Hill P A, Reynolds J J, Meikle M C
Cell and Molecular Biology Department, Strangeways Research Laboratory, Worts Causeway, Cambridge, UK.
Biochim Biophys Acta. 1994 Nov 11;1201(2):193-202. doi: 10.1016/0304-4165(94)90041-8.
Matrix proteins were extracted from bovine cortical bone with EDTA/Tris-HCl under non-dissociative conditions at neutral pH. Four distinct bone resorptive proteins with molecular masses of 14, 25, 29 and 40 kDa were purified and partially characterized using an in vitro neonatal mouse calvarial assay and a growth factor assay using BALB/c/3T3 cells. The 14 kDa protein was purified by anion exchange chromatography (Mono Q) and gel filtration (Superdex 75HR) using FPLC (fast protein liquid chromatography); this factor stimulated the proliferation of MCF-7 human breast cancer cells, a bioassay which is specific for the insulin-like growth factors (IGFs). The 25, 29 and 40 kDa proteins were purified by sequential chromatography as follows: anion-exchange (Mono Q), heparin-Sepharose, hydroxyapatite, concanavalin A-Sepharose, phenyl-Superose, reversed phase high performance liquid chromatography (HPLC) and sodium dodecylsulfate polyacrylamide gelelectrophoresis (SDS-PAGE). The 25 kDa protein was identified as TGF-beta by its inhibitory effect on the proliferation of mink lung cells. The 40 kDa protein enhanced the formation of multinucleate tartrate-resistant acid phosphatase positive cells in a murine bone marrow differentiation assay, but was without effect in an isolated osteoclast assay and had no growth factor activity; this protein is likely to be a colony stimulating factor. The 29 kDa protein was also without growth factor activity; it was, however, able to stimulate bone resorption in the isolated osteoclast assay, suggesting a direct action in osteoclast function. The 29 and 40 kDa proteins may be osteoblast gene products that have been sequestrated by the bone matrix in a similar fashion to TGF-beta and the IGFs. This is the first report of proteins isolated from bone matrix which directly stimulate osteoclast differentiation and activity.
在中性pH的非解离条件下，用乙二胺四乙酸/三羟甲基氨基甲烷盐酸盐（EDTA/Tris-HCl）从牛皮质骨中提取基质蛋白。使用体外新生小鼠颅骨试验和利用BALB/c/3T3细胞的生长因子试验，对四种分子量分别为14、25、29和40 kDa的不同骨吸收蛋白进行了纯化和部分特性鉴定。14 kDa的蛋白通过快速蛋白质液相色谱（FPLC），利用阴离子交换色谱（Mono Q）和凝胶过滤（Superdex 75HR）进行纯化；该因子刺激MCF-7人乳腺癌细胞的增殖，这是一种对胰岛素样生长因子（IGF）特异的生物测定法。25、29和40 kDa的蛋白通过以下顺序色谱法进行纯化：阴离子交换（Mono Q）、肝素琼脂糖、羟基磷灰石、伴刀豆球蛋白A琼脂糖、苯基-Superose、反相高效液相色谱（HPLC）和十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）。25 kDa的蛋白因其对貂肺细胞增殖的抑制作用而被鉴定为转化生长因子-β（TGF-β）。在小鼠骨髓分化试验中，40 kDa的蛋白增强了抗酒石酸酸性磷酸酶阳性多核细胞的形成，但在分离的破骨细胞试验中无作用且无生长因子活性；该蛋白可能是一种集落刺激因子。29 kDa的蛋白也无生长因子活性；然而，它能够在分离的破骨细胞试验中刺激骨吸收，表明其对破骨细胞功能有直接作用。29和40 kDa的蛋白可能是以与TGF-β和IGF类似的方式被骨基质隔离的成骨细胞基因产物。这是首次报道从骨基质中分离出直接刺激破骨细胞分化和活性的蛋白。
