Cell surface gamma-glutamyl transpeptidase in live cultures.
A physiological assay for measuring surface accessible gamma-glutamyl transpeptidase activity in adherent, living cultures is described. Cell surface transpeptidase activity remained linear throughout a 60-min time course over a wide range of cell densities. In addition, the assay conditions have neither acute nor long-term effects on cell growth potential, cellular morphology, or cell surface transpeptidase activity levels. As a result, cell surface transpeptidase activity can be continually evaluated in the same cultures during proliferation. The assay appears to be specific for cell surface transpeptidase and can be used to study the partitioning of the enzyme between substrate-accessible and substrate-inaccessible pools. This method utilizes an automated microtiter plate reader for the spectrophotometric quantification of small aliquots removed from cultures incubated with the chromogenic substrate L-gamma-glutamyl-p-nitroanilide. The use of a microtiter plate autoreader and the minimal handling of the cells permit a large number of cultures to be assayed with a substantial reduction in the time required to measure surface transpeptidase activity. The assay described is a nondestructive means for studying cell surface-accessible gamma-glutamyl transpeptidase catalytic activity within the microenvironment of the living culture.
Morgenstern
K
K
Department of Pathology, University of Oklahoma Health Sciences Center, Oklahoma City 73190.
Hanson-Painton
O
O
De Bault
L
L
D000818:Animals; D001253:Astrocytes / Q000201:enzymology; D001322:Autoanalysis; D002452:Cell Count; D002462:Cell Membrane / Q000201:enzymology* / Q000648:ultrastructure; D002471:Cell Transformation, Neoplastic; D007700:Kinetics; D051381:Rats; D013053:Spectrophotometry; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured; D005723:gamma-Glutamyltransferase / Q000378:metabolism*
D005723:gamma-Glutamyltransferase