Hooper D C, Alpers D H, Burger R L, Mehlman C S, Allen R H
J Clin Invest. 1973 Dec;52(12):3074-83. doi: 10.1172/JCI107506.
Elucidation of the mechanism of intrinsic factor (IF)-mediated vitamin B(12) (B(12)) binding to ileal binding sites has been hampered by the use of crude or only partially purified preparations of IF in previous studies. We have used homogeneous human IF and hog IF isolated by affinity chromatography to study [(57)Co]B(12) binding to ileal mucosal homogenates. The following observations were made: (a) Human IF-B(12) and hog IF-B(12) were bound to human, monkey, hog, dog, rabbit, mouse, hamster, and guinea pig ileal, but not jejunal, homogenates in amounts significantly greater than free B(12) or B(12) bound to five other homogeneous B(12)-binding proteins; (b) only IF-mediated B(12) binding was localized to ileal homogenates and was inhibited by EDTA; (c) values for the association constant (K(a)) for the various ileal homogenates mentioned above and human IF-B(12) and hog IF-B(12) ranged from 0.3 x 10(9) M(-1) to 13.0 x 10(9) M(-1). Apparent differences in the K(a) for human IF-B(12) and hog IF-B(12) existed in most species; (d) the number of ileal IF-B(12) binding sites per gram (wet weight) of ileal mucosa ranged from 0.3 x 10(12) to 4.9 x 10(12). The same value was always obtained with human IF-B(12) and hog IF-B(12) for any given homogenate preparation; (c) 100-fold excesses of free B(12) or human IF and hog IF devoid of B(12) did not significantly inhibit human IF-B(12) and hog IF-B(12) binding to human and hog ileal homogenates. THESE EXPERIMENTS PERFORMED WITH HOMOGENEOUS IF INDICATE THAT: (a) gastric factors other than IF are not required for B(12) binding to ileal IF-B(12)-binding sites: (b) the mechanism of ileal IF-B(12) binding is different from that of free B(12) or of B(12) bound to non-IF-B(12)-binding proteins; (c) human IF and hog IF have different structures; (d) human IF-B(12) and hog IF-B(12) bind to the same ileal binding sites; and (c) human and hog ileal IF-B(12) binding sites bind free B(12) and human and hog IF devoid of B(12) poorly, if at all.
以往研究中使用粗制或仅部分纯化的内因子（IF）制剂，阻碍了对IF介导的维生素B12（B12）与回肠结合位点结合机制的阐明。我们使用通过亲和层析分离得到的纯合人IF和猪IF，研究[57Co]B12与回肠黏膜匀浆的结合。得到以下观察结果：（a）人IF-B12和猪IF-B12与人、猴、猪、狗、兔、小鼠、仓鼠和豚鼠的回肠匀浆结合，但不与空肠匀浆结合，结合量显著高于游离B12或与其他五种纯合B12结合蛋白结合的B12；（b）只有IF介导的B12结合定位于回肠匀浆，且受EDTA抑制；（c）上述各种回肠匀浆与人IF-B12和猪IF-B12的缔合常数（Ka）值范围为0.3×109 M-1至13.0×109 M-1。大多数物种中，人IF-B12和猪IF-B12的Ka存在明显差异；（d）每克（湿重）回肠黏膜的回肠IF-B12结合位点数范围为0.3×1012至4.9×1012。对于任何给定的匀浆制剂，用人IF-B12和猪IF-B12总是得到相同的值；（e）100倍过量的游离B12或不含B12的人IF和猪IF，对人IF-B12和猪IF-B12与人及猪回肠匀浆的结合没有显著抑制作用。用纯合IF进行的这些实验表明：（a）B12与回肠IF-B12结合位点结合不需要IF以外的胃因子；（b）回肠IF-B12结合机制不同于游离B12或与非IF-B12结合蛋白结合的B12的结合机制；（c）人IF和猪IF结构不同；（d）人IF-B12和猪IF-B12与相同的回肠结合位点结合；（e）人和猪回肠IF-B12结合位点与游离B12以及不含B12的人IF和猪IF结合很差，甚至根本不结合。
