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从小牛胸腺分离纯化末端脱氧核苷酰转移酶

ISOLATION AND PURIFICATION OF TERMINAL DEOXYNUCLEOTIDYL TRANSFERASE FROM CALF THYMUS

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【作者】 李敏孙芝琳蓝天鹤

【Author】 Li Ming;Sun Zhilin;Lan Tianhe Department of Biochemistry

【机构】 华西医科大学生物化学教研室华西医科大学生物化学教研室

【摘要】 采用离子交换层析和Oligo(dT)-纤维素亲和层析相结合的方法,从小牛胸腺分离纯化末端脱氧核苷酰转移酶(TdT),500g胸腺TdT的比活性为1076~2030u/mg蛋白,总活性为4400~7296u,聚丙烯酰胺凝胶电泳一条带,SDS-凝胶电泳测其分子量得到7900和23700道尔顿的两个亚基,不含DNA聚合酶。

【Abstract】 The calf thymus is an excellent sourcefor enzyme preparation and purification. Inthis study, using a combined method of ex-change columns and Oligo (dT)-celluloseaffinity chromatography, TdT was purifiedand prepared from calf thymus. The proce-dure consisted of: (1) calf thymus tissue washomogenized and allowed to thaw over-night, to precipitate the acidic proteins at pH6.5; (2) followed by the phosphocelluloseabsorption; (3)the DEAE-cellulose chromato-graphy; (4)35-55% (NH42SO4 cut; and (5)Oligo(dT)-cellulose affinity chromatography.Thus, it was possible to obtain 7-10 mp TdTfrom 500g of the calf thymus with a totalTdT activity of 4400-7296 units and a specificactivty of 1076-2130 u/mg of protein. Theenzyme exhibited a single zone in 10% poly-acrylamide gel electrophoresis and sodiumdodecyl sulfate (SDS)-polyacrylamide gelelectrophoresis contained two polypeptidewith molecular weight of about 7900 an?23700 respectively, corresponding to the α(10000)and β(23000) subunit of TdT reportedpreviously by Okamura.

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