The role and fate of rabbit and human transcobalamin II in the plasma transport of vitamin B12 in the rabbit.

Abstract

A B S T R A C T Previous studies have shown that plasma transcobalamin II (TCII) facilitates the cellular uptake of [57Co]vitamin B13 (Be) by a variety of tissues, but the lack of an intrinsic label on the protein moiety of the TCII-Bn complex has made it impossible to determine the role and fate of TCII during this process. We have labeled homogeneous rabbit and human TCII with 'I-labeled N-succinimidyl-3-(4-hydroxyphenyl) propionate and have performed in vivo experiments in rabbits.When 'M-labeled rabbit TCII-['"Co] B1s and 'I-labeled bovine albumin were simultaneously injected intravenously, we observed that 'I and 'Co were cleared from plasma at a faster rate (ti = 11 h) than 'I and that 12J and 57Co were present in excess of 'I in the kidney, liver, spleen, heart, lung, and small intestine j h after injection. Later, "7Co remained in excess of PI, but the ratio of 'I to 'I decreased progressively in all of these tissues; 'I fragments (mol wt less than 1,000) appeared in the plasma and were rapidly excreted in the urine. After 1 h following injection, 'Co was present in excess of 'I in the plasma. Additional experiments revealed that both isotopic moieties of human TCII-Bn were cleared from rabbit plasma 30% faster than their rabbit TCII-Bn3 counterparts and that apo-rabbit TCII and apo-human TCII were cleared from rabbit plasma 30% faster than holo-rabbit TCII and holo-human TCII, respectively.These studies, and appropriate control experiments, indicate that TCII and the TCII-Bu2 complex are cleared

Binding and Uptake of Transcobalamin II by Human Fibroblasts

“…Human TC II was purified from normal human plasma and labeled with 1251 as described (10,16 tuCi/nmol).…”

“…The unlabeled, purified human and rabbit TC 11 (10), and granulocyte R-binder (17) were stored at -25°C in 0.75 M NaCl containing 0.05 M potassium-phosphate, pH 7.5.…”

“…Subsequently, Cbl is converted to its coenzyme forms, adenosylcobalamin and methylcobalamin. Because of a lack of a label on the protein moiety of the TC II-Cbl complex, the evidence for internalization and degradation of the TC II has been indirect, although recent in vivo studies using [57Co]Cbl bound to 125I-labeled TC II (10) have shown that there is initially a coordinate clearance of both labels from rabbit plasma by a variety of tissues and that the TC II is rapidly degraded in this process.…”

RIA for Serum Holo-Transcobalamin: Method Evaluation in the Clinical Laboratory and Reference Interval

“…However, the physiologic cycle of holoTC is complex and may be affected by several pathologic mechanisms. HoloTC in plasma is in a dynamic state, the half-life being only ϳ60 min (7,8 ). The holoTC concentration may be reduced by impaired generation of holoTC either because of Cbl malabsorption or because of depletion of Cbl reserves in general Cbl deficiency.…”

Effect of Proteolytic Enzymes on the Binding of Cobalamin to R Protein and Intrinsic Factor

“…equilibrate at 4°C with 0.01 M Tris-HCl, pH 8.0, containing 0.15 M NaCl and 50 ug/ml of bovine serum albumin (Sigma Chemical Co.). 5-ml fractions were collected and assayed for total Cbl-binding ability (18) and Cbl-binding ability due to IF (18 (16) and labeled with 1251 and "3Il by the method of Bolton and Hunter (19) as previously described (20). Rabbit anti-human IF (21) and anti-human R protein (16) sera were obtained as previously described.…”