The immplication of crystal surface binding substances (CSBS) on urinary stone formation.

尿大分子被认为在结石形成中起重要作用。本研究的目的是对影响结石形成的尿蛋白进行表征。1.通过在人全尿中加入钙和草酸盐，获得晶体表面结合物质（CSBS）。CSBS是一种吸附在草酸钙晶体表面的尿大分子，对草酸钙结晶有很强的抑制作用。2.通过DEAE-SepharoseCL-6 B离子色谱分离，得到了草酸钙晶体生长抑制活性较强的富集蛋白组分。经羟基磷灰石柱层析和磷酸钠分步洗脱后，得到的蛋白质组分经SDS-PAGE电泳后显示出多条蛋白质条带。用氨基酸序列分析仪分析了两种主要蛋白质，分子量分别为30 Kd和67 Kd。结果表明，30 Kd蛋白与人凝血酶原蛋白结合，67 Kd蛋白与人骨桥蛋白结合。4.对羟基磷灰石柱层析分离的蛋白组分进行免疫印迹分析。检测人血清白蛋白、α 1-酸性糖蛋白、α 1-微球蛋白、α 2-HS糖蛋白、视黄醇结合蛋白、转铁蛋白、Tamm-Horsfall粘蛋白和凝血酶原。但是，这些蛋白质对草酸钙晶体的形成没有明显的抑制作用，因为每种蛋白质的组分对晶体生长的抑制作用都很弱，因此我们认为CSBS中的其他未知蛋白质是主要的抑制蛋白质。

Urinary macromolecules are considered to play a important role in stone formation. The aim of our study is to characterize the urinary protein affecting stone formation.1.We obtained crystal surface binding substance (CSBS) by adding calcium and oxalate to human whole urine. The CSBS is urinary macromolecules absorbed onto the surface of calcium oxalate crystals and yields strong inhibitory activity on calcium oxalate crystallization. Proteins contained in the CSBS are analyzed.2.Fractions enriched in proteins with strong calcium oxalate crystal growth inhibitory activity are obtained by DEAE-Sepharose CL-6B ion-chromatography. These fractions are fractionated by hydroxyapatite column with stepwise elution of sodium phosphate.3.The SDS-PAGE shows many bands of protein. Two major proteins, molecular weight of 30Kd and 67Kd, are analyzed by a amino acid sequencer. As a result, 30Kd protein is identified with human prothrombin and 67Kd protein with human osteopontin.4.Protein composition of fractions obtained by hydroxyapatite column is also analyzed by immunoblotting. Human serum albumin, alpha 1-acid glycoprotein, alpha 1-microglobulin, alpha 2-HS glycoprotein, retinol-binding protein, transferrin, Tamm-Horsfall mucoprotein and prothrombin are detected. However, it is considered that these proteins does not play a signifficant role on calcium oxalate crystal formation because each fractions containing each proteins reveal weak inhibitory power on crystal growth.We conclude that other unknown proteins included in CSBS are thought to be dominant protein inhibitors.