Young M, Sammons R D, Mueller W T, Benkovic S J
Biochemistry. 1984 Aug 14;23(17):3979-86. doi: 10.1021/bi00312a027.
Antibody probes of Western blots [Renart, J., Reiser, J., & Stark, G. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 3116] of chicken liver homogenates under various conditions revealed that glycinamide ribonucleotide transformylase can be rapidly proteolyzed in such homogenates. These findings, along with molecular weight measurements by ultracentrifugation, identify the true form of glycinamide ribonucleotide transformylase as a monomeric protein of 117000 daltons. This protein has been purified 400-fold in 44% yield from chicken liver in one step on an affinity column of 10-formyl-5,8-dideazafolate-Sepharose. Native glycinamide ribonucleotide transformylase retains full activity after proteolytic cleavage to a form (Mr 55000) similar to fragments seen in the Western blot of the homogenates. This phenomenon may be responsible for the previous identification of glycinamide ribonucleotide (GAR) transformylase as a dimer of 55000-dalton subunits. Similar analyses using antibodies to 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) transformylase [Mueller, W. T., & Benkovic, S. J. (1981) Biochemistry 20, 337] and trifunctional enzyme [Smith, G. K., Mueller, W. T., Wasserman, G. F., Taylor, W. D., & Benkovic, S. J. (1980) Biochemistry 19, 4313] confirm that these two proteins were isolated in their native forms.
在不同条件下对鸡肝匀浆进行蛋白质印迹分析（[雷纳特，J.，赖泽尔，J.，& 斯塔克，G.（1979年）《美国国家科学院院刊》76，3116]）的抗体探针显示，甘氨酰胺核苷酸转甲酰基酶在这种匀浆中可被快速蛋白酶解。这些发现，连同通过超速离心进行的分子量测量，确定甘氨酰胺核苷酸转甲酰基酶的真实形式是一种117000道尔顿的单体蛋白。该蛋白在10 - 甲酰 - 5,8 - 二去氮叶酸 - 琼脂糖亲和柱上一步从鸡肝中纯化了400倍，产率为44%。天然甘氨酰胺核苷酸转甲酰基酶在被蛋白酶解为一种形式（Mr 55000）后仍保留全部活性，这种形式类似于在匀浆蛋白质印迹中看到的片段。这种现象可能是之前将甘氨酰胺核苷酸（GAR）转甲酰基酶鉴定为55000道尔顿亚基二聚体的原因。使用针对5 - 氨基咪唑 - 4 - 甲酰胺核苷酸（AICAR）转甲酰基酶（[米勒 W. T.，& 本科维奇，S. J.（1981年）《生物化学》20，337]）和三功能酶（[史密斯，G. K.，米勒，W. T.，瓦瑟曼，G. F.，泰勒，W. D.，& 本科维奇，S. J.（1980年）《生物化学》19，4313]）的抗体进行的类似分析证实，这两种蛋白质是以其天然形式分离得到的。
