Assay of apical membrane enzymes based on fluorogenic substrates.
A series of enzymatic rate assays are described. The assays are based on coumarin derivatives that are fluorogenic substrates for the enzymes dipeptidase IV, aminopeptidase N, alkaline phosphatase, and gamma-glutamyltransferase. These simple assays are rapid and offer improved sensitivity over established colorimetric methods. The substrates have apparent affinities for the enzymes of 5-250 microM. L-Glutamic acid gamma-(7-amido-4-methylcoumarin) is characterized as a substrate of gamma-glutamyltransferase on the basis of inhibition of enzymatic cleavage when the glycylglycine acceptor molecule is omitted and inhibition of the enzymatic reaction by addition of glycine. Assay conditions for the four enzymes are established such that less than 0.6% of the substrate is consumed, fluorescence is proportional to enzymatic product, and results may be directly compared to established colorimetric assays. Intestinal epithelial cells are used both to establish appropriate assay conditions and to demonstrate the utility of the assays.
Blackmon
D L
DL
Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
Watson
A J
AJ
Montrose
M H
MH
D000469:Alkaline Phosphatase / Q000032:analysis* / Q000378:metabolism; D000626:Aminopeptidases / Q000032:analysis* / Q000378:metabolism; D000818:Animals; D018826:CD13 Antigens; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology*; D003106:Colon / Q000201:enzymology; D003124:Colorimetry / Q000379:methods; D003374:Coumarins; D018819:Dipeptidyl Peptidase 4; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases / Q000032:analysis* / Q000378:metabolism; D004848:Epithelium / Q000201:enzymology; D005456:Fluorescent Dyes; D006801:Humans; D007082:Ileum / Q000201:enzymology; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D008871:Microvilli / Q000201:enzymology; D013050:Spectrometry, Fluorescence / Q000379:methods; D013379:Substrate Specificity; D005723:gamma-Glutamyltransferase / Q000032:analysis* / Q000378:metabolism
D003374:Coumarins; D005456:Fluorescent Dyes; D005723:gamma-Glutamyltransferase; D000469:Alkaline Phosphatase; D000626:Aminopeptidases; D018826:CD13 Antigens; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; D018819:Dipeptidyl Peptidase 4