Mathan V I, Babior B M, Donaldson R M
J Clin Invest. 1974 Sep;54(3):598-608. doi: 10.1172/JCI107797.
To determine whether the molecular configuration of vitamin B(12) influences the attachment of intrinsic factor-vitamin B(12) complex to ileal microvillous membrane receptor sites, we have examined the kinetics of uptake of intrinsic factor-bound cyanocobalamin by brush borders and microvillous membranes isolated from guinea pig ileum, and have compared this uptake with that of intrinsic factor alone and with that of intrinsic factor complexed with various analogs of cyanocobalamin. We first studied the kinetics of binding of cyanocobalamin and other cobamides to human gastric intrinsic factor. The binding of cyanocobalamin showed saturation kinetics and, at relatively high concentrations of cyanocobalamin, a Scatchard plot of binding was linear. The dissociation constant for the intrinsic factor-cyanocobalamin complex was 0.066 nM. When the binding of various vitamin B(12) analogs to intrinsic factor was determined by competition experiments, the analogs could be separated into two categories: those with affinities similar to that of cyanocobalamin and those with affinities much lower than that of cyanocobalamin. The affinity of cyanocobalamin for intrinsic factor was not altered by various substitutions at the -CN position, while removal of a single amido group on the corrin ring of substitution of the dimethylbenzimidazole base greatly reduced affinity. Removal of the base totally abolished binding. These findings, confirming those reported by others, are consistent with the concept that the cyanocobalamin molecule fits into a "pocket" in the intrinsic factor molecule, with the nucleotide base facing inward and the -CN side of the planar corrin ring facing outward. We then investigated the attachment of intrinsic factor-bound cyanocobalamin to ileal receptor. Attachment to microvillous membranes showed saturation kinetics with a dissociation constant of 0.25 nM. Attachment was rapid and was 70% complete within 5 min; the second-order rate constant for attachment was 1.3 x 10(6) M(-1) s(-1). The half-time for dissociation of intrinsic factor-bound cyanocobalamin from the ileal receptor was approximately 35 min. Free intrinsic factor inhibited the attachment of intrinsic factor-bound cyanocobalamin, but the rate of attachment of free intrinsic factor was slower than that of intrinsic factor bound to cyanocobalamin. When intrinsic factor was complexed with various analogs of cyanocobalamin, the affinities of these complexes for ileal microvillous membranes were similar to that of intrinsic factor-bound cyanocobalamin. These findings suggest that the molecular configuration of vitamin B(12) is not a major determinant in the interaction between intrinsic factor-bound vitamin B(12) and its ileal receptor site.
为了确定维生素B12的分子构型是否会影响内因子 - 维生素B12复合物与回肠微绒毛膜受体位点的结合，我们研究了从豚鼠回肠分离的刷状缘和微绒毛膜摄取内因子结合型氰钴胺素的动力学，并将这种摄取与单独的内因子以及与氰钴胺素各种类似物复合的内因子的摄取进行了比较。我们首先研究了氰钴胺素和其他钴胺素与人类胃内因子的结合动力学。氰钴胺素的结合呈现饱和动力学，并且在相对高浓度的氰钴胺素下，结合的Scatchard图呈线性。内因子 - 氰钴胺素复合物的解离常数为0.066 nM。当通过竞争实验测定各种维生素B12类似物与内因子的结合时，这些类似物可分为两类：亲和力与氰钴胺素相似的和亲和力远低于氰钴胺素的。氰钴胺素在 -CN位置的各种取代不会改变其与内因子的亲和力，而在咕啉环上除去单个酰胺基或取代二甲基苯并咪唑碱基会大大降低亲和力。除去碱基则完全消除结合。这些发现证实了其他人报道的结果，与氰钴胺素分子适合内因子分子中的一个“口袋”的概念一致，其中核苷酸碱基向内，平面咕啉环的 -CN侧向外。然后我们研究了内因子结合型氰钴胺素与回肠受体的结合。与微绒毛膜的结合呈现饱和动力学，解离常数为0.25 nM。结合迅速，在5分钟内完成70％；结合的二级速率常数为1.3×10⁶ M⁻¹ s⁻¹。内因子结合型氰钴胺素从回肠受体解离的半衰期约为35分钟。游离内因子抑制内因子结合型氰钴胺素的结合，但游离内因子的结合速率比与氰钴胺素结合的内因子慢。当内因子与氰钴胺素的各种类似物复合时，这些复合物对回肠微绒毛膜的亲和力与内因子结合型氰钴胺素相似。这些发现表明，维生素B12的分子构型不是内因子结合型维生素B12与其回肠受体位点之间相互作用的主要决定因素。
