哺乳动物细胞系的嘌呤突变体。II. 中国仓鼠肺细胞中磷酸核糖焦磷酸酰胺转移酶缺陷型突变体的鉴定。

Purine mutants of mammalian cell lines. II. Identification of a phosphoribosylpyrophosphate amidotransferase-deficient mutant of Chinese hamster lung cells.

作者信息

Feldman R I, Taylor M W

出版信息

Biochem Genet. 1975 Apr;13(3-4):227-34. doi: 10.1007/BF00486016.

Abstract

A class of purine auxotrophs blocked early in the purine biosynthetic pathway was examined. The inability of these mutants to accumulate formylglycinamide ribotide (FGAR) in the presence of azaserine suggested that one or more of the first three enzymes of the pathway were either missing or defective. By direct enzyme assay, phosphoribosylpyrophosphate (PRPP) amidotransferase (E.C. 2.4.2.14) was found to be absent in extracts of mutant cells. Thus these cells were unable to convert PRPP to phosphoribosylamine (PRA). By reacting ribose 5-phosphate with ammonium ions, PRA was generated nonenzymatically in the incubation mixture, thus enabling us to test for the presence of the two enzymes required to convert PRA to FGAR. It was demonstrated that sufficient amounts of these enzymes, phosphoribosylglycineamide synthetase (E.C. 6.3.1.3) and phosphoribosylglycineamide formyltransferase (E.C. 2.1.2.2), were present in mutant extracts to allow synthesis of FGAR to occur once PRA was so provided.

摘要

对一类在嘌呤生物合成途径早期受阻的嘌呤营养缺陷型菌株进行了研究。在重氮丝氨酸存在的情况下，这些突变体无法积累甲酰甘氨酰胺核苷酸（FGAR），这表明该途径的前三种酶中的一种或多种缺失或存在缺陷。通过直接酶活性测定，发现突变体细胞提取物中缺乏磷酸核糖焦磷酸（PRPP）酰胺转移酶（E.C. 2.4.2.14）。因此，这些细胞无法将PRPP转化为磷酸核糖胺（PRA）。通过使5-磷酸核糖与铵离子反应，在孵育混合物中通过非酶促反应生成了PRA，从而使我们能够检测将PRA转化为FGAR所需的两种酶的存在情况。结果表明，突变体提取物中存在足够量的这些酶，即磷酸核糖甘氨酰胺合成酶（E.C. 6.3.1.3）和磷酸核糖甘氨酰胺甲酰转移酶（E.C. 2.1.2.2），一旦提供PRA，就能够合成FGAR。