Abstract
The heavy subunit (M(r), 72,614) of rat kidney gamma-glutamylcysteine synthetase, the enzyme that catalyzes the first step of glutathione (GSH) synthesis, mediates the catalytic activity of this enzyme and its feedback inhibition by GSH. There is evidence that the light subunit has a regulatory function (Huang, C.-S., Chang, L.-S., Anderson, M.E., and Meister, A. (1993) J. Biol. Chem. 268, 19675-19680). In the present work the cDNA for the light subunit was isolated, sequenced, and expressed in Escherichia coli. The cDNA was found to code for a protein of 274 amino acid residues (M(r) 30, 548). Recombinant holoenzyme was obtained by co-expression of the heavy and light subunits and by mixing of the separately expressed proteins. These recombinant holoenzyme preparations exhibit catalytic and GSH feedback inhibitory properties that are virtually identical to those of the isolated holoenzyme. These studies establish that the light subunit is an integral part of the enzyme and that the light and heavy subunits, are coded for separately. Possibly significant similarity of sequence of amino acids was found between the light subunit and E. coli gamma-glutamylcysteine synthetase, which is a single polypeptide.
Highlights
Theheavysubunit (Mr 72,614) ofratkidney y- heavy subunit obtained from the isolated holoenzyme) exglutamylcysteinesynthetase,the enzyme thacat talyzes the first step of glutathione (GSHs)ynthesis, mediates the catalytic activity of this enzyme and its feedback inhibition byGSH
Recombinant holoenzyme was obtained by co-expression of the heavy and light hibitsa much higher K, value for glutamateand greater sensitivity to feedback inhibition by GSH than does the isolated holoenzyme [5].These resultssuggestedthat thelight subunit, not itself enzymatically active, may be an essential component of the holoenzyme by promoting high affinity for glutamate and appropriate sensitivity to feedback inhibition by GSH
After all of the significant similarity of sequence of amino acids was heavy subunit antibody was adsorbed, the column was washed with found between thelight subunit andE. coli y-glutamylcysteine synthetase,which is a single polypeptide
Summary
Pi iodoacetic acid (3 mg, 16 @mol)for 20 min. The reduced and carboxymethylated protein was applied to a C-4 reversed phase HPLC’. The isolated heavy subunit, which is catalytically active and inhibited by GSH, was cloned and by HPLC using a C-18 reversed phase column (4.2 X 250 mm); a 070% linear gradient was used, over 70 min, between 0.1% trifluoroacetic acid and 0.1% trifluoroacetic acid containing 95% acetonitrile at a flow rate of 1ml/min. The resulting 2-kb DNA fragment that contains a T7 promoter, andthe heavy subunit cDNAwas ligated to pRGCSL previously treated with ClaI (filled-in) and HindIII. The membrane was incubated with prehybridization buffer (5 X SSPE containing lox Denhardt's solution, 50% formamide, 2% SDS, and 100 Fg/ml denatured salmon sperm DNA) at 42 "C with the 736-base pair NcoI-BglI fragment of the light subunit cDNA labeled with 32Pby nick translation(about 2 X 10' cpmlpg). The membrane was hybridized with the 32P-labeledprobe corresponding to the 764-base pair PstI fragment of the heavy subunit cDNA [7].Autoradiography was performed at -70 "C for 4 days
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
