Development and application of a fluorometric assay for mammalian membrane dipeptidase.
Membrane dipeptidase (EC 3.4.13.19) is a widely distributed mammalian cell surface enzyme involved in the metabolism of glutathione, leukotriene D4, and certain beta-lactam antibiotics. In this study we have developed a sensitive and rapid assay for membrane dipeptidase based on the fluorometric detection of the D-Phe released from the model substrate Gly-D-Phe. The released D-Phe is first acted on by D-amino acid oxidase in the presence of flavin adenine dinucleotide. The resulting hydrogen peroxide is then metabolized by peroxidase in the presence of the acceptor substrate p-hydroxyphenylacetic acid which is converted to a highly fluorescent compound. The assay configuration is sensitive down to 0.1 nmol D-Phe and can accurately measure membrane dipeptidase activity even in the presence of large amounts of contaminating protein. The membrane dipeptidase assay and the subsequent fluorometric detection of the released D-Phe can be performed in microtiter plates, thus taking less than 1 h to process 96 samples. This sensitive and rapid assay will be useful for the routine measurement of membrane dipeptidase activity in a number of different applications.
Heywood
S P
SP
Department of Biochemistry and Molecular Biology, University of Leeds, United Kingdom.
Hooper
N M
NM
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D015377:Cilastatin / Q000494:pharmacology; D003605:D-Amino-Acid Oxidase / Q000378:metabolism; D004150:Dipeptidases / Q000378:metabolism*; D004151:Dipeptides / Q000378:metabolism*; D005470:Fluorometry; D006861:Hydrogen Peroxide / Q000378:metabolism; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D009195:Peroxidase / Q000378:metabolism; D010648:Phenylacetates / Q000378:metabolism; D010649:Phenylalanine / Q000032:analysis; D013552:Swine
D004151:Dipeptides; D010648:Phenylacetates; D015377:Cilastatin; C008070:4-hydroxyphenylacetic acid; D010649:Phenylalanine; D006861:Hydrogen Peroxide; D009195:Peroxidase; D003605:D-Amino-Acid Oxidase; D004150:Dipeptidases; C038937:dipeptidase