It has been established that three binders of vitamin B,, can be separated by DEAEcellulose column chromatography following addition of labelled vitamin to normal serum (1-4). All 3 binders are present in trace amounts. They are known as transcobalamin I (TC I ) , transcobalamin I1 (TC 11), and transcobalamin I11 (TC 111) or "main protein peak binder" (MPPB) . TC I1 and TC I11 have an electrophoretic mobility of a-globulin, while TC I has the mobility of ,&globulin (1, 2).Labelled vitamin B,, is cleared rapidly from intravenously administered TC I1 5 7 C~ B,, but much more slowly from TC I 5 7 C~ B,, (2,5,6). The differences in clearance are less marked, however, when the transcobalamins are labelled with orally administered labelled vitamin BI2. The rapid clearance of 57C0 B12 from intravenously administered B,,--TC I1 has been attributed to partial denaturation of the protein during its purification in vitro (7). Finkler and Hall (8) reported that HeLa cells take up TO B,, from TC I1 in vitro but not from TC I while Retief et al. (9) observed that TC I1 delivers vitamin B,z to reticulocytes at a faster rate than TC I. Uptake by perfused rat liver of labelled vitamin B,, from 5 7 C~ B,, labelled TC I1 and TC I11 from serum of normal donors and pernicious anemia patients was substantially greater than from 5 7 C~ B,, labelled TC I (10). The exact role of TC 111 vita-1In receipt of a grant from the Leukemia Research Fund.747 min B,, in plasma is still uncertain. Chanarin and co-workers have recently shown that following oral administration of 5 7 C~ BIZ, the labelled vitamin is taken up simultaneously by all 3 serum binders (7, 11). These authors also showed immunological identity of TC I and TC 111. However, the ability of TC I11 to deliver vitamin B,, to cells is still unknown.H o a r a n d et al. (12) used phytohemagglutinin (PHA) -transformed lymphocytes as a model cell system to investigate uptake of serum bound radioactive vitamin B,, by normal proliferating human hemopoietic cells. In the present study we have used this system to compare the ability of human TC I11 with that of TC I and TC I1 to deliver labelled vitamin B,, to human cells. Materials and Methods. Preparation of vitamin B,, binders.This has been reported in detail elsewhere (13). Thirty ml of normal serum were used in each column chromatographic separation, Serum used in each experiment was drawn from an individual donor considered to be hematologically normal. Five hundred pg of 5 7 C~ B,, (specific activity 120 mCi/mg; purchased from the Radiochemical Centre, Amersham, Amersham, England) were added per ml to 30 ml of serum. The solution was allowed to stand at 37" for 20 min, then dialysed at 4" against 4 litres of 0.0175 M sodium phosphate buffer, pH 6.3, for 48 hr. The buffer was changed twice during this period.DEAE-cellulose ( Schleicher and Schuell, No. 70, of ion exchange capacity between 0.90 and 0.95 mEq/g) was packed after
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