The action of Taiwan cobra venom on methionine enkephalin: a useful assay for oligopeptidase content.
The pentapeptide methionine enkephalin is readily hydrolysed by the oligopeptidase activity contained in Taiwan cobra (Naja naja atra) venom. It is a significantly better substrate than the peptides previously used to identify the presence of this enzyme, but it retains many of the sequence characteristics shared by these other peptides. Analysis of the manner of hydrolysis by means of high-performance liquid chromatography and electrospray mass spectrometry revealed the simultaneous actions of at least two types of oligopeptidase on the neuropeptide, producing two routes of initial breakdown. By one route, an endopeptidase cleaved the Gly-Phe bond of enkephalin first to release Tyr-Gly-Gly and Phe-Met. By the other route, an aminopeptidase was able to release Tyr and Gly-Gly-Phe-Met by cleaving the Tyr-Gly bond first. From amongst the various peptide fragments produced, Tyr-Gly-Gly was subject to immediate aminopeptidase action to release Tyr and Gly-Gly. The free Tyr produced in these reactions was in turn quickly transformed by the L-amino acid oxidase in the venom. The kinetic qualities of the enkephalin hydrolysis, and the conversions of the fragments Tyr-Gly-Gly and Tyr, were measured. Methionine enkephalin has potential as a routine assay for venom oligopeptidases, either in testing the venoms from other species or in attempts to purify these enzymes. Moreover, the ease of hydrolysis of this bioactive peptide, coupled with the revelation of the other enzymic steps involving the fragments generated, may provide important clues as to the possible role of the oligopeptidases (and L-amino acid oxidase) in the venom.
Anderson
L A
LA
Department of Pure and Applied Chemistry, University of Strathclyde, Glasgow, U.K.
Dufton
M J
MJ
D000594:Amino Acid Oxidoreductases / Q000378:metabolism; D000626:Aminopeptidases / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D004546:Elapid Venoms / Q000494:pharmacology*; D010450:Endopeptidases / Q000378:metabolism; D004744:Enkephalin, Methionine / Q000378:metabolism*; D006868:Hydrolysis; D050830:L-Amino Acid Oxidase; D013058:Mass Spectrometry; D010447:Peptide Hydrolases / Q000378:metabolism*; D013379:Substrate Specificity
D004546:Elapid Venoms; D004744:Enkephalin, Methionine; D000594:Amino Acid Oxidoreductases; D050830:L-Amino Acid Oxidase; D010450:Endopeptidases; D010447:Peptide Hydrolases; C028759:oligopeptidase; D000626:Aminopeptidases