Amino acid sequence of rat kidney gamma-glutamylcysteine synthetase.

Abstract

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.