Specificity studies on enteropeptidase substrates related to the N-terminus of trypsinogen.
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.
Jenö
P
P
Green
J R
JR
Lentze
M J
MJ
D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D010450:Endopeptidases / Q000378:metabolism*; D004765:Enteropeptidase / Q000302:isolation & purification / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D005260:Female; D007421:Intestine, Small / Q000201:enzymology; D009842:Oligopeptides / Q000378:metabolism; D010455:Peptides / Q000138:chemical synthesis / Q000378:metabolism* / Q000494:pharmacology; D051381:Rats; D013379:Substrate Specificity; D014362:Trypsinogen / Q000037:antagonists & inhibitors / Q000378:metabolism*
D009842:Oligopeptides; D010455:Peptides; C023519:glycyl-aspartyl-aspartyl-aspartyl-aspartyl-lysine-2-naphthylamide; D014362:Trypsinogen; D010450:Endopeptidases; D004765:Enteropeptidase