A fluorimetric assay for aminopeptidase W.
A novel two-step enzyme-linked assay for aminopeptidase W is described and validated by comparison with other assays. L-alpha-Glutamyl-L-tryptophan (Glu-Trp) is a favoured substrate for this enzyme. With the use of glutamate dehydrogenase (EC 1.4.1.2) in a second step, the assay measured the release of free glutamate from L-alpha-glutamyl-L-tryptophan by the increase in NADH fluorescence. In the presence of 5 mM-1,10-phenanthroline and 50 microM-cilastatin the contribution of other membrane peptidases, in particular aminopeptidases N and A and microsomal dipeptidase in kidney, was very small. Residual cytosolic activities hydrolysing Glu-Trp were sensitive to inhibition by 2.5 mM-N-ethylmaleimide. The activity of aminopeptidase W was unaffected by these inhibitors. There was good correlation between the fluorimetric assay and those in which the free tryptophan released by kidney membrane fractions was determined by h.p.l.c. or the aminopeptidase W was measured immunoradiometrically with a monoclonal antibody.
Department of Biological Sciences, University of Keele, Staffs, U.K.
M C
MC
Jackson
Y
Y
Choudry
A
A
Bourne
J F
JF
Woodley
A J
AJ
Kenny
D000626:Aminopeptidases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D003374:Coumarins / Q000378:metabolism; D004150:Dipeptidases / Q000378:metabolism; D004151:Dipeptides / Q000378:metabolism; D005969:Glutamate Dehydrogenase / Q000378:metabolism; D006868:Hydrolysis; D066298:In Vitro Techniques; D007583:Jejunum / Q000201:enzymology; D007672:Kidney Cortex / Q000201:enzymology; D008565:Membrane Proteins / Q000378:metabolism; D008871:Microvilli / Q000201:enzymology; D051381:Rats; D013050:Spectrometry, Fluorescence; D013552:Swine
D003374:Coumarins; D004151:Dipeptides; D008565:Membrane Proteins; C057081:alpha-glutamyltryptophan; C057094:7-(alpha-glutamyl)-4-methylcoumarylamide; D005969:Glutamate Dehydrogenase; D000626:Aminopeptidases; C047029:aminopeptidase W; D004150:Dipeptidases; D002118:Calcium