HUMAN CYTOMEGALOVIRUS-ASSOCIATED DNA-POLYMERASE AND PROTEIN-KINASE ACTIVITIES
HUMAN CYTOMEGALOVIRUS-ASSOCIATED DNA-POLYMERASE AND PROTEIN-KINASE ACTIVITIES
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DOI：
10.1099/0022-1317-57-1-149
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发表时间：
1981-01-01
影响因子：
3.8
通讯作者：
HUANG, ES
中科院分区：
文献类型：
--
作者：
MAR, EC;PATEL, PC;HUANG, ES
人巨细胞病毒（HCMV），完全从细胞外介质中纯化，除了蛋白激酶活性外，还含有DNA聚合酶活性。DNA聚合酶在5-10 mM MgCl 2存在下表达其最大活性。该酶能够有效地利用活化的小牛胸腺DNA，poly（dA）. oligo（dT）12-18和poly（dC）. oligo（dG）12-18作为模板引物。用0.18- 0.2MgKCl从磷酸纤维素柱上洗脱DNA聚合活性。即使在100 μ g/ml的高浓度下，用活化的小牛胸腺DNA作为模板引物，它对膦酰基乙酸的抑制也具有相对的抗性，但是当聚（dA）·cnt·时，DNA聚合酶活性在该浓度下被完全抑制。oligo（dT）12-18用作模板引物。用0.01- 0.3M的（NH 4）2SO 4与活化的小牛胸腺DNA或poly（dA）抑制酶活性。oligo（dT）12-18作为模板引物。蛋白激酶在10-20 mM MgCl 2存在下具有最大活性，并且优选病毒体蛋白作为硫酸鱼精蛋白的磷酸受体。组蛋白，酪蛋白和牛血清白蛋白被发现是穷人的底物。体内[32 P] Pi标记的病毒粒子的磷酸化蛋白质模式与体外磷酸化Nonidet P40解离的病毒粒子的磷酸化蛋白质模式不相同，尽管在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳中7个磷酸化多肽确实共迁移。已知的溶解病毒体的方法表明，DNA聚合酶和蛋白激酶是病毒体的内部成分。
Human cytomegalovirus (HCMV), purified exclusively from the extracellular media, contained a DNA polymerase activity in addition to a protein kinase activity. The DNA polymerase expressed its maximum activity in the presence of 5-10 mM MgCl2. The enzyme was able to use effectively activated calf thymus DNA, poly(dA) .cntdot. oligo(dT)12-18 and poly(dC) .cntdot. oligo(dG)12-18 as the template primers. The DNA polymerizing activity was eluted with 0.18-0.2 MgKCl from a phosphocellulose column. It was relatively resistant to phosphonoacetic acid inhibition even at a high concentration of 100 .mu.g/ml with activated calf thymus DNA as the template primer, but the DNA polymerase activity was totally suppressed at this concentration when poly(dA) .cntdot. oligo(dT)12-18 was used as the template primer. The enzyme activity was inhibited by (NH4)2SO4 at 0.01-0.3 M with either activated calf thymus DNA or poly(dA) .cntdot. oligo(dT)12-18 as the template primer. The protein kinase has maximum activity in the presence of 10-20 mM MgCl2 and preferred virion proteins as phospho-acceptor to protamine sulfate. Histone, casein and bovine serum albumin were found to be poor substrates. The phosphorylated protein pattern of the in vivo [32P]Pi-labeled virions was not identical to that of the in vitro phosphorylated Nonidet P40-dissociated virions, although 7 phosphorylated polypeptides did co-migrate in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Procedures known to solubilize virions showed that the DNA polymerase and protein kinase were internal components of the virion.