Yan N, Meister A
Department of Biochemistry, Cornell University Medical College, New York, New York 10021.
J Biol Chem. 1990 Jan 25;265(3):1588-93.
gamma-Glutamylcysteine synthetase catalyzes the first step in the synthesis of glutathione. The enzyme isolated from rat kidney has two subunits (heavy, Mr 73,000; and light, Mr 27,700) which may be dissociated by treatment with dithiothreitol. The heavy subunit exhibits all of the catalytic activity of the isolated enzyme and also feedback inhibition by glutathione. The light subunit has no known function and may not be an integral part of the enzyme. cDNA clones encoding rat kidney gamma-glutamylcysteine synthetase were isolated from a lambda gt11 cDNA library by immunoscreening with antibody against the isolated enzyme and further screening with oligonucleotide probes derived from several peptides whose sequences were determined by the Edman method. The nucleotide sequence of the mRNA for the heavy subunit was deduced from the sequences of the cDNA of three such clones. The sequence, which codes for 637 residues (Mr 72,614), contains all four of the independently determined peptide sequences (approximately 100 residues). This amino acid sequence shows extremely low overall similarity to that of gamma-glutamylcysteine synthetase isolated from Escherichia coli.
γ-谷氨酰半胱氨酸合成酶催化谷胱甘肽合成的第一步。从大鼠肾脏分离出的该酶有两个亚基（重亚基，分子量73,000；轻亚基，分子量27,700），用二硫苏糖醇处理可使其解离。重亚基表现出分离出的酶的所有催化活性，并且受谷胱甘肽的反馈抑制。轻亚基的功能未知，可能不是该酶的必需组成部分。通过用针对分离出的酶的抗体进行免疫筛选，并进一步用源自通过埃德曼法测定了序列的几种肽的寡核苷酸探针进行筛选，从λgt11 cDNA文库中分离出编码大鼠肾脏γ-谷氨酰半胱氨酸合成酶的cDNA克隆。从重亚基的三个此类克隆的cDNA序列推导出mRNA的核苷酸序列。该序列编码637个残基（分子量72,614），包含所有四个独立测定的肽序列（约100个残基）。该氨基酸序列与从大肠杆菌分离出的γ-谷氨酰半胱氨酸合成酶的氨基酸序列总体相似性极低。
