No abstract

No abstract

Approximately equal cellular, receptor or tissue uptake of either holo TC I1 (TC I1 which is carrying Cbl) or apo TC I1 (the carrier protein only, free of Cbl) has been reported (1, 2). This seemed to be an unlikely behavior for TC I1 since its function is to deliver Cbl. If one accepts that about 90% of serum TC I1 is apo TC I1 (3), and some would put the figure even higher, then equal competition between apo and holo TC I1 would create a 9:l disadvantage for an essential transport function. Looking at analogous transport systems, the promotion of iron uptake by transferrin for example, the holo form is much better taken up than the apo (4).The models of the uptake process for the present study were HeLa cells and cultured lymphocytes. The HeLa cell is an established model for testing the function of TC I1 and although the peripheral lymphocyte is known to be responsive to TC 11-Cbl when stimulated ( 5 ) , the use of a cultured, established human cell line constitutes a new model.The cellular uptake of Cbl can be divided into several phases and through preliminary work we defined the events encompassed by the conditions for 3 hr uptake at 37" used in the main part of the study. (a) Uptake of TC 11-Cbl reaches a plateau and TC 11-Cbl is at a maximum within the cell. (b) The incorporated Cbl has the potentiality for conversion to coenzymes but the process is just beginning. (c) No free Cbl has been released by the HeLa cell and only small amounts b.ave begun to leave the lymphocyte.This work has been published in abstract form (6).Materials and methods. Materials. The lymphocyte line, RPMI 6410, and the culture media, RPMI 1640, were purchased from Associated Biomedic Systems Inc. Other materials for cell culture and the HeLa cell were ~_ _ _ _ _ _ ~~ This work was supported by the Medical Research Service of the Veterans Administration.as described (7). The semipurified TC I1 was prepared from human Cohn fraction I11 (generously supplied by Dr. J. Fenton of the Division of Laboratories, New York State Dept. of Health) as before (7). The CN ["'Co] Cbl (7) was reduced to a specific activity of about 45 mCi/mg.Cultures. The lymphocytes were maintained in 75 ml Falcon Culture flasks in RPMI 1640 medium pH 7.0 containing 20% fetal calf serum, 100 units/ml of penicillin and 50 pg/ml of streptomycin. The cultures were adjusted to 3.0-5.0 x 10" viable cells after feeding and allowed to grow to 1.0-1.3x 10" viable cells/ml. The viability of both cells was determined by the Trypan blue exclusion met hod.Preparation of the holo and apo T C II. The starting material was always from a single lot of Cohn fraction 111. Workable batches were made by batch absorption using CM-Sephadex (7). Each batch was checked for Cbl content and for unsaturated binding capacity for Cbl (3, 8) giving the total amount of TC I1 as well as the apo and holo fractions. Aliquots of a size suitable for a day's work were frozen and thawed as needed. Both holo and apo TC I1 were prepared from the same batches. To obtain a preparation of only holo TC 11,...

No abstract

No abstract