A rapid radioassay and some properties for leukotriene C4 synthase.
Incubation of glutathione, containing 35S-glutathione (GSH), and leukotriene (LTA4) in the presence of LTC4 synthase produced 35S-LTC4. The LTC4 was ether extracted and detected by ultraviolet absorbance (280 nm) after reverse phase-high performance liquid chromatography (RP-HPLC) separation. The 35S-LTC4 was quantitated by scintillation counting. LTC4 production increased in a dose-dependent manner for GSH concentrations from 10(-8) to 10(-2) M. On the contrary, the 35S detected in the ether phase and the 35S-LTC4 recovered from RP-HPLC decreased inversely with the concentration of unlabeled GSH added but did not with the addition of L-cysteine. This decrease in 35S-LTC4 production versus increased GSH levels results from a direct competition between unlabeled GSH and 35S-GSH reacting with the LTA4. Incubation of GSH (0.5 mM), containing 35S-GSH (0.3 uCi, 10 nM) and LTA4 (10 microM) with LTC4 synthase produced 35S-LTC4 at a linear rate over 5 min. The 35S-LTC4 increased in proportion to the LTC4 synthase added. Therefore, we have demonstrated that LTC4 synthase activity can be monitored by a simple radioassay procedure that measures 35S counts in ether extracts from incubation mixtures. This reliable and rapid assay procedure promises to facilitate characterization and purification of LTC4 synthase, a membrane-bound GSH S-transferase of unique specificity.
Abe
M
M
Research Institute for Diseases of the Chest, Faculty of Medicine, Kyushu University, Fukuoka.
Hara
N
N
Hugli
T E
TE
D002851:Chromatography, High Pressure Liquid / Q000379:methods; D005978:Glutathione / Q000378:metabolism; D005982:Glutathione Transferase / Q000032:analysis*; D011863:Radioimmunoassay / Q000379:methods; D011869:Radioligand Assay / Q000379:methods; D013462:Sulfur Radioisotopes
D013462:Sulfur Radioisotopes; D005982:Glutathione Transferase; C041116:leukotriene-C4 synthase; D005978:Glutathione