Purification and properties of bovine nasal hyaline cartilage collagenase.
1. Collagenase from bovine nasal hyaline cartilage was extracted with 1 and 3 M NaCl in Tris-CaCl2 buffer. 2. Two peaks of collagenase activity were revealed on DE52 ion exchange column, collagenase 1 and collagenase 2. 3. The apparent mol. wt of collagenase 1 and 2 as determined by SDS-PAGE were 68 and 43 kDa, respectively. 4. Both enzymes degrade native collagen type II into two characteristic products, TCA(3/4) and TCB(1/4), at 25 degrees C and pH 7.6. 5. Trypsin and aminophenylmercuric acetate were capable of increasing the collagenase 1 activity. 6. The two enzymes can be characterized as metalloproteinases since they were inhibited by EGTA and 1,10-phenanthroline. The use of proteinase inhibitors (N-ethylmaleimide, iodoacetic acid, phenylmethylsulphonyl fluoride, soybean trypsin inhibitor, pepstatin, dithiothreitol) showed that the enzymes do not contain serine, cysteine or aspartic acid in their active sites.
Boukla
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Department of Genetics, Aristotelian University of Thessaloniki, Greece.
D000818:Animals; D002356:Cartilage / Q000201:enzymology*; D002417:Cattle; D002850:Chromatography, Gel; D003094:Collagen / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D003012:Microbial Collagenase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D008970:Molecular Weight; D009666:Nose
D003094:Collagen; D003012:Microbial Collagenase