Chen Y, Bal B S, Gorski J P
Division of Molecular Biology and Biochemistry, School of Biological Sciences, University of Missouri, Kansas City 64110-2499.
J Biol Chem. 1992 Dec 5;267(34):24871-8.
Calcium binding properties of bone acidic glycoprotein-75, osteopontin, and bone sialoprotein were determined in 10 mM imidazole buffer (pH 6.8), containing either 60 mM KCl or 150 mM NaCl. Proteins assayed were first bound to nitrocellulose to mimic substrate-bound forms in vivo; retention of phosphoproteins was determined through use of radioiodinated tracers. Binding studies were carried out both as a function of calcium concentration and the amount of phosphoprotein. In the presence of 60 mM KCl, bone acidic glycoprotein-75 exhibited the largest calcium binding capacity (139 atoms/molecule at saturation), with bone sialoprotein intermediary (83 atoms/molecule) and osteopontin lowest (50 atoms/molecule). Sites detected for each phosphoprotein exhibited overall binding constants in the 0.5-1.0 mM extracellular range. In 150 mM NaCl and 1-2 mM total calcium, phosphoproteins bound between 72 and 19 mol of calcium/mol with the same relative order. Binding was proportional to amount of phosphoprotein in either salt condition. The presence of 5 mM calcium had a different effect on concentration-dependent binding to type I collagen for each phosphoprotein. Bone acidic glycoprotein-75 alone was found to undergo an unusual calcium-enhanced polymerization reaction, confirmed by light scattering measurements, wherein collagen binding was greatest with polymeric forms. These findings demonstrate that acidic phosphoproteins from bone bind calcium atoms with a range of capacities. Calcium appears to induce conformational changes in bone acidic glycoprotein-75 which influences its self-association and binding to different substrata.
在含有60 mM KCl或150 mM NaCl的10 mM咪唑缓冲液（pH 6.8）中测定了骨酸性糖蛋白-75、骨桥蛋白和骨唾液酸蛋白的钙结合特性。所检测的蛋白质首先与硝酸纤维素结合，以模拟体内底物结合形式；通过使用放射性碘化示踪剂测定磷蛋白的保留情况。结合研究是作为钙浓度和磷蛋白量的函数进行的。在60 mM KCl存在下，骨酸性糖蛋白-75表现出最大的钙结合能力（饱和时为139个原子/分子），骨唾液酸蛋白居中（83个原子/分子），骨桥蛋白最低（50个原子/分子）。为每种磷蛋白检测到的位点在细胞外0.5 - 1.0 mM范围内表现出总体结合常数。在150 mM NaCl和总钙浓度为1 - 2 mM时，磷蛋白以相同的相对顺序结合72至19摩尔钙/摩尔。在任何一种盐条件下，结合都与磷蛋白的量成正比。5 mM钙的存在对每种磷蛋白与I型胶原的浓度依赖性结合有不同影响。单独的骨酸性糖蛋白-75被发现会发生一种不寻常的钙增强聚合反应，这通过光散射测量得到证实，其中胶原与聚合形式的结合最强。这些发现表明，来自骨的酸性磷蛋白具有一系列的钙结合能力。钙似乎会诱导骨酸性糖蛋白-75的构象变化，这会影响其自身缔合以及与不同底物的结合。
