A B S T R A C T Extracts of human articular cartilage contain proteases capable of degrading the proteoglycan component of cartilage matrix at neutral and acid pH. These enzymes have been partially purified by ion exchange chromatography and characterized by disc electrophoresis, inhibition patterns, and action on proteoglycan. Three distinct metalloproteases are described. A neutral protease that digests proteoglycan subunit optimally at pH 7.25 has been purified up to 900-fold. It is strongly inhibited by o-phenanthroline, a-2-macroglobulin, and egg white, and to a lesser extent by D-penicillamine and EDTA. Inhibition by chelating agents is reversed by cobalt, zinc, and ferrous ions. Two acid metalloproteases, distinct from cathepsins Bi, D, and F, digest proteoglycan subunit at pH 4.5 and 5.5. Both are inhibited by o-phenanthroline and activity is restored by cobalt, zinc, or ferrous ions. With electron microscopy, it was found that cartilage slices were depleted of ruthenium red-staining matrix proteoglycan after incubation in vitro with a partially purified cartilage extract at neutral pH. Sedimentation, gel chromatography, sodium dodecyl sulfate-gel electrophoresis, and immunodiffusion studies of digests of isolated proteoglycan fraction produced by the partially purified cartilage extract at neutral and acid pH confirmed that the cartilage enzymes act only on the protein component of proteoglycan subunit, producing fragments with 5 to 12 chondroitin sulfate chains. The link proteins were not digested.

A B S T R A C T Proteolytic enzymes have been studied in extracts of human articular cartilage by the use of micromethods. The digestion of hemoglobin at pH 3.2 and of cartilage proteoglycan at pH 5 was shown to be due chiefly to cathepsin D. Cathepsin D was purified 900-fold from human patellar cartilage. Its identity was established by its specific cleavage of the B chain of insulin. At least six multiple forms of cathepsin D are present in cartilage; these corresponded to bovine forms 4-9. Cathepsin D had no action on proteins at pH 7.4. However, cartilage extracts digested proteoglycan, casein, and histone at this pH. The proteolytic activities against these three substrates were purified about 170-, 160-, and 70-fold, respectively. Each activity appeared in multiple forms on DEAE-Sephadex chromatography. The three activities appear to be different since cysteine inhibited casein digestion, aurothiomalate inhibited histone digestion, and neither inhibited proteoglycan digestion. Tests with a wide range of inhibitors and activators suggest that these three activities differ from other neutral proteases described in the literature.

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The neutral metalloprotease extracted from 1,200 gm of human articular cartilage was purified 1,400‐ to 2,400‐fold by diethylaminoethyl‐ and carboxymethyl‐Sephadex chromatography. Disc electrophoresis and an isoelectric focusing method resolved the neutral enzyme activity into 4 bands. All bands had a similar amino acid analysis and a similar molecular weight by sodium dodecylsulfate electrophoresis and gel filtration: 24,000–27,000 daltons. The enzyme degraded proteoglycan subunit and proteoglycan aggregate to products with a sedimentation coefficient of 3S, but at low dilutions the enzyme produced 19.3S fragments. It is postulated that this protease may contribute to the development of osteoarthritis from within the cartilage matrix.