KINETICS OF B12, INTRINSIC FACTOR AND OTHER PROTEINS

维生素B12(钴胺，Cbl)是人类(和 哺乳动物)代谢同型半胱氨酸的酶甲基化 甲硫氨酸的生物合成和分子重排 人(和动物)细胞中甲基丙二酰辅酶A转化为琥珀酰辅酶A 新陈代谢。缺乏这种维生素会导致严重的巨幼细胞 贫血和神经精神障碍。两个完整的生理学 为了向组织提供足够的CbL，需要进行一些处理。这个 首先是肠道吸收，这一过程是由胃介导的 与Cbl复合的内因数(IF)在上小 并将其像If-Cbl一样输送到回肠末端，在那里它结合 上皮粘膜微绒毛膜上的IF受体 并通过内吞作用内化。回肠粘膜内 细胞，释放的Cbl单向移动到门静脉血液，在那里它 似乎与血浆转运蛋白转钴胺II(TCII)结合。 TCII介导第二个基本的生理过程，即运输 血浆中的维生素，如TCII-Cbl复合体，到组织中 在那里它与细胞膜上的特定TCII受体结合 然后被受体介导的内吞作用内化。 导致Cbl亏缺的这一复杂过程的扰动 由于肠道吸收受损(缺乏IF或肠道 吸收不良)和缺乏TCII。因此，长期目标是 本课题的研究目的是：1)研究基因表达的调控。 TCII；II)TCII缺乏症先天疾病的特征； 三)阐明TCII和IF在促进 Cbl的肠道吸收。 为了实现这些目标，我们将表征基因组顺式元件 TCII基因(我们已克隆)和细胞质反式活性 调节TCII的结构性和组织特异性表达的因子， 并确定导致TCII基因突变的原因 三种先天性形式的TCII表达异常。这些基因研究 TCII应确定上调TCII合成的因素 自身免疫性疾病，B细胞增殖和一些癌症，这些可能 也下调TCII的合成，在一些受试者中观察到。 为了实现第三个目标，我们将克隆表达于 豚鼠回肠黏膜。我们将首先提纯分析 用IF-Cbl亲和层析法测定受体的量 亲和矩阵，然后从N端获得氨基酸序列 和内肽，并使用衍生的寡核苷酸来筛选 以豚鼠回肠黏膜组织中的mRNA为模板，构建了全长cdna文库。CDNA3 将提供有关受体的一级结构的信息，即 跨膜结构域(S)和功能，将有助于克隆和 确定IF受体基因的特征。对IF受体和If受体的研究 TCII在分子水平上可以阐明TCII在细胞周期中的作用。 Cbl的同化作用及Cbl吸收障碍的病因探讨 在没有缺乏的情况下会导致Cbl缺乏 内在因素。

Vitamin B12 (cobalamin, Cbl) is an essential cofactor in human (and mammalian) metabolism for the enzymatic methylation of homocysteine in the biosynthesis of methionine, and for the molecular rearrangement of methylmalonyl-CoA to succinyl-CoA in human (and animal) cellular metabolism. A deficiency of this vitamin results in severe megaloblastic anemia and neuropsychiatric disorders. Two integrated physiological processes are required for sufficient Cbl to be provided to tissues. The first is intestinal absorption, and this process is mediated by gastric intrinsic factor (IF) which complexes with Cbl in the upper small intestine and transports it as IF-Cbl to the distal ileum where it binds to an IF receptor on the microvillus membranes of the epithelial mucosa and is internalized by a process of endocytosis. Within the ileal mucosal cell, the released Cbl moves unidirectionally to the portal blood where it appears bound to the plasma transport protein, transcobalamin II (TCII). TCII mediates the second essential physiologic process, which is transport of the vitamin in the plasma, as the TCII-Cbl complex, to the tissues where it binds to a specific TCII receptor on the plasma membrane of cells and is then internalized by receptor mediated endocytosis. Perturbations of this complex process that lead to Cbl deficiency result from impaired intestinal absorption (lack of IF or intestinal malabsorption) and lack of TCII. Accordingly, the long-term objectives and aims of this project are: i) to study the regulation of expression of TCII; ii) to characterize the congenital disorders of TCII deficiency; iii) to elucidate the integrated function of TCII and IF in facilitating the intestinal absorption of Cbl. To achieve these aims, we will characterize the genomic cis elements of the TCII gene (which we have cloned) and the cytoplasmic trans-active factors that regulate constitutive and tissue specific expression of TCII, and identify the mutations of the TCII gene that are responsible for the three congenital forms of abnormal TCII expression. These genetic studies of TCII should identify the factors that up-regulate synthesis of TCII in autoimmune diseases, B-cell proliferations and some cancers, and which may also down-regulate TCII synthesis as observed in some subjects. To achieve the third objective we will clone the IF receptor expressed on the ileal mucosa of guinea pig ileum. We will first purify analytical amounts of the receptor by affinity chromatography using an IF-Cbl affinity matrix and then obtain an amino acid sequence from the N-terminus and internal peptides and use the derived oligonucleotides to screen a cDNA library prepared from the mRNA of guinea pig ileal mucosa. The cDNA will provide information about the primary structure of the receptor, the transmembrane domain(s) and function, and will be useful to clone and characterize the IF-receptor gene. Such studies of the IF-receptor and TCII at the molecular level can clarify the role of TCII in the assimilation of Cbl and clarify the etiology of impaired Cbl absorption that can result in Cbl deficiency in the absence of deficiency of intrinsic factor.