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  • Title: Application of N-carboxyalkyl peptides to the inhibition and affinity purification of the porcine matrix metalloproteinases collagenase, gelatinase, and stromelysin.
    Author: Stack MS, Emberts CG, Gray RD.
    Journal: Arch Biochem Biophys; 1991 Jun; 287(2):240-9. PubMed ID: 1654808.
    Abstract:
    Several N-carboxyalkyl peptides were synthesized and tested as inhibitors of pig synovial collagenase, 72-kDa gelatinase and stromelysin (matrix metalloproteinases MMP-1, MMP-2, and MMP-3). The most potent of the series, CH3CH2CH2(R,S)CH(COOH)-NH-Leu-Phe-Ala-NH2, competitively inhibited cleavage of dinitrophenyl-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH2 at the Gly-Leu bond by MMP-1 and MMP-2 (KI = 30 and 40 microM, respectively). A similar inhibitory potency was found for MMP-1 with soluble Type I collagen and MMP-3 with substance P as substrate. The inhibitor was coupled to EAH-Sepharose 4B through a C-terminal amide. In the presence of 2 M NaCl at pH 7.2, this matrix bound MMP-1, MMP-2, and MMP-3 from concentrated culture medium of pig synovial membranes. The enzymes coeluted at pH 4.1 and subsequently were resolved by chromatography on DEAE-Sephacel and heparin-Sepharose. Purified MMP-1 catalyzed the o-phenanthroline-sensitive cleavage of collagen into TCA and TCB fragments as well as slower hydrolysis of the alpha 2 chain. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of MMP-1 indicated a predominant polypeptide of approximately 44 kDa and minor species of approximately 24 and 21 kDa. The 44-kDa species and one of the smaller polypeptides reacted with an antiserum to residues 195-207 of human fibroblast MMP-1, indicating that porcine MMP-1 contains a similar sequence and that the smaller components were probably derived from MMP-1. Neither MMP-2 nor MMP-3 reacted with this antiserum. Purified porcine MMP-2 degraded gelatin but not collagen and exhibited an apparent Mr of approximately 71 kDa. Additional smaller polypeptides were present, one of which may correspond to tissue inhibitor of metalloproteinases. MMP-3 showed doublets of approximately 47/46 and 26/25 kDa and cleaved substance P at the Gly6-Phe7 bond. This procedure provides a rapid means of obtaining all three MMPs from one source in approximately 15% yield each.
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