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Purification of a cysteine endopeptidase which is secreted with bioactive peptides from the epidermal glands of Xenopus laevis

1991; Wiley; Volume: 195; Issue: 1 Linguagem: Inglês

10.1111/j.1432-1033.1991.tb15676.x

1432-1033

Nigel J. Darby, David Bruce Lackey, Derek G. Smyth,

Biochemical and Structural Characterization

The purification is reported of an endopeptidase, XSCEP1 ( Xenopus skin cysteine endopeptidase), present in skin secretions of Xenopus . The procedure involved an initial concentration of the enzyme by batchwise anion‐exchange chromatography and ammonium sulphate precipitation. The proteolytic activity, determined with Z‐Phe‐Arg‐Amc (Z, benzyloxycarbonyl; Amc, 7‐amidomethylcoumarin) as substrate, was fractionated by gradient ion‐exchange chromatography, yielding a major component which was purified to homogeneity by chromatography on an organomercury‐agarose column. SDS/PAGE demonstrated the presence of a single protein with a molecular mass of 27 kDa. The purified enzyme, which possesed a pH optimum of 5.5, exhibited the properties of a cysteine endopeptidase: it was activated by dithiothreitol and EDTA and inhibited by the mechanism‐based inhibitor trans ‐epoxysuccinyl‐ l ‐leucylamido(4‐guanidino)butane. XSCEP1 exhibited a marked preference for substrates with a hydrophobic residue in the P 1 position and arginine in the P 2 position as opposed to a substrate with arginine residues in both positions. The enzyme was also able to cleave a Val‐Arg‐Gly sequence in a model substrate, reflecting cleavages undergone by a number of peptides present in Xenopus skin. The results point to a functional role for XSCEP1 as a putative processing enzyme.