Purification of a Gelatin-Degrading Type IV Collagenase Secreted by ras Oncogene-Transformed Fibroblasts

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Abstract
Connective tissue matrix-degrading metalloproteinases play an important role in cancer invasion. In this report we describe the isolation of a metalloproteinase exhibiting both type IV collagenolytic and gelatinolytic activities from the conditioned medium of NIH-3T3 fibroblasts transformed with DNA containing an activated C-Harvey-ras oncogene from T24 bladder cancer cells. This tumor pro-teinase was purified by anion exchange chromatography, zinc-chelate Sepharose chromatography, and gel permeation chromatography. The final product was homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (relative molecular mass =67, 000). Gelatin zymography revealed two bands of gelatinolytic activity, corresponding to molecular weights of 67, 000 and 62, 000. Upon immunoblotting with the use of an affinity-purified polyclonal rabbit antibody to a peptide region of type IV collagenase that lacks homology with interstitial collagenase or stromelysin, the purified tumor enzyme was identified as type IV collagenase. [J Natl Cancer Inst 1988;80:1416–1420]

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