A highly sensitive fluorometric assay for "enkephalinase," a neutral metalloendopeptidase that releases tyrosine-glycine-glycine from enkephalins |
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Authors: | D Florentin A Sassi B P Roques |
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Institution: | 1. Département de Chimie Organique, ERA 613 du CNRS 4 avenue de l''Observatoire, Paris 75006, France;2. U266 de l''INSERM, UER des Sciences Pharmaceutiques et Biologiques, 4 avenue de l''Observatoire, Paris 75006, France |
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Abstract: | A fluorogenic peptide, dansyl-D-Ala-Gly-Phe(pNO2)-Gly (DAGNPG), was synthesized as a selective substrate for the neutral metalloendopeptidase (EC 3.4.24.11) involved in enkephalin metabolism. This enzyme, designated "enkephalinase," cleaves the Gly-Phe(pNO2) peptide bond of DAGNPG (V = 0.65 mumol/mg protein/min and Km = 45 microM) leading to a fluorescence increase related to the disappearance of intramolecular quenching of the dansyl fluorescence by the nitrophenyl residue. This change was used for quantitative measurements of "enkephalinase" activity in different tissues and determination of inhibitory potency of various compounds. The substrate is not cleaved by aminopeptidase or dipeptidylaminopeptidase activities and the assay itself is rapid, convenient, and sensitive. |
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Keywords: | dansyl “enkephalinase” fluorometric assay kinetics enzyme neuropeptides organic synthesis |
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