Abstract
A fluorogenic substrate for assay of leukotriene D4hydrolase (LTDase; EC 3.4.13.19) has been prepared and evaluated, using enzyme purified from porcine kidney. The compound is based on internal quenching of the synthetic, fluorescent amino acidd,l-2-amino-3-(7-methoxy-4-coumaryl)propanoic acid (d,l-Amp) by a 2,4-dinitrophenyl (DNP) group. The compound is ϵ-DNP-l-Lys-d-Amp which incorporates the D-isomer of Amp to exploit the unique ability among mammalian peptidases for LTDase to hydrolyze peptides containing ad-amino acid in the C-terminal position. ϵ-DNP-l-Lys-d-Amp was found to be an excellent substrate for LTDase, withKmvalue of 370 μM. Under the conditions of assay, the substrate was without noticeable quenching effect on the fluorescence of the product (d-Amp) liberated by the action of LTDase. Using porcine kidney microvillar membranes, which contain a battery of peptidases, the specific inhibitor of LTDase, cilastatin, completely inhibited the breakdown of ϵ-DNP-l-Lys-d-Amp, indicating that the substrate is selective for LTDase.
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