ASSESSMENT OF THE ROLE OF THE FIBRONECTIN-LIKE DOMAIN OF GELATINASE-ABY ANALYSIS OF A DELETION MUTANT

The properties of a deletion mutant Delta(V191-Q364) of gelatinase A, which represents the removal of the fibronectin like type II repeats d efined by exons 5-7, were compared with those of full length gelatinas e A. Both enzymes underwent self-activation over a similar time course in the presence of 4-aminophenylmercuric acetate. The fully active en zymes had similar k(cat)/K-m values for the cleavage of an octapeptide substrate, but the deletion mutant had 50% of the activity of wild ty pe gelatinase A against beta-casein and 10% of the activity against ge latin. The cleavage pattern for gelatin was similar for both enzymes b ut differed for type IV collagen. Comparison of the rates of associati on of the tissue inhibitors of metalloproteinase (TIMP)-1 and TIMP-2 a nd their N-terminal domains to both forms of gelatinase indicated that the fibronectin-like domain plays little role in TIMP binding. The de letion mutant failed to bind to collagen, while the wild type gelatina se bound tightly, indicating that the fibronectin-like domain is the s ole site of collagen binding. Both gelatinases could be activated by c oncanavalin A-activated fibroblasts, suggesting that the fibronectin-l ike domain is not required for the membrane-mediated activation proces s.