Document Detail

Subcellular localization and partial purification of the 25-hydroxyvitamin D3 1-hydroxylation reaction in the chick myelomonocytic cell line HD-11.
MedLine Citation:
PMID:  8384398     Owner:  NLM     Status:  MEDLINE    
Hypercalcemia in human granuloma-forming diseases like sarcoidosis results from the endogenous overproduction of 1,25-dihydroxyvitamin D [1,25-(OH)2D] by disease-activated tissue macrophages. The recent identification of an immortalized chick myelomonocytic cell line, HD-11, that constitutively expresses a 25-hydroxyvitamin D (25-OHD) 1-hydroxylation reaction has alleviated dependence on studying primary macrophage cultures with no replicative potential in vitro. In these experiments we established conditions for the maximal expression of the HD-11 cell 25-OHD3-1-hydroxylation reaction and localized this activity to the mitochondrial fraction. On a per cell basis, the activity of HD-11 cell 25-OHD3 1-hydroxylation reaction was comparable to that in primary cultures of chick renal tubular epithelial cells, which express the authentic renal 25-OHD3 1-hydroxylase. Maximal product yield was achieved after incubation of HD-11 cells with 200 nM 25-OHD3 for 3 h. Although adherent monolayers possessed 3- to 4-fold more capacity for hormone production than cells in suspension, suspended cells exhibited easily detectable 25-OHD3 catalytic activity (0.58 +/- 0.08 pmol per 10(6) cells per h; +/- SEM), 50% of which remained solubilized in a sonicate of suspended cells cleared of nuclei and plasma membrane. Subcellular localization disclosed 91% of the residual activity to be concentrated in the mitochondrial subfraction. A detergent-solubilized extract of this mitochondrial subfraction contained 1.9 +/- 0.3 pmol 1,25-(OH)2D3 synthetic capacity per mg protein. The catalytic activity (1-hydroxylase activity) was concentrated 20.2-fold after chromatography on octyl-amino agarose and was associated with 0.054 nmol cytochrome P450 per mg protein.(ABSTRACT TRUNCATED AT 250 WORDS)
S Shany; S Y Ren; J E Arbelle; T L Clemens; J S Adams
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Publication Detail:
Type:  Journal Article; Research Support, U.S. Gov't, P.H.S.    
Journal Detail:
Title:  Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research     Volume:  8     ISSN:  0884-0431     ISO Abbreviation:  J. Bone Miner. Res.     Publication Date:  1993 Mar 
Date Detail:
Created Date:  1993-04-21     Completed Date:  1993-04-21     Revised Date:  2007-11-14    
Medline Journal Info:
Nlm Unique ID:  8610640     Medline TA:  J Bone Miner Res     Country:  UNITED STATES    
Other Details:
Languages:  eng     Pagination:  269-76     Citation Subset:  IM    
Department of Medicine, Cedars-Sinai Research Institute, University of California Los Angeles School of Medicine.
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MeSH Terms
Avian leukosis virus
Calcitriol / isolation & purification*,  metabolism
Chromatography, Affinity
Cytochrome P-450 Enzyme System / chemistry
Kidney Tubules / cytology,  metabolism
Macrophages / metabolism*
Tumor Cells, Cultured
Grant Support
Reg. No./Substance:
32222-06-3/Calcitriol; 9035-51-2/Cytochrome P-450 Enzyme System

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine

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