An oxalate-binding protein with crystal growth promoter activity from human kidney stone matrix
An oxalate-binding protein with crystal growth promoter activity from human kidney stone matrix
复制标题
DOI：
10.1046/j.1464-410x.2002.02849.x
复制
发表时间：
2002-08-01
影响因子：
4.5
通讯作者：
Selvam, R
中科院分区：
文献类型：
--
作者：
Govindaraj, A;Selvam, R
目的从以草酸钙（CaOx）为主要成分的肾结石基质中分离蛋白质，鉴定草酸盐结合蛋白的存在及其在CaOx晶体生长系统中的作用。溶解的蛋白质通过用Tris-HCl缓冲液中增加的氯化钠浓度洗脱进行纤维素柱层析。洗脱出三个蛋白级分峰，即缓冲液中的级分I、缓冲液中的0.05 mol/L NaCl中的级分II和缓冲液中的0.3 mol/L NaCl中的级分III。结果所有三个组分在pH 7.4时具有最大的CaOx结合活性，但组分II在pH 4.5时也具有活性。组分I促进体外CaOx晶体生长，而组分II和III具有抑制作用。当组分I在Sephadex G-200柱上进一步分离时，得到两个蛋白组分（Ia和Ib）。组分Ia蛋白具有高的CaOx结合活性，组分Ib具有低的CaOx结合活性。组分Ia在凝胶电泳和Western印迹上具有48 kDa的分子量。48 kDa蛋白不与晶体基质蛋白抗体、带3蛋白抗体或白蛋白发生交叉反应。该蛋白质促进CaOx晶体生长，最适温度为37 ℃，pH为6.5。柠檬酸盐对晶体生长的抑制作用在48 kDa蛋白的存在下显著降低。蛋白质促进成核和聚集的CaOx晶体在体外结晶系统中，在pH 6.5，而馏分Ib（29 kDa）抑制成核和聚集。使用48 kDa的抗体，从结石基质的蛋白质的产率为32%，通过EDTA提取和只有3%，与其他方法。该蛋白质也被检测到在细胞核和线粒体，并在其他基质组分的磷酸钙和尿酸stone.Conclusions 48 kDa的蛋白质分离的结石基质是一个有效的促进剂的CaOx晶体生长具有高结合活性，它是丰富的细胞核和线粒体。
Objective To fractionate renal-stone matrix proteins, identify the presence of oxalate-binding protein and assess its effect in a calcium oxalate (CaOx) crystal growth system.Materials and methods Proteins were isolated from the matrix of kidney stones containing CaOx as the major constituent, using EDTA as a demineralizing agent. The solubilized proteins were subjected to cellulose-column chromatography by eluting with increasing sodium chloride concentrations in Tris-HCl buffer. Three protein fraction peaks were eluted, i.e. fraction I in buffer, fraction II in 0.05 mol/L NaCl in buffer and fraction III in 0.3 mol/L NaCl in buffer. The protein fractions were tested for their effects on CaOx crystal growth.Results All three fractions had maximum CaOx binding activity at pH 7.4 but fraction II also had activity at pH 4.5. Fraction I promoted in vitro CaOx crystal growth, while fractions II and III were inhibitory. When fraction I was further separated on a Sephadex G-200 column, two protein fractions (Ia and Ib) were obtained. Fraction Ia protein had high and fraction Ib low CaOx-binding activity. Fraction Ia had a molecular weight of 48 kDa on gel electrophoresis and Western blotting. The 48 kDa protein did not cross-react with crystal matrix protein antibody, band-3 protein antibody, or albumin. The protein promoted CaOx crystal growth, with an optimum temperature of 37 degreesC and pH 6.5. The inhibitory effect of citrate on crystal growth was significantly lower in the presence of the 48 kDa protein. The protein promoted nucleation and aggregation of CaOx crystals in the in vitro crystallization system at pH 6.5, whereas fraction Ib (29 kDa) inhibited both nucleation and aggregation. Using the 48 kDa antibody, the yield of the protein from the stone matrix was 32% by EDTA extraction and only 3% with other methods. The protein was also detected in the nucleus and mitochondria, and in other matrix fractions of calcium phosphate and uric acid stones.Conclusions The 48 kDa protein isolated from stone matrix is a potent promoter of CaOx crystal growth with high oxalate-binding activity; it is enriched in the nucleus and mitochondria.