Isolation and characterization of a metalloprotease associated with chicken epiphyseal cartilage matrix vesicles.
A metalloprotease has been isolated from matrix vesicles of chicken epiphyseal cartilage and subsequently characterized. Matrix vesicles obtained by collagenase digestion and differential centrifugation were further purified by Sepharose CL2B gel filtration. The protease was solubilized from the vesicles by treatment with deoxycholate and freeze-thawing, and then isolated by Sephadex G150 gel filtration. Disc electrophoresis of the enzyme, which displayed protease activity toward azocasein substrate, gave a single protein band. Based on molecular weight (MW) determination, lack of immunocross reactivity, and differences in electrophoretic migration, there is little possibility of any contamination with external protease from the commercial collagenase used for vesicle preparation. The matrix vesicle protease had a MW of 33,000 and a pH optimum of 7.2 and was completely inhibited by 0.1 mM EDTA and 0.2 mM o-phenanthroline. alpha 2-Macroglobulin, ovalbumin, cysteine, penicillamine, ethane-1-hydroxy-1, 1-diphosphonate (EHDP) and pyrophosphate at higher concentrations were also inhibitory. The inhibition by omicron-phenanthroline was reversed by Co2+, Zn2+, Fe2+, and Cu2+. Protease activity was most abundant in the heavy fraction of matrix vesicles fractionated by discontinuous sucrose density gradient centrifugation. Release of this protease at the calcifying front could degrade noncollagenous protein moieties that inhibit precipitation of minerals in the extravesicular matrix and thus facilitate mineralization.
D000818:Animals; D001857:Bone Matrix / Q000201:enzymology* / Q000378:metabolism; D002356:Cartilage / Q000201:enzymology* / Q000378:metabolism; D002645:Chickens; D010450:Endopeptidases / Q000302:isolation & purification*; D004838:Epiphyses / Q000201:enzymology* / Q000378:metabolism; D008666:Metalloendopeptidases; D008667:Metalloproteins / Q000378:metabolism*
D008667:Metalloproteins; D010450:Endopeptidases; D008666:Metalloendopeptidases