EXPRESSION OF COLLAGENASE GELATINASE ACTIVITY FROM BASEMENT-MEMBRANE FIBRONECTIN - ISOLATION AFTER LIMITED PROTEOLYSIS OF A BOVINE LENS CAPSULE AND MOLECULAR DEFINITION OF THIS THIOL-DEPENDENT ZINC METALLOPROTEINASE/

We have isolated the collagenase/gelatinase activity of fibronectin fr om a bovine lens capsule hydrolysate, using heparin-agarose, gelatin-a garose, immunopurification with polyclonal antibodies directed against bovine plasma fibronectin, and immunopurification with a monoclonal a ntibody directed against the extra-domain A of cellular fibronectin. T he expression of collagenase/gelatinase activity by the purified fibro nectin fragment was dependent on the incubation time at 37 degrees C a nd the addition of gelatin to the purified sample. Under these conditi ons, the purified fibronectin fragment exhibited collagenase/ gelatina se activity, as measured by means of gelatin zymography and the intram olecularly quenched fluorogenic substrate of collagenases cyl-[3-(2,4- dinitrophenyl)-L-2,3-diaminopropionyl] -alanylarginylamide. This activ ity was due to proteins of 47 kDa and 37 kDa, as indicated by the gela tin-zymography pattern. When the processing was analyzed, by means of SDS/PAGE under reducing conditions, purified starting material of 66 k Da and 55 kDa was observed, and molecular masses of 45, 30 and 27 kDa were found for the processed samples. Under these conditions, the proc essing was more significant when a substrate, i.e the fluorogenic pept ide or gelatin, was added to the processing mixture. An inhibition-pro file study showed a zinc-dependent collagenase activity. Using the 45- kDa chymotryptic fragment from human plasma fibronectin, which contain s the collagen-binding site, the same results were obtained. These res ults allow us to define a thiol-dependent zinc metalloproteinase expre ssed after limited proteolysis of both basement membrane and plasma fi bronectins. This proteinase contains a collagen-binding domain, a zinc -binding sequence, and a cysteine involved in catalysis. This enzyme i s a member of the thimet family of zinc metalloproteinases.