Relative deficiency of acidic isoforms of osteopontin from stone former urine.
Relative deficiency of acidic isoforms of osteopontin from stone former urine.
复制标题
DOI：
10.1007/s00240-012-0459-1
复制
发表时间：
2012-10
影响因子：
--
通讯作者：
Wesson JA
中科院分区：
文献类型：
--
作者：
Kolbach AM;Afzal O;Halligan B;Sorokina E;Kleinman JG;Wesson JA
我们用高分辨率等电聚焦(等电点，等电点3.5-4.5)和Western印迹检测了从正常人(NU)尿液中分离的骨桥蛋白(OPN)与来自结石形成者(SFU)尿液中类似样品的相对凝胶迁移率。我们还报告了对这些样本进行竞争性ELISA分析的结果。我们证明了人尿OPN有一个离散的四条带分离模式，与先前报道的四种OPN亚型一致。较低的两种MR异构体比较高的两种MR异构体向凝胶的酸性端迁移的程度更大。根据对以0.1PI为单位增量分组的光点强度的分析，信号的密度测量表明，与NU相比，OPN从SFU到NU的迁移模式有显著差异。提出了一种基于OPN 2D Western印迹中相对信号强度来计算加权平均PI的新方法。分析表明，与正常人群相比，结石患者中高MR形式的OPN的加权平均PI值显著增加。此外，碱性磷酸酶处理的NU样品导致平均PI沿碱性方向移动了0.05个单位，这表明平均磷酸化程度的降低可能是NU和SFU PI之间差异的原因。
We have tested the relative electrophoretic mobility of osteopontin (OPN) isolated from urine obtained from normal individuals (NU) against similar samples derived from the urine of stone formers (SFU) using high-resolution isoelectric focusing (isoelectric point, pI range 3.5–4.5) in 2D electrophoresis, with Western blot detection. We also report the results from competitive ELISA analyses of these samples. We demonstrated that human urinary OPN has a discrete four band separation pattern that conforms to four previously documented OPN isoforms. The lower two Mr isoforms migrate to a greater degree toward the acidic end of the gel than do the higher two Mr isoforms. Densitometry of the signal reveals significant difference in the migration pattern of OPN from SFU as compared to that from NU based on an analysis of the spot intensities grouped in 0.1 pI unit increments. A novel method for the calculation of a weight-averaged pI based on the relative signal strength in an OPN 2D Western blot was developed. The analysis revealed a significantly increased weight-averaged pI values for the higher Mr forms of OPN in the stone former compared to normal population. Additionally, alkaline phosphatase-treated NU samples resulted in a significant average pI shift of 0.05 units in the alkaline direction, suggesting that a decrease in the average degree of phosphorylation could be responsible for the difference between NU and SFU pI.