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 10⁶ 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. These data suggest that HD‐11 cell 25‐OHD₃ 1‐hydroxylation reaction may be similar to the well‐characterized 25‐OHD₃ 1‐hydroxylase expressed in the kidney.