Cytosol Preparations are Inadequate for Quantitating Unoccupied Receptors for 1,25-Dihydroxyvitamin D3

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Abstract
Recent studies in this laboratory have indicated that 90% of the unoccupied receptors for 1,25-dihydroxyvitamin D3 [1,25(OH)2-D3] are associated with nuclear components when chick intestinal mucosa is homogenized in low salt buffer (TED: 10 mM Tris, 1.5 mM EDTA, 1.0 mM dithiothreitol, pH 7.4). This observation suggested that previously reported cytosol 1,25(OH)2D3 receptors could result instead from salt extraction of nuclear receptors. The studies herein indicate that tissue 1,25(OH)2D3 receptor recovery is 30–50% lower in cytosol prepared from KTED (0.3 M KC1 + TED) or STKM (0.25 M sucrose, 50 mM Tris, 25 mM KC1, 5 mM MgCl2, pH 7.4) than in TED-prepared chromatin. Thus tissue concentrations of unoccupied 1,25(OH)2D3 receptors can be closely estimated in TED-chromatin; full quantitation can be achieved by summing the number of receptors in TED-chromatin plus TED-cytosol. Incubation at different temperatures for varying times yielded maximal receptor recovery (6.1 pmol/g mucosal wet weight) at 4°C for 4–24 h or at 23° for 30 min. Scatchard analyses confirmed that only a single class of high affinity (Kd 0.4 nM) binding sites was present under all incubation conditions. Dithiothreitol significantly improved receptor recovery both in cytosol and in chromatin preparations. Conversely, inclusion of 20% glycerol caused an artificial increase in specific H-1,25(OH)2D3 binding due to a second class of chromatin binding sites with ten-fold higher Kd (8.1 nM) and a greater number of binding sites than the 1,25(OF)2D3 receptor. In conclusion, the TED-chromatin assay procedure provides better quantitation of the tissue content of unoccupied 1,25(OH)2D3 receptors than do previously described techniques. The presence of unoccupied nuclear-associated 1,25(OH)2D3 receptors in other target tissues emphasizes the potential for erroneous physiological conclusions if these chromatin-associated receptors are overlooked.