跳转至内容
Merck
CN
  • A fluorescence-based microtiter plate assay for γ-glutamylcyclotransferase.

A fluorescence-based microtiter plate assay for γ-glutamylcyclotransferase.

Analytical biochemistry (2011-10-18)
Philip G Board, Inca Hutchinson
摘要

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.

材料
货号
品牌
产品描述

Sigma-Aldrich
γ-Glu-Cys, ≥80% (HPLC)
Sigma-Aldrich
2,3-萘二甲醛, suitable for fluorescence
Supelco
四[3,5-双(1,1,1,3,3,3-六氟代-2-甲氧基-2-丙基)苯基]硼酸钠 三水合物, Selectophore