Beauchamp B B, Richardson C C
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115.
Proc Natl Acad Sci U S A. 1988 Apr;85(8):2563-7. doi: 10.1073/pnas.85.8.2563.
Escherichia coli optA1, a mutant unable to support the growth of T7 phage containing mutations in gene 1.2, contains reduced amounts of dGTP. Extracts of E. coli optA1 catalyze the hydrolysis of dGTP at a rate 50-fold greater than do extracts of E. coli optA+. The dGTPase responsible for the increased hydrolysis has been purified to apparent homogeneity. Purification of the protein is facilitated by its high affinity for single-stranded DNA. By using this purification scheme an identical dGTPase has been purified from E. coli optA+. The purified proteins catalyze the hydrolysis of dGTP to yield deoxyguanosine and tripolyphosphate. The products of hydrolysis, chromatographic properties, denatured molecular mass of 56 kDa, N-terminal amino acid sequence, substrate specificity, and heat inactivation indicate that the proteins purified from optA1 and from optA+ cells are identical and identify the enzyme as the deoxyguanosine 5'-triphosphate triphosphohydrolase purified to homogeneity from wild-type E. coli [Seto, D., Bhatnagar, S. K. & Bessman, M. J. (1988) J. Biol. Chem. 263, 1494-1499]. OptA1 cells contain approximately equal to 50-fold more active molecules of the 56-kDa dGTPase than do E. coli optA+ cells.
大肠杆菌optA1是一种无法支持含有1.2基因发生突变的T7噬菌体生长的突变体，其dGTP含量降低。大肠杆菌optA1的提取物催化dGTP水解的速率比大肠杆菌optA+提取物高50倍。负责增加水解作用的dGTP酶已被纯化至表观均一性。该蛋白质对单链DNA的高亲和力有助于其纯化。通过使用这种纯化方案，已从大肠杆菌optA+中纯化出相同的dGTP酶。纯化后的蛋白质催化dGTP水解，生成脱氧鸟苷和三磷酸。水解产物、色谱性质、变性分子量56 kDa、N端氨基酸序列、底物特异性和热失活表明，从optA1和optA+细胞中纯化的蛋白质是相同的，并将该酶鉴定为从野生型大肠杆菌中纯化至均一性的脱氧鸟苷5'-三磷酸三磷酸水解酶[濑户，D.，巴特纳格尔，S.K.和贝斯曼，M.J.（1988年）《生物化学杂志》263，1494 - 1499]。与大肠杆菌optA+细胞相比，OptA1细胞中56 kDa的dGTP酶活性分子大约多50倍。
