Specific in vitro adenylylation of the simian virus 40 large tumor antigen.
Specific in vitro adenylylation of the simian virus 40 large tumor antigen.
复制标题
猿猴病毒 40 大肿瘤抗原的体外特异性腺苷酸化。
DOI：
10.1073/pnas.81.21.6574
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发表时间：
1984
影响因子：
11.1
通讯作者：
Livingston,DM
中科院分区：
文献类型：
--
作者：
Bradley,MK;Hudson,J;Villanueva,MS;Livingston,DM
在存在Mg 2+的情况下，用腺苷[8- 3 H]-、[α-32 P]-或[α-[35 S]硫代]-三磷酸盐孵育来自转化或裂解感染细胞的猴病毒40（SV 40）大肿瘤抗原（T），导致其标记，如在NaDodSO 4/聚丙烯酰胺凝胶中出现完整的适当免疫反应性条带所定义。在含有3% NaDodSO 4和2-巯基乙醇的缓冲液中煮沸后以及在0.1 M HCl、0.1 M NH 4 OH或羟胺中加热后，放射性仍与蛋白质相关，但在37 ℃下在0.1 M NaOH中孵育后，其解离。凝胶纯化的[α-32 P] ATP + T复合物在5.6 M HCl中有限煮沸后，释放出o-[32 P]磷酸丝氨酸，蛇毒磷酸二酯酶或0.5 M哌啶处理这种复合物导致[α-32 P]AMP的释放。当使用纯化的可溶性T或不溶性的特异性免疫沉淀抗原作为底物时，反应进行。ATP和dATP是优选的核苷酸底物相比，与其他六个标准的核糖核苷或脱氧核苷三磷酸。T + [α-32 P]ATP复合物的部分胰蛋白酶消化显示存在单个标记肽，其Mr约等于12 - 14 X 10（3），并且在彻底消化后，指纹中存在单个放射性斑点。这些数据表明T可以在蛋白质表面的有限部分中的特定丝氨酰残基处被腺苷酰化。此外，腺苷酸化似乎是可逆的，并通过焦磷酸化机制进行，因为在腺苷酸化T与Mg 2+、焦磷酸钠和聚（dT）孵育后，核苷酸从蛋白质中释放。
Incubation of the simian virus 40 (SV40) large tumor antigen (T) from either transformed or lytically infected cells with adenosine [8-3H]-, [alpha-32P]-, or [alpha-[35S]thio]-triphosphate in the presence of Mg2+ resulted in its labeling as defined by the appearance of an intact, appropriately immunoreactive band in NaDodSO4/polyacrylamide gels. Radioactivity remained associated with the protein after boiling in buffer containing 3% NaDodSO4, and 2-mercaptoethanol as well as after heating in 0.1 M HCl, 0.1 M NH4OH, or hydroxylamine, but it was dissociated after incubation in 0.1 M NaOH at 37 degrees C. After limited boiling of gel-purified [alpha-32P] ATP + T complex in 5.6 M HCl, o-[32P]phosphoserine was released, and snake venom phosphodiesterase or 0.5 M piperidine treatment of such a complex resulted in the liberation of [alpha-32P]AMP. The reaction proceeded when either purified, soluble T or insoluble, specifically immunoprecipitated antigen was used as substrate. ATP and dATP were the preferred nucleotide substrates by comparison with the other six standard ribonucleoside or deoxynucleoside triphosphates. Partial tryptic digests of T + [alpha-32P]ATP complexes revealed the presence of a single labeled peptide of Mr approximately equal to 12 - 14 X 10(3), and after exhaustive digestion, there was a single radioactive spot in the fingerprint. These data indicate that T can be adenylylated at a specific seryl residue(s) in a limited portion of the protein surface. Furthermore, adenylylation appears to be reversible and to proceed by a pyrophosphorylytic mechanism, since the nucleotide was released from the protein following incubation of adenylylated T with Mg2+, sodium pyrophosphate, and poly(dT).