trypsinogen and glycyl-aspartyl-aspartyl-aspartyl-aspartyl-lysine-2-naphthylamide

The specificity of the synthetic substrate Gly-[L-Asp]4-L-Lys 2-naphthylamide originally developed for the assay of enteropeptidase (EC 3.4.21.9), was investigated with partially purified aminopeptidase. Our results indicate that, not only enteropeptidase, but also the concerted action of the aminopeptidases of the rat small intestine, can rapidly release 2-naphthylamine from the substrate. A previously undescribed, highly active, dipeptidylaminopeptidase, which hydrolyses a Gly-Asp dipeptide from the N-terminus of the substrate, was detected in rat small intestine. The resulting [L-Asp]3-L-Lys 2-naphthylamide fragment is then degraded by a combination of aminopeptidase A and N to yield free 2-naphthylamine. Thus the present substrate cannot be regarded as being specific for enteropeptidase, and its use leads to an over-estimation of enteropeptidase activity in homogenates and extracts of intestinal tissue. In order to prevent this non-specific hydrolysis by aminopeptidases, stereoisomeric substrates with the sequence L-Ala-D-Asp-[L-Asp]3-L-Lys methyl ester, D-Ala-[L-Asp]4-L-Lys methyl ester and L-Ala-[Asp]4-L-Lys methyl ester were synthesized and tested as alternative substrates by their ability to inhibit the enteropeptidase-catalysed activation of trypsinogen.

Topics: Animals; Cattle; Chromatography, High Pressure Liquid; Endopeptidases; Enteropeptidase; Enzyme Activation; Female; Intestine, Small; Oligopeptides; Peptides; Rats; Substrate Specificity; Trypsinogen

Topics: Acute Disease; Adult; Animals; Cacodylic Acid; Callitrichinae; Cattle; Celiac Disease; Child; Chlorocebus aethiops; Chronic Disease; Dianisidine; Duodenum; Endopeptidases; Enteropeptidase; Guinea Pigs; Histocytochemistry; Humans; Intestine, Small; Mice; Mice, Inbred Strains; Oligopeptides; Pancreatitis; Rats; Swine; Swine, Miniature; Trypsinogen

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.

Topics: 2-Naphthylamine; Animals; Child; Endopeptidases; Enteropeptidase; Female; Humans; Hydrogen-Ion Concentration; Infant; Intestine, Small; Kinetics; Male; Oligopeptides; Rats; Spectrometry, Fluorescence; Substrate Specificity; Trypsin; Trypsinogen