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 γ-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 μm. l-Glutamic acid γ-(7-amido-4-methylcoumarin) is characterized as a substrate of γ-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 <0.6% of the substrate is consumed, fluorescence is proportional to enzymatic product, and results may be directly compared to established colorimetric assays. Interstinal epithelial cells are used both to establish appropriate assay conditions and to demonstrate the utility of the assays. © 1992.