ISOLATION AND CHARACTERIZATION OF A CHICKEN GELATINASE (TYPE-IV COLLAGENASE)

The proform of chick gelatinase (type IV collagenase) was isolated and purified to a high specific activity of 12 071 U/mg from cultured embryonic skin fibroblasts stimulated with cytochalasin-B. The enzyme was activated in the presence of 4-aminophenylmercuric acetate with a fall in molecular weight from 66 000-58 000 on non-reducing polyacrylamide gel electrophoresis and was active over the pH range of 6.0-8.9 against a number of substrates. Further biochemical characterisation showed that the organomercurial activated form of the enzyme behaved like a typical mammalian gelatinase, actively degrading gelatin, soluble type I collagen, collagenase generated type I fragments, type IV collagen (producing 3/4 and 1/4 fragments) and type V collagen, whilst having little effect on laminin. The enzyme was inhibited by metal chelators such as EDTA and 1,10-phenanthroline, but not by inhibitors of other mechanistic classes. A 23000 M(r) binding protein was found to be tightly associated with the proenzyme; it is suggested that this may be TIMP-2. An antiserum was raised to the proenzyme and was found to localise intra-and extra-cellularly in both tissue sections and cell cultures.