The application of a new synthetic substrate to the determination of enteropeptidase in rat small intestine and human intestinal biopsies.
The application of a new synthetic substrate to the direct determination of enteropeptidase is described. The substrate Gly-(L-Asp)4-L-Lys-2-naphthylamide contains the amino acid sequence of the activation peptides of trypsinogen linked via an amide bond to the fluorophore 2-naphthylamine. The sequence of amino acids is responsible for the specificity and substrate recognition of the enteropeptidase-catalyzed activation of trypsinogen. Interference in the assay by trypsin is prevented by the addition of soybean trypsin inhibitor to the substrate solution. The fluorimetric determination of the liberated 2-naphthylamine allows the direct observation of the reaction kinetics. For the hyrolysis of the synthetic substrate by purified enteropeptidase the pH optimum was 8.2 and the Km 0.17 mmol/l. The new substrate was used to determine the distribution of enteropeptidase along the rat small intestine and also to measure enteropeptidase activity in human intestinal biopsies.
D015081:2-Naphthylamine / Q000032:analysis; D000818:Animals; D002648:Child; D010450:Endopeptidases / Q000032:analysis*; D004765:Enteropeptidase / Q000032:analysis*; D005260:Female; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007223:Infant; D007421:Intestine, Small / Q000201:enzymology*; D007700:Kinetics; D008297:Male; D009842:Oligopeptides / Q000138:chemical synthesis; D051381:Rats; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D014357:Trypsin; D014362:Trypsinogen
D009842:Oligopeptides; C023519:glycyl-aspartyl-aspartyl-aspartyl-aspartyl-lysine-2-naphthylamide; D014362:Trypsinogen; D015081:2-Naphthylamine; D010450:Endopeptidases; D014357:Trypsin; D004765:Enteropeptidase