The identification, purification and characterisation of an inhibitor of collagenase (20K) produced by neoplastic epithelial cells.
A rat carcinoma cell line (T2/H7) constitutively synthesised interstitial collagenase. When these cells were incubated with 12-O-tetradecanoylphorbol 13-acetate (TPA) they secreted an inhibitor of collagenase, which resulted in a net decrease of collagenolytic activity being detected in conditioned medium. Using reverse zymography, the Mr of the inhibitor was found to be 20,000 which suggests that it may be the rat homologue of inhibitor of metalloproteinase 2 (IMP2; TIMP-2), as it inhibited both the gelatinolytic and collagenolytic activities of rat collagenase. The inhibitor was separated from collagenase by filtration through a YM30 membrane. The inhibitor was purified further by sequential chromatography on heparin-Sepharose and Con A-Sepharose. It bound to heparin-Sepharose in 75 mM NaCl and was eluted with 300 mM NaCl. It did not bind to Con A-Sepharose, suggesting that it was a non-glycosylated molecule. The inhibitor was resistant to treatment with either trypsin, APMA or heat.
Whitelock
J M
JM
Department of Cellular Pathology, University of Technology, Sydney, NSW Australia.
O'Grady
R L
RL
Gibbins
J R
JR
D000818:Animals; D002277:Carcinoma / Q000201:enzymology*; D002846:Chromatography, Affinity; D004848:Epithelium / Q000201:enzymology; D006358:Hot Temperature; D003012:Microbial Collagenase / Q000037:antagonists & inhibitors*; D008970:Molecular Weight; D011480:Protease Inhibitors / Q000737:chemistry / Q000302:isolation & purification*; D011489:Protein Denaturation; D051381:Rats; D014407:Tumor Cells, Cultured / Q000201:enzymology
D011480:Protease Inhibitors; D003012:Microbial Collagenase