Dns-Gly-(p-NO2)Phe-beta Ala, a specific fluorogenic substrate for neutral endopeptidase 24.11.
A novel fluorogenic peptide, dansyl-Gly-(p-NO2) Phe-beta Ala (DGNPA), was synthesized as a selective substrate for neutral endopeptidase 24.11, an enzyme involved in enkephalin and atrial natriuretic peptide degradation and a marker of differentiation (CD10) on the surface of lymphohematopoietic cells. Cleavage of the substrate Gly-(p-NO2)Phe amide bond leads to an increase in fluorescence related to the disappearance of the intramolecular quenching of the dansyl fluorescence by the nitrophenyl residue. This new fluorogenic substrate is an improvement over the commercially available dansyl-D-Ala-Gly-(p-NO2)Phe-Gly, as the Gly4 residue of the latter has been replaced by a beta-alanine, therefore eliminating a residual sensitivity of the peptide toward angiotensin converting enzyme. Moreover, deletion of the D-Ala2 residue was shown to increase the quenching efficiency, thus raising the sensitivity of the assay, which was further improved by stopping the reaction with dioxane. The present substrate has improved affinity (Km = 37 microM, V = 0.72 mumol min-1 mg protein-1), selectivity, and sensitivity over its precursor and was used in automated assays using 96-well microplates and a fluorescence plate reader.
Goudreau
N
N
Département de Pharmacochimie Moléculaire et Structurale, U 266 INSERM, URA D1500 CNRS, Faculté de Pharmacie, Paris.
Guis
C
C
Soleilhac
J M
JM
Roques
B P
BP
D000595:Amino Acid Sequence; D000818:Animals; D015415:Biomarkers; D001921:Brain / Q000201:enzymology*; D002462:Cell Membrane / Q000201:enzymology; D003619:Dansyl Compounds / Q000138:chemical synthesis*; D007202:Indicators and Reagents; D007668:Kidney / Q000201:enzymology*; D008969:Molecular Sequence Data; D015260:Neprilysin / Q000032:analysis*; D009842:Oligopeptides / Q000138:chemical synthesis*; D011817:Rabbits; D013050:Spectrometry, Fluorescence / Q000379:methods; D013056:Spectrophotometry, Ultraviolet / Q000379:methods; D013379:Substrate Specificity
D015415:Biomarkers; D003619:Dansyl Compounds; D007202:Indicators and Reagents; D009842:Oligopeptides; C088815:dansyl-glycyl-4-nitrophenylalanyl-beta-alanine; D015260:Neprilysin