶F一样，伪狂犬病病毒和火鸡疱疹病毒的TdR激酶缺乏可检测到的CdR磷酸化活性。最后，一种对溴脱氧尿苷耐药的狨猴疱疹病毒突变体、马疱疹病毒1型和食蟹猴疱疹病毒在生产性感染期间既不诱导TdR也不诱导CdR磷酸化酶。
