Katsura N, Yamada K
Bone. 1986;7(2):137-43. doi: 10.1016/8756-3282(86)90686-1.
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.
一种金属蛋白酶已从鸡骨骺软骨的基质小泡中分离出来并进行了表征。通过胶原酶消化和差速离心获得的基质小泡经Sepharose CL2B凝胶过滤进一步纯化。用脱氧胆酸盐处理并冻融后，蛋白酶从这些小泡中溶解出来，然后通过Sephadex G150凝胶过滤进行分离。该酶对偶氮酪蛋白底物具有蛋白酶活性，其圆盘电泳显示为单一蛋白条带。基于分子量（MW）测定、缺乏免疫交叉反应以及电泳迁移差异，由用于制备小泡的商业胶原酶导致外部蛋白酶污染的可能性很小。基质小泡蛋白酶的分子量为33,000，最适pH为7.2，被0.1 mM乙二胺四乙酸（EDTA）和0.2 mM邻菲罗啉完全抑制。较高浓度的α2 -巨球蛋白、卵清蛋白、半胱氨酸、青霉胺、乙烷-1-羟基-1,1-二膦酸盐（EHDP）和焦磷酸盐也具有抑制作用。邻菲罗啉的抑制作用可被Co2 +、Zn2 +、Fe2 +和Cu2 +逆转。通过不连续蔗糖密度梯度离心分级分离的基质小泡重组分中蛋白酶活性最为丰富。在钙化前沿释放这种蛋白酶可能会降解抑制细胞外基质中矿物质沉淀的非胶原蛋白部分，从而促进矿化。
