1 Department of Medical Biochemistry, Faculty of Health Sciences, Aarhus University, Aarhus University2 Laboratorium for Proteinkemi, Faculty of Science, Aarhus University, Aarhus University3 Department of Chemistry, University of Utah, Utah.4 Department of Molecular Biology, University of Aarhus, Denmark5 Clinical Biochemistry, Aarhus University Hospital, Denmark6 Department of Medical Biochemistry, University of Aarhus, Denmark
The transport of vitamin B12 (B12)/cobalamin in the tissue-fluids is facilitated by three different binding-proteins: Intrinsic factor (IF), transcobalamin (TC) and haptocorrin. Especially the first two are important for the cellular uptake of B12. Intrinsic factor is produced in the ventricle and is essential for the B12 uptake in the distal part of ileum by the receptor complex cubilin/amionless. TC is important for the uptake of B12 from plasma. In the kidney, megalin is the receptor for the TC- B12 complex, whereas uptake of TC- B12 in the extrarenal tissue occurs by means of a still unknown receptor structure. This receptor is suggested to be regulated by the vitamin B12 level in the cells, which is interesting in relation to cancer growth. The cellular endocytosis of TC- B12 complex by this unknown receptor is being investigated, using confocal microscopy. Fluorescently labeled B12 molecules (Oregon green linked to B12) have been synthesized to determine the B12 uptake level in normal and various tumour-derived cells (e.g. Hela cells from cervix epithelioid carcinoma and BN- cells from rat yolk sac sarcoma). Costaining of the B12 binders has been performed using fluorescently labelled secondary antibodies recognising primary antibodies against IF and TC. The data show a cell growth-regulated uptake of free fluorescent B12 but a strong inducement of uptake by TC and IF. After uptake the B12 fluorochrome colocalizes with the B12 binders.