Purification and identification of factors involved in osteoclastic maturation from bovine bone matrix

点击翻译按钮获取中文摘要

Several studies have shown that formation of mature osteoclasts significantly increases when osteoclast progenitors are incubated on bone matrix compared with the formation on a plastic substratum. These observations have suggested that some components of bone matrix play a functional role in formation of mature osteoclasts. However, detailed characterization of these components derived from bone matrix has not yet been made.In the present grant-aid for scientific research, we purified the osteoclastic maturation-inducing factors from bovine bone, and obtained the following results.(1) Although tartrate-resistant acid phosphatase (TRAP) -positive cells formed from calvarial cells in the presence of 1alpha, 25- (OH) _2D_3 on plastic culture dishes could not resorb dentine slices, 1alpha, 25- (OH) _2D_3-induced TRAP-positive cells formed in combination with EDTA-extracts from bovine bone could resorb it.(2) The OMIFs were extracted from bovine bone powder during demineralization with EDTA,and fractionated and purified by gel filtration over Superdex 75 prep grade. The peak of OMIFs activity was eluted as an approximately 12KD species from the gel filtration column.(2) The peak fraction having OMIFs activity eluted as a major peak at about 60mM sodium phosphate (pH6.8) by the hydroxyapatite column.(3) The OMIFs eluted as a major peak at about 250mM NaCl by Mono Q equilibrated with 0.02M Bis-Tris propane (pH7.0).(4)The OMIFs were separated into several peaks by reversed-phase HPLC.the hydroxyapatite column. The molecular weights of OMIFs estimated from high-resolution 16.5% polyacrylamide gel electrophoresis were 5.1KD,6.8KD and 8.4KD respectively.In further studies, we will analyze the amino-acid sequence and define physicochemical characteristics of the purified OMIFs.