Purification of a cysteine endopeptidase which is secreted with bioactive peptides from the epidermal glands of Xenopus laevis.
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 possessed 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 P1 position and arginine in the P2 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.
Darby
N J
NJ
National Institute for Medical Research, London, England.
Lackey
D B
DB
Smyth
D G
DG
D000595:Amino Acid Sequence; D000818:Animals; D002846:Chromatography, Affinity / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000379:methods; D003546:Cysteine Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism; D012627:Sebaceous Glands / Q000201:enzymology* / Q000378:metabolism; D013379:Substrate Specificity; D014982:Xenopus laevis
D010455:Peptides; D003546:Cysteine Endopeptidases; C067163:cysteine endopeptidase, Xenopus skin