Purification and characterization of bovine interstitial collagenase and tissue inhibitor of metalloproteinases.
In this report we describe the purification of bovine interstitial collagenase and provide information on its substrate specificity, kinetic parameters of catalytic activity, and amino terminal protein sequence. In addition, we present a simplified protocol for the purification of bovine tissue inhibitor of metalloproteinases (TIMP). Collagenase was purified by sequential chromatography through heparin-Sepharose, DEAE-Sepharose, and green-agarose, resulting in a product that was greater than 95% pure as judged by polyacrylamide electrophoresis. Typical of other interstitial collagenases, the isolated bovine protein was activated by protease and organomercurial treatment. It also demonstrated a kinetics and substrate specificity similar to those of human collagenase. TIMP was purified by sequential chromatography through heparin-Sepharose and DEAE-Sepharose followed by reverse-phase HPLC. The purified protein had a size, N-terminal sequence, and inhibitor activity similar to those of other mammalian TIMPs. Partial peptide sequences suggested that bovine collagenase and TIMP have strong sequence homology to their human homologues.
Sudbeck
B D
BD
Dermatology Division, Jewish Hospital, Washington University, St. Louis, MO 63110.
Jeffrey
J J
JJ
Welgus
H G
HG
Mecham
R P
RP
McCourt
D
D
Parks
W C
WC
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D003429:Cross Reactions; D005347:Fibroblasts / Q000201:enzymology; D006023:Glycoproteins / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D006801:Humans; D020781:Matrix Metalloproteinase 1; D003012:Microbial Collagenase / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D051381:Rats; D012867:Skin; D019714:Tissue Inhibitor of Metalloproteinases
D006023:Glycoproteins; D019714:Tissue Inhibitor of Metalloproteinases; D003012:Microbial Collagenase; D020781:Matrix Metalloproteinase 1