Subcellular localization and partial purification of the 25-hydroxyvitamin D3 1-hydroxylation reaction in the chick myelomonocytic cell line HD-11.
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)
Shany
S
S
Department of Medicine, Cedars-Sinai Research Institute, University of California Los Angeles School of Medicine.
Ren
S Y
SY
Arbelle
J E
JE
Clemens
T L
TL
Adams
J S
JS
D000818:Animals; D001354:Avian Leukosis Virus; D002117:Calcitriol / Q000302:isolation & purification* / Q000378:metabolism; D002645:Chickens; D002846:Chromatography, Affinity; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry; D006900:Hydroxylation; D007684:Kidney Tubules / Q000166:cytology / Q000378:metabolism; D008264:Macrophages / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D003577:Cytochrome P-450 Enzyme System; D002117:Calcitriol