Binding of Vitamin B₁₂‐Rat Transcobalamin II and Free Vitamin B₁₂ to Plasma Membranes Isolated from Rat Liver

Abstract

When dialysed rat serum which contains a single, low molecular weight binder for vitamin B12, rat transcobalamin II (rat TC-II), was labelled in vitro with 57Co-vitamin B12 and then incubated at 30 degrees C (pH 7-5) with vesicles of highly purified plasma membranes separated from microsomal fractions of rat liver by density gradient centrifugation, the 57Co-vitamin B12-rat TC-II complex bound to high affinity sites on the vesicles via a specific (binding after correction for 'non-specific' binding in the presence of a large excess of the non-radioactive complex), saturable, and reversible interaction. The apparent affinity constant for the binding reaction was 5-5 X 10(9) M-1. Using the same incubation conditions, free vitamin B12 also bound to the vesicles of plasma membranes via a specific, saturable, but apparently irreversible interaction. Preincubation of the membranes with free vitamin B12 did not interfere with the subsequent binding of the vitamin B12-rat TC-II complex to the membranes; however, preincubation with the vitamin B12-rat TC-II complex did interfere, to some extent, with the subsequent binding of free vitamin B12. Dialysed rat serum, perhaps the free rat TC-II in the dialysed serum, also inhibited the binding of the vitamin B12-rat TC-II complex to the plasma membranes. The relationship of the binding sites identified in this report to the absorption of vitamin B12 by rat liver, and thus their physiological significance remains unknown until further work is done, perhaps using intact hepatocytes.

A saturable high affinity binding site for transcobalamin II-vitamin B12 complexes in human placental membrane preparations.

“…The reason for the greater binding of free B12 by the rat liver membrane preparation is not apparent. As stated by Fiedler-Nagy et al (15), since virtually all (99%) of the B12, in the blood of rats is bouind to TCII (28), rat liver cells contact little free B12 and, while there may well be a second mechanism for the uptake of free B12 it is quantitatively unimportant. That such a mechanism also exists in humans is indicated by the fact that patients with congenital deficiency of TCII can be successfully treated with large doses of free B12 (10).…”

“…That it is a close approximation is stuggested by the fact that a less detailed binding cturve, obtained with the TCII eltuted from Sepharose-B12 (and thtus, free of endogenous TCII-B12), gave a K, of about 7-10 nM-'. An approximate K = 6 nM-' for the binding of IF-B12 to the receptor in human intestinal mtucosa (3) and one of 5.5 nM-1 for the binding of rat TCII-B12 to rat liver plasma membranes have been reported (15).…”

“…A number of studies have demonstrated that TCII promotes the uptake of B12 by human cells in vitro (11)(12)(13)(14). Recently, specific saturable binding sites for rat TCII-B12 complexes on rat liver plasma membranes have been described (15).…”

Binding and Uptake of Transcobalamin II by Human Fibroblasts

“…The data pre-degradation markedly. Finally, preliminary experisented by Fiedler-Nagy et al (6) also suggest that rat ments from our laboratory show that chloroquine also liver membrane preparations have a high affinity bind-inhibits the release of complex.4 All of these results support the contention that TC II is degraded to free amino acids within lysosomes, that these amino acids then leave the lysosome (and appear as 125I-labeled amino acids in experiments like ours) and that such degradation is a necessary precondition for release of Cbl from the lysosome.…”

“…Cellular uptake studies have suggested that the TC II-Cbl binds to the cell surface before the internalization of the Cbl (4,5). A specific receptor for the TC II-Cbl complex in rat liver and human placental membranes has been identified and is assumed to mediate this TC IICbl binding (6,7). Based on sub-cellular fractionation studies after intravenous injection of Cbl in rats, Newmark et al (8) and Pletsch and Coffey (9) proposed that the TC II-Cbl complex then enters the cell via endocytosis and is transiently localized in secondary lysosomes where the TC II'is degraded and the Cbl is released into the cytoplasm.…”