Intrinsic Factor-mediated Attachment of Vitamin B12 to Brush Borders and Microvillous Membranes of Hamster Intestine*

Abstract

Summary. Hamster intrinsic factor (IF) preparations markedly enhanced the uptake of 57cobalt-labeled cyanocobalamin (B_2-57Co) by brush borders and microvillous membranes isolated from villous absorptive cells obtained from the distal but not the proximal half of hamster intestine. A similar effect was observed with rat and rabbit IF preparations, but IF preparations obtained from man, dog, and hog were ineffective. After -fractionation of hamster IF preparations by gel filtration or ion exchange chromatography, the extent to which each fraction enhanced B_2-57Co uptake by brush borders correlated closely with the vitamin B12 binding capacity of the fraction. IFmediated attachment of B12-57Co to brush borders occurred rapidly, was not diminished by removal of glucose or oxygen from the incubation medium, and was not significantly altered when incubation temperatures were reduced from 370 C to 70 C. Marked reduction in uptake occurred, however, in the absence of divalent cations.IF enhanced B12-57Co uptake by brush borders isolated from the proximal half of the intestine when these proximal brush borders were preincubated with supernatant fluid obtained after centrifugation of homogenates of distal intestinal mucosa at 28,500 g. The factor in this supernate responsible for the effect on proximal brush borders was shown to be particulate in nature upon centrifugation at speeds of 54,500 g or greater. The resultant pellet contained ribosomes and membranous fragments.Prolonged incubation of brush borders with crude saline extracts of hamster gastric mucosa resulted in decreased uptake of B_2-57Co and marked lysis of brush borders with concomitant release of tissue nitrogen. Neither lysis of brush borders nor decreased uptake of B12-57Co with prolonged incubation was observed when hamster IF was partially purified. Furthermore, uptake of 12-57Co by brush borders increased with increasing purity of the IF preparation used.These results demonstrate IF-mediated attachment of B,2-57Co to brush borders and microvillous membranes of hamster intestinal cells and provide further support for the presence of a specific receptor for IF-bound vitamin

Identification of a macromolecular factor in the ileum which binds intrinsic factor and immunologic identification of intrinsic factor in ileal extracts

“…Such a localization of this binding factor is consistent with the observations of Donaldson, Mackenzie, and Trier (28) that human gastric juice enhanced the uptake of B12 to epithelial cell brush borders and microvillous membranes of the distal small intestine of the hamster. Mackenzie, Kopp, Donaldson, and Trier (29) recently reported that antiserum produced against isolated brush border preparations inhibited the uptake of vitamin B1,.…”

Characterization of Ileal Vitamin B12 Binding Using Homogeneous Human and Hog Intrinsic Factors

“…The mechanism of IF-mediated B12 absorption is not well understood, but a number of studies have demonstrated that IF facilitates B12 binding to ileal sacs (1)(2)(3)(4)(5)(6), mucosal homogenates (4,(7)(8)(9)(10), microvillus membrane preparations (11,12), and to a component solubilized from ileal mucosa with Triton X-100 (13). IF-facilitated B12 binding of this type is dependent on pH, is inhibited by EDTA, and is postulated to represent the first step in the complex process by which B12 passes from the intestinal lumen into the portal blood.…”

“…IF-facilitated B12 binding of this type is dependent on pH, is inhibited by EDTA, and is postulated to represent the first step in the complex process by which B12 passes from the intestinal lumen into the portal blood. (2,7,9,11,14,15) or not (3,6,8,16) (17). Binding studies using crude gastric preparations are difficult to interpret because of the presence of B12 binding proteins that lack in vivo IF activity as well as the possible presence of additional gastric inhibitors or stimulators.…”

Attachment of the B12-Binding Principles Obtained from Lactobacillus leichmannii to Intestinal Mucosa Homogenates

“…Non-IF B12-binders obtained from various animal tissues did not augment the B12 uptake by guinea pig IMH (25,44), isolated brush borders (33) and seg ments (52) of hamster small intestine.…”

“…The IMH took up B12 in the absence of either Ca2+ or Mg2+ almost equally well as in the complete system ( Table 2). The deletion of Ca2+ resulted in a reduction of uptake aug mented by IF, indicating that Ca2+ plays an important role in the uptake of B12 mediated by IF (3,(31)(32)(33)(34)(35)(36)(37)(38). In contrast to IF, the aug mentation by the B12-binding principle was not affected by the deletion of divalent cations.…”