Jarvis M F, Burns C J, Pauls H W, Assal A, Kim J S, Cheney D L, Youssefyeh R D
Department of Pharmacology, Rhone-Poulenc Rorer Central Research, Collegeville, Pennsylvania 19426.
Calcif Tissue Int. 1993 May;52(5):372-7. doi: 10.1007/BF00310202.
The present studies characterize the binding of [14C]citric acid to synthetic hydroxyapatite (HA) crystals. [14C]Citric acid specifically bound to HA and was dependent upon the concentration of HA in the assay. The binding of [14C] citric acid to HA reached equilibrium within 20 min and remained stable for at least 90 min. Dissociation of bound [14C]citric acid was biphasic in nature since both rapid and more slowly reversible binding components were detected. Saturation experiments also indicated that [14C]citric acid labeled two recognition sites with different affinity (KdH = 42 nM and KdL = 24,000 nM) and density (BmaxH = 161 fmol/micrograms HA and BmaxL = 8.8 pmol/micrograms HA). Ligand competition experiments revealed that compounds that are known to readily bind bone (e.g., sodium pyrophosphate, methylene diphosphonic acid, etidronate) potently inhibited the binding of [14C]citric acid to HA, whereas compounds known to have poorer affinity for bone (e.g., oxalic acid and GABA) did not. Computer analysis of these inhibition curves revealed specific ligand interactions at two different affinity recognition sites. The present results indicate that [14C]citric acid binds discrete sites on synthetic HA in a fashion consistent with a specific labeling of the bisphosphonate recognition site. Analysis of the binding of [14C]citric acid to HA provides a useful method to further explore the structure activity relationships of novel compounds that have binding affinity for bone.
本研究对[14C]柠檬酸与合成羟基磷灰石（HA）晶体的结合特性进行了表征。[14C]柠檬酸特异性结合到HA上，且依赖于检测中HA的浓度。[14C]柠檬酸与HA的结合在20分钟内达到平衡，并至少保持稳定90分钟。结合的[14C]柠檬酸的解离本质上是双相的，因为检测到了快速和更缓慢可逆的结合成分。饱和实验还表明，[14C]柠檬酸标记了两个具有不同亲和力（KdH = 42 nM和KdL = 24,000 nM）和密度（BmaxH = 161 fmol/微克HA和BmaxL = 8.8 pmol/微克HA）的识别位点。配体竞争实验表明，已知容易结合骨的化合物（如焦磷酸钠、亚甲基二膦酸、依替膦酸）能有效抑制[14C]柠檬酸与HA的结合，而对骨亲和力较差的化合物（如草酸和γ-氨基丁酸）则不能。对这些抑制曲线的计算机分析揭示了在两个不同亲和力识别位点上的特异性配体相互作用。目前的结果表明，[14C]柠檬酸以与双膦酸盐识别位点特异性标记一致的方式结合到合成HA上的离散位点。分析[14C]柠檬酸与HA的结合为进一步探索对骨具有结合亲和力的新型化合物的构效关系提供了一种有用的方法。
