A continuous fluorometric assay for gamma-glutamyltranspeptidase.
The synthesis of gamma-glutamyl-7-amino-4-(trifluoromethyl)coumarin and its use as a substrate for gamma-glutamyltranspeptidase is described. The reaction product 7-amino-4-(trifluoromethyl)coumarin was fluorescent at neutral pH values and with excitation and emission wavelengths of 400 and 490 nm, respectively, concentration was linearly related to fluorescence over the range of 10 to 300 pmol/3 ml reaction mixture. At pH 8.4, the optimum for purified gamma-glutamyltranspeptidase, continuous fluorometric determination of enzyme activity with time could be carried out. This permitted accurate estimations to be made of initial reaction rates. Low levels of gamma-glutamyltranspeptidase activity could be shown in isolated rat hepatocytes (1 x 10(5) cells). Such activity could be inhibited by the addition acivicine (alpha-amino-3-chloro-5-isoxazoleacetic acid), suggesting the absence of peptidase-like activity. Following 2-week pretreatment of rats with the antiestrogen, tamoxifen, a fourfold increase in gamma-glutamyltranspeptidase activity was observed. The present method offers a convenient, sensitive method for the determination of gamma-glutamyltranspeptidase activity without the need for elaborate workup procedures.
White
I N
IN
MRC Toxicology Unit, University of Leicester, United Kingdom.
Razvi
N
N
Lawrence
R M
RM
Manson
M M
MM
D000818:Animals; D003374:Coumarins / Q000138:chemical synthesis; D004965:Estrogen Antagonists / Q000494:pharmacology; D005260:Female; D005971:Glutamates / Q000138:chemical synthesis; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007700:Kinetics; D008099:Liver / Q000187:drug effects / Q000201:enzymology; D051381:Rats; D011916:Rats, Inbred F344; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013379:Substrate Specificity; D013629:Tamoxifen / Q000494:pharmacology; D005723:gamma-Glutamyltransferase / Q000032:analysis* / Q000378:metabolism
D003374:Coumarins; D004965:Estrogen Antagonists; D005971:Glutamates; C099645:gamma-glutamyl-7-amino-4-(trifluoromethyl)coumarin; D013629:Tamoxifen; D005723:gamma-Glutamyltransferase