KINETICS OF B12, INTRINSIC FACTOR & OTHER PROTEINS

从饮食中吸收钴胺素（Cbl），其随后的 转运和细胞摄取，需要Cbl与两个 可溶性蛋白质和这些蛋白质中的每一种的特异性膜受体 proteins. 该项目的目标是纯化和表征 这些Cbl结合蛋白及其膜受体，并描绘 它们在Cbl代谢中的作用。 该项目现在将重点放在 具体目标如下：1）研究结构、合成和 分泌转钴胺素II（TCII），所需的转运蛋白 细胞摄取Cbl，并检查TCII 促进Cbl的摄取。 将开发单克隆抗体 针对TCII分子上的特异性表位和 与Cbl结合的特异性抗体交叉反应的蛋白质 将鉴定和测序受体结合位点和受体结合位点的序列 以确定包含以下功能结构域的氨基酸： TCII，这将与完整的氨基酸序列相关联 从互补DNA（cDNA）推导（参见下文）。 的 TCII的生物合成和分泌将在人脐静脉中进行研究。 静脉内皮细胞使用[35 S]甲硫氨酸标记新生TCII。 TCII在Cbl的肠吸收中的作用将通过以下方法进行检查： 研究Cbl吸收过程中TCII的mRNA表达， 免疫化学技术; 2）纯化TCII膜受体， 人胎盘，表征其结构并产生抗血清， 3）从人内皮细胞中分离TCII cDNA， 细胞文库，确定cDNA的核苷酸序列， 可以推导出TCII的完整氨基酸序列。 确定 重组TCII的功能特性。 研究限制 长度多态性，并确定TCII的染色体定位 基因 这些研究将提供有关蛋白质的基本信息 细胞摄取Cbl所需的蛋白质， TCII，所有这些都是重要的，在了解获得性和 Cbl代谢的先天性疾病。

The absorption of cobalamin (Cbl) from the diet, its subsequent transport and cellular uptake, requires the interaction of Cbl with two soluble proteins and specific membrane receptors for each of these proteins. The objectives of this project have been to purify and characterize these Cbl binding proteins and their membrane receptors and to delineate their role in Cbl metabolism. This project will now focus on the following specific objectives: 1) Study the structure, snythesis and secretion of transcobalamin II (TCII), the transport protein required for the cellular uptake of Cbl and examine the process by which TCII facilitates the uptake of Cbl. Monoclonal antibodies will be developed to specific epitopes on the TCII molecule and peptide fragments of the protein that cross react with specific antibodies to the Cbl binding site and to the receptor binding site will be identified and sequenced to determine the amino acids which comprise the functional domains of TCII and this will be correlated with the complete amino acid sequence deduced from the complementary DNA (cDNA) (vide infra). The biosynthesis and secretion of TCII will be studied in human umbilical vein endothelial cells using [35S] methionine labeling of nascent TCII. The role of TCII in the intestinal absorption of Cbl will be examined by studying the expression of mRNA for TCII during Cbl absorption and by immunochemical techniques; 2) Purify the membrane receptor for TCII from human placenta, characterize its structure and generate an antiserum to this purified protein; 3) Isolate the TCII cDNA from a human endothelial cell library, determine the nucleotide sequence of the cDNA from which the complete amino acid sequence of TCII can be deduced. Determine the functional properties of the recombinant TCII. Study the restriction length polymorphism and determine the chromosomal location of the TCII gene. These studies will provide fundamental information about the proteins required for the cellular uptake of Cbl and the molecular genetics of TCII, all of which are important in the understanding of acquired and congenital disorders of Cbl metabolism.