TY - JOUR
T1 - Purification and characterization of bovine interstitial collagenase and tissue inhibitor of metalloproteinases
AU - Sudbeck, Barry D.
AU - Jeffrey, John J.
AU - Welgus, Howard G.
AU - Mecham, Robert P.
AU - McCourt, David
AU - Parks, William C.
N1 - Funding Information:
This work was supported by Grants HD-05291, HL-29594, 35805 from the National Institutes of Health. This work is
PY - 1992/3
Y1 - 1992/3
N2 - 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 >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 reversephase 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.
AB - 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 >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 reversephase 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.
UR - http://www.scopus.com/inward/record.url?scp=0026606742&partnerID=8YFLogxK
U2 - 10.1016/0003-9861(92)90408-O
DO - 10.1016/0003-9861(92)90408-O
M3 - Article
C2 - 1311165
AN - SCOPUS:0026606742
SN - 0003-9861
VL - 293
SP - 370
EP - 376
JO - Archives of Biochemistry and Biophysics
JF - Archives of Biochemistry and Biophysics
IS - 2
ER -