The properties of the neutral proteinase released by primary chondrocyte cultures and its action on proteoglycan aggregate.

The enzymatic mechanism of proteoglycan breakdown is of major interest, since it has been proposed that osteoarthritis involves increased proteolytic breakdown of proteoglycans. This paper describes the properties of the proteoglycan-degrading enzymes released into the extracellular milieu by chondrocyte cultures that produce cartilage-specific type II collagen but no detectable type I collagen. Attention has been focused on enzymes active at neutral pH, since the pH of the extracellular matrix is around neutrality. Biogel P-60 chromatography of concentrated culture medium showed a major peak of enzyme activity on proteoglycan monomer entrapped in polyacrylamide beads as well as on native proteoglycan aggregates. The enzyme yields a specific limit digestion peptide from the aggregate of approximately 55,000 daltons (in the presence of SDS). This limit peptide is probably derived from the hyaluronic acid-binding region of proteoglycan. The proteolytic enzyme is latent but can be activated by aminophenylmercuric acetate or trypsin. The molecular weight of both the active and latent forms, determined by gel filtration, is approximately 33,000. The activity is not inhibited by phenylmethylsulfonyl fluoride or pepstatin but is completely inhibited by o-phenanthroline; the activity is restored by Zn or Co ions in the presence of calcium chloride. Removal of calcium by dialysis results in a reversible loss of activity. The release of such a metalloproteinase by chondrocytes into the extracellular milieu, its activity at physiological pH and its ability to degrade native proteoglycans are consistent with a role of the enzyme in proteoglycan metabolism.