Silverstone A, Sun L, Witte O N, Baltimore D
J Biol Chem. 1980 Jan 25;255(2):791-6.
An immunoprecipitation assay for measuring synthesis of murine terminal deoxynucleotidyltransferase (EC 2.7.7.31) has been developed using rabbit antiserum to calf terminal transferase. The antiserum precipitates a single Mr = 60,000 polypeptide (TdT-60) from all cell lines and tissues that contain enzymologically demonstrable terminal transferase. This polypeptide is not precipitated from labeled extracts of cells that lack terminal transferase by enzymological criteria. TdT-60 fractionates with terminal transferase during phosphocellulose chromatography and sediments with it in a sucrose gradient. TdT-60 is not detectably processed to lower molecular weight polypeptides, and terminal transferase activity sediments as a Mr = 60,000 activity; thus, we believe it to be the active form of terminal transferase. Using this assay we have demonstrated that terminal transferase is synthesized in both the murine thymus and the bone marrow at a rate proportional to its biochemically measured steady state level. After cortisone treatment of mice, the Mr = 60,000 polypeptide disappears from the thymus and then reappears as the thymus begins to be repopulated.
利用兔抗小牛末端转移酶血清，开发了一种用于测量小鼠末端脱氧核苷酸转移酶（EC 2.7.7.31）合成的免疫沉淀测定法。该抗血清能从所有含有可通过酶学方法检测到的末端转移酶的细胞系和组织中沉淀出一条单一的分子量为60,000的多肽（TdT-60）。根据酶学标准，这种多肽不会从缺乏末端转移酶的细胞的标记提取物中沉淀出来。在磷酸纤维素色谱分离过程中，TdT-60与末端转移酶一起分级分离，并在蔗糖梯度中与之一起沉降。TdT-60没有被检测到加工成分子量更低的多肽，并且末端转移酶活性以分子量为60,000的活性形式沉降；因此，我们认为它是末端转移酶的活性形式。使用这种测定法，我们已经证明末端转移酶在小鼠胸腺和骨髓中均有合成，其合成速率与其生化测量的稳态水平成正比。用可的松处理小鼠后，分子量为60,000的多肽从胸腺中消失，然后随着胸腺开始重新填充而再次出现。
