Shimura F, Wasserman R H
Endocrinology. 1984 Nov;115(5):1964-72. doi: 10.1210/endo-115-5-1964.
The vitamin D-induced intestinal calcium-binding protein (CaBP) was quantitated in membranous components of the intestinal mucosa by a specific and sensitive RIA. Inclusion of detergent (Triton X-100) in extraction buffer and in the RIA system was required to release and measure membrane-associated CaBP. Purified brush borders were shown to contain CaBP with a specific activity (micrograms per mg protein) about 12% of that in the total homogenate. By transferring proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to nitrocellulose blots electrophoretically, CaBP was immunologically detected in brush borders from vitamin D3-treated chicks, but not in those from vitamin D3-deficient chicks. CaBP was also detected in isolated brush border membrane vesicles by the gel electrophoresis-blot transfer technique. Brush border CaBP was inaccessible to proteolytic hydrolysis by trypsin unless trypsinized in the presence of detergent. CaBP-binding substances were found to be present in purified brush borders, using the gel overlay technique. A specific binding protein with a mol wt in the range of 50,000-70,000 daltons was identified, as well as an avid CaBP binder at less than 14,000 mol wt. These observations provide evidence for the association of a significant fraction of total intestinal CaBP with brush borders in vivo, which might have physiological relevance.
通过一种特异且灵敏的放射免疫分析法（RIA）对维生素D诱导的肠钙结合蛋白（CaBP）在肠黏膜膜性成分中的含量进行了测定。在提取缓冲液和RIA系统中加入去污剂（Triton X - 100），以释放并测量与膜相关的CaBP。结果显示，纯化的刷状缘所含CaBP的比活性（每毫克蛋白质微克数）约为全匀浆中的12%。通过将十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳分离的蛋白质电泳转移至硝酸纤维素印迹膜上，在经维生素D3处理的雏鸡的刷状缘中免疫检测到了CaBP，但在维生素D3缺乏的雏鸡的刷状缘中未检测到。通过凝胶电泳印迹转移技术，在分离的刷状缘膜泡中也检测到了CaBP。除非在去污剂存在的情况下用胰蛋白酶处理，刷状缘CaBP对胰蛋白酶的蛋白水解作用不敏感。利用凝胶覆盖技术发现纯化的刷状缘中存在CaBP结合物质。鉴定出一种分子量在50,000 - 70,000道尔顿范围内的特异性结合蛋白，以及一种分子量小于14,000道尔顿的强CaBP结合剂。这些观察结果为体内大部分肠CaBP与刷状缘的关联提供了证据，这可能具有生理意义。
