Purification of rabbit bone inhibitor of collagenase.
1. Rabbit bones in tissue culture synthesize an inhibitor of collagenase during the first 4 days of culture. 2. The inhibitor was purified by a combination of gel filtration, concanavalin A--Sepharose chromatography, ion-exchange chromatography and zinc-chelate affinity chromatography. 3. The purified inhibitor migrated as a single band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and had a mol.wt. of 28000. 4. The inhibitor blocked the activity of the metalloproteinases collagenase, gelatinase, neutral proteinase III (proteoglycanase), human leucocyte collagenase and gelatinase, but not thermolysin or bacterial collagenase. The serine proteinases plasmin and trypsin were not inhibited. 5. The inhibitor interacted with purified rabbit bone collagenase with 1:1 stoichiometry. 6. The inhibitory activity was lost after incubation for 1 h at 90 degrees C, after treatment with trypsin (250 micrograms/ml) at 37 degrees C for 30 min and after reduction and alkylation.
D000818:Animals; D001842:Bone and Bones / Q000201:enzymology*; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D046508:Culture Techniques; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000302:isolation & purification* / Q000494:pharmacology; D006358:Hot Temperature; D008666:Metalloendopeptidases; D003012:Microbial Collagenase / Q000037:antagonists & inhibitors*; D011480:Protease Inhibitors; D011817:Rabbits; D019714:Tissue Inhibitor of Metalloproteinases; D014357:Trypsin
D004791:Enzyme Inhibitors; D011480:Protease Inhibitors; D019714:Tissue Inhibitor of Metalloproteinases; D014357:Trypsin; D008666:Metalloendopeptidases; D003012:Microbial Collagenase