Can flow cytoenzymology be applied to measure membrane-bound enzyme kinetics? Assessment by analysis of gamma-glutamyl transpeptidase activity.
We describe an improved technique which allows the analysis of enzyme reaction kinetics for gamma-glutamyl transpeptidase (gamma-GT) by flow cytometry. This is technically difficult because of the location of the enzyme on the external surface of the cell membrane leading to the rapid escape of the product. The reaction is determined by monitoring the conversion of gamma-glutamyl aminomethylcoumarin to aminomethylcoumarin. Reaction kinetics are described for BL8 hepatocyte and JB1 hepatoma cells lines, together with inhibition kinetics for the active site-directed glutamine analogue L-(alpha-S,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid. We show that it is possible to follow the reaction dynamics in a heterogeneous mixture of BL8 and JB1 cells allowing discrimination of the two cell types based on gamma-GT activity. Improvements for further optimizing the assay of this important enzyme are suggested.
Dive
C
C
MRC Clinical Oncology and Radiotherapeutics Unit, Cambridge, U.K.
Workman
P
P
Watson
J V
JV
D000818:Animals; D006528:Carcinoma, Hepatocellular; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D005434:Flow Cytometry / Q000295:instrumentation / Q000379:methods*; D007700:Kinetics; D008099:Liver / Q000166:cytology / Q000201:enzymology; D051381:Rats; D013050:Spectrometry, Fluorescence; D014407:Tumor Cells, Cultured; D005723:gamma-Glutamyltransferase / Q000037:antagonists & inhibitors / Q000378:metabolism*
D005723:gamma-Glutamyltransferase