Abstract
Human articular cartilage contains very low levels of metalloprotease activity; the activity in 1 g of cartilage is approximately equivalent to the activity of 1 microgram of trypsin. Development of a sensitive assay, based on the digestion of radioactive proteoglycan, has made it possible to study protease activity in 1-2-g specimens of cartilage. Cartilage was extracted with Tris buffer in the cold and with Tris buffer containing 10 mM CaCl2 at 60 degrees C. The extracts were passed through Sepharose 6B; two major and two minor metalloprotease activities were detected. A neutral metalloprotease activity, pH optimum 7.4, was found as a latent form of Mr = 56,000. It could be activated with aminophenylmercuric acetate or trypsin with a resultant decrease of Mr to 40,000. An acid metalloprotease, pH optimum 5.3, also occurred as a latent form of Mr = 50,000. Activation converted this to Mr = 35,000. Removal of calcium ions by dialysis reduced the activity of the neutral enzyme by 80-85% and of the acid enzyme by 100%. Both activities were restored by 10 mM Ca2+. Both enzymes were completely inhibited by 1 mM o-phenanthroline in the presence of excess calcium. This inhibition was overcome by 1 mM Zn2+ and, to a lesser extent, by Co2+. These proteases may be important in the metabolism of the cartilage matrix and in its destruction in osteoarthritis.
Highlights
Human articular cartilage contains very low levels treatment
It was discovered that both neutral and acid metalloproteases are present in cartilage in latent forms
1 mM o-phenanthroline in the presence of excess cal- Enzyme Assay-We have previously described an assay for procium. This inhibition was overcomeby l mM Zn2+and, teases based on the digestion of proteoglycan monomer prepared from toa lesser extent, by Co2+
Summary
From the Departmentsof Biochemistry and Medicine, Universityof Miami School of Medicine, Miami,Florida 33101. Amide gel beads, the pore size is adjusted to retain monomers, but to permit the escape of digestion products smaller than approximately 200,000 daltons This assay is used in order to identify proteases in cartilage that can attack the protein core of proteoglycans in the. In 1976 [1] we discovered that human articular cartilage contains metalloprotease activity capable of cleaving the core protein of cartilage proteoglycan at physiological pH. It would be very useful to be able to measure enzyme activities in small samples of cartilage (1-2 g) This would permit the study of tissue from a single patient to detemine the effect of factors such as age, disease, and drug.
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