Seetharam B, Levine J S, Ramasamy M, Alpers D H
Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110.
J Biol Chem. 1988 Mar 25;263(9):4443-9.
High levels of receptor for intrinsic factor-cobalamin (vitamin B12) were detected in human, canine, and rat kidneys. The ratio of specific activity (picomoles/mg of protein) for kidney relative to intestine was 116, 20, and 797, respectively, in these species. The receptor was purified about 3000-fold from 200 g of rat kidney with a recovery of 16% and exhibited a single band on nondenaturing gel electrophoresis. Quantitative amino acid analysis of the receptor gave a value of 457,310 g of amino acid/mol of intrinsic factor-cobalamin binding activity. The pure receptor revealed an Mr of 430,000, as assessed by filtration with Bio-Gel A-5m. Treatment with papain resulted in the production of active monomers of Mr to about 205,000-210,000. Electrophoresis in the presence of sodium dodecyl sulfate confirmed the monomer Mr to be 230,000. The monomer receptor did not reveal the presence of any further subunits upon reductive alkylation. Following cyanogen bromide cleavage the kidney receptor revealed three peptides of Mr 115,000, 60,000, and 54,000. The pI of these peptides was 5.17, 6.17, and 6.17, respectively. Western blot analysis using antiserum raised to the receptor demonstrated a protein with an Mr of 175,000 and 230,000 for intestinal and kidney membrane receptors, respectively. Immunologically, the rat kidney receptor was identical to the rat ileal receptor but was distinct from the canine ileal receptor. Ultrastructural localization revealed the presence of the receptor in the apical surface membrane of proximal tubular cells of the kidney and absorptive cells of the ileum. The kidney is the best source for obtaining this receptor in reasonable quantities.
在人、犬和大鼠的肾脏中检测到高水平的内因子 - 钴胺素（维生素B12）受体。在这些物种中，肾脏相对于肠道的比活性（皮摩尔/毫克蛋白质）分别为116、20和797。从200克大鼠肾脏中纯化该受体约3000倍，回收率为16%，在非变性凝胶电泳上呈现单一条带。对该受体进行定量氨基酸分析，得出每摩尔内因子 - 钴胺素结合活性的氨基酸含量为457,310克。通过Bio - Gel A - 5m过滤评估，纯受体的Mr为430,000。用木瓜蛋白酶处理导致产生Mr约为205,000 - 210,000的活性单体。在十二烷基硫酸钠存在下进行电泳证实单体Mr为230,000。还原烷基化后，单体受体未显示出任何其他亚基的存在。溴化氰裂解后，肾脏受体显示出Mr为115,000、60,000和54,000的三种肽段。这些肽段的pI分别为5.17、6.17和6.17。使用针对该受体产生的抗血清进行的蛋白质印迹分析表明，肠道和肾脏膜受体的蛋白质Mr分别为175,000和230,000。在免疫学上，大鼠肾脏受体与大鼠回肠受体相同，但与犬回肠受体不同。超微结构定位显示该受体存在于肾脏近端小管细胞和回肠吸收细胞的顶端表面膜中。肾脏是获取适量该受体的最佳来源。
