Purification of Vitamin D Binding Protein and Its Role in the Uptake of 25-Hydroxyvitamin D by Cultured Kidney Cells (Mdck Cells, Tissue Culture, Dbp)
Keenan, Michael James
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Permalink
https://hdl.handle.net/2142/77450
Description
Title
Purification of Vitamin D Binding Protein and Its Role in the Uptake of 25-Hydroxyvitamin D by Cultured Kidney Cells (Mdck Cells, Tissue Culture, Dbp)
Author(s)
Keenan, Michael James
Issue Date
1984
Department of Study
Food Science
Discipline
Food Science
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biology, General
Language
eng
Abstract
This thesis reports the isolation of vitamin D binding protein (DBP) and its use in studies investigating the uptake of 25(OH)D(,3) by kidney cell lines. DBP was isolated from pig plasma in three steps: chromatography on DEAE cellulose and DEAE Sephadex, followed by chromatofocusing. The procedure represents a shorter purification scheme than those previously reported. Antibodies to DBP were produced in rabbits and coupled to cyanogen bromide-activated Sepharose 4B. With this antibody affinity column, DBP was isolated fron pig plasma to a purity similar to that in the initial purification in a one-step procedure. Holo- and apo-DBP did not co-chromatograph upon chromatofocusing, which was used to separate the two forms. Holo-DBP was used as the source of 25(OH)D(,3) to study its uptake by kidney cells.
Two established kidney cell lines, MDCK (dog) and LLC-MK(,2) (monkey) were used in uptake studies. A comparison of the uptake of free 25(OH)D(,3) over time with the uptake of 25(OH)D(,3), bound to DBP, indicated that DBP was involved in the specific uptake of 25(OH)D(,3) by the MDCK cell line. The biphasic total uptake curve for 25(OH)D(,3) generated when MDCK cells were incubated with increasing amounts of holo-DBP appeared to consist of two components: one was nonspecific while the other was saturable and specific. In these studies with LLC-MK(,2) cells, a significant specific uptake was not readily identified. These cells may lack a specific uptake mechanism or it is possible that maintenance of these cells in a serum-free medium disturbed their metabolism or that receptors from monkey kidney cells do not interact with DBP isolated from pig plasma.
From the results reported here, the MDCK cell line would appear preferable to the LLC-MK(,2) line for use in studies examining the uptake of 25(OH)D(,3). The results with the MDCK line are compatible with the holo-DBP (with bound 25(OH)D(,3)) interacting with a cell surface receptor. Because the implications of the uptake of vitamin D and its metabolites occurring by a receptor-mediated mechanism are significant, further research to characterize the process is required.
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