Application of N-Carboxyalkyl Peptides to the Inhibition and Affinity Purification of the Porcine Matrix Metalloproteinases Collagenase,Gelatinase,and Stromelysin.

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)-N H-Le u- Phe- Ala­ NH2,competitively inhibited cleavage of dinitrophenyl­ Pro-Leu-Gly-Leu-Trp-Ala-o-Arg-NH2 at the Gly-Leu bond by MMP-1 and MMP-2 (K 1 = 30 and 40 µM,re­ spectively). 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-,and MMP-3 from concentrated culture me­ dium 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-sensi­ tive cleavage of collagen into TCA and T C8 fragments as well as slower hydrolysis of the a2 chain. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of MMP-1 indicated a predominant polypeptide of approx­ imately 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 resi­ dues 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 ap­ proximately 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 Pat the Gly 6- P he 7 bond. This procedure provides a rapid means of obtaining all three MMPs from one source in approximately 15% yield each.