title: A fluorescence-based microtiter plate assay for γ-glutamylcyclotransferase.
abstract: γ-Glutamylcyclotransferase (GGCT) is a component of the γ-glutamyl cycle and specifically cleaves γ-glutamyl-amino acid dipeptides to release the free amino acid and 5-oxoproline. The action of GGCT in glutathione synthetase-deficient patients results in the accumulation of high levels of 5-oxoproline. In addition, GGCT may be a biomarker in some cancers. GGCT inhibitors, therefore, may be of value in the treatment of glutathione synthetase deficiency or in the elucidation of the role of GGCT in cancer cells. Published GGCT assays are not suitable for high-throughput screening for inhibitory molecules. We have developed a fluorescence-based 96-well plate assay for the determination of the rate of γ-glutamylcysteine cleavage by GGCT. After the reaction, the residual γ-glutamylcysteine is determined fluorometrically after derivatization with 2,3-naphthalenedicarboxaldehyde. The method has sufficient sensitivity to detect low-affinity competitive inhibitors and, as a result of its simplicity and microtiter plate format, can be readily used in high-throughput inhibitor screens.
authors: John Curtin School of Medical Research, Australian National University, Canberra, ACT 2601, Australia. philip.board@anu.edu.au
authors: Philip G
authors: PG
authors: Board
authors: Inca
authors: I
authors: Hutchinson
mesh_terms: D004121:Dimethyl Sulfoxide / Q000737:chemistry; D004151:Dipeptides / Q000378:metabolism; D004353:Drug Evaluation, Preclinical; D057075:Enzyme Assays / Q000379:methods*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D057166:High-Throughput Screening Assays; D006801:Humans; D013050:Spectrometry, Fluorescence; D005722:gamma-Glutamylcyclotransferase / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: D004151:Dipeptides; D004791:Enzyme Inhibitors; D005722:gamma-Glutamylcyclotransferase; C017341:gamma-glutamylcysteine; D004121:Dimethyl Sulfoxide