Type IV collagenase is a basement membrane degrading metalloproteinase that tumor cells secrete to facilitate migration through vascular channels and during the transition from in situ to invasive carcinoma. Human type IV collagenase was isolated and purified from culture supernatants of A2058 melanoma cells. After ammonium sulfate precipitation and gel filtration chromatography, the final purification of the enzyme was accomplished through type IV collagen affinity chromatography. The type IV collagenase has a molecular weight of 68,000. Amino acid analysis revealed high glycine and serine content. Two dimensional gel electrophoresis of the protein revealed a single polypeptide with an isoelectric point of about 5.8. Polyclonal antibodies were raised in rabbits. After 3-4 months of weekly injections, a low titer of collagenase specific antibody was demonstrated by ELISA. Co-expression of the metastatic phenotype and type IV collagenase was demonstrated and type IV collagenase was demonstrated in two gene transfer systems, i.e. in H-ras transfected NIH/3T3 cells and in human bronchial epithelial cells that were transformed with v-H-ras through protoplast fusion. These data as well as previous observations showing good correlation between type IV collagenolytic activity and metastatic behavior suggest that this enzyme may represent an important in vitro and in vivo marker for metastatic potential. Preparatory steps have therefore been taken for cloning of the gene for human type IV collagenase using cDNA libraries from a human breast carcinoma or teratocarcinoma cell lines that express type IV collagenase.
