KINETICS OF B12, INTRINSIC FACTOR & OTHER PROTEINS

从饮食中吸收钴胺(Cbl)，其后续的 转运和细胞摄取，需要Cbl与两个 其中每一种的可溶性蛋白质和特异膜受体 蛋白质。 这个项目的目标一直是提纯和表征 这些Cb1结合蛋白及其膜受体的研究 它们在Cbl代谢中的作用。本项目现在将重点放在 以下具体目标：1)研究结构、合成和 分泌转钴胺II(TCII)，这是所需的运输蛋白 细胞对Cbl的摄取，并研究TCII 促进Cbl的摄取。将开发出单克隆抗体 TCII分子上的特定表位和TCII的多肽片段 与Cbl结合的特定抗体发生交叉反应的蛋白质 将对结合位点和受体结合位点进行鉴定和测序 测定组成其功能结构域的氨基酸 TCII，这将与完整的氨基酸序列相关 从互补DNA(CDNA)推导出来的(见下文)。这个 TCII的生物合成和分泌将在人脐上进行研究 血管内皮细胞新生TCII的[35S]蛋氨酸标记。 TCII在Cbl肠道吸收中的作用将通过 Cbl吸收过程中TCII基因表达的研究 免疫化学技术；2)纯化TCII膜受体 人胎盘，鉴定其结构并产生抗血清 3)从人内皮细胞中分离TCII基因 细胞库，确定其来源的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。研究制约因素 长度多态与TCII基因的染色体定位 吉恩。 这些研究将提供有关蛋白质的基本信息。 细胞摄取Cbl所必需的物质及其分子遗传学 TCII，所有这些都对理解获得性和 先天性Cbl代谢障碍。

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.