Normal rabbit intestinal cytosol as a source of binding protein for the 1,25-dihydroxyvitamin D3 assay

Cytosol prepared from small intestine of vitamin D-sufficient rabbits contains a specific high-affinity binding protein for 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). This binding protein sediments at 3.0-3.5 S in sucrose density gradients containing 0.3 M KCl. Scatchard analysis using intestinal cytosol demonstrated a Kd of 0.05 nM and a maximum binding capacity of 92 fmol/mg cytosol protein for 1,25(OH)2D3 at 4 degrees C. Competitive binding studies with various metabolites of vitamin D showed a relative binding affinity of this protein for 1,25(OH)2D3 greater than 25-hydroxyvitamin D3 greater than vitamin D3. With 200 micrograms of rabbit intestinal cytosol protein, as little as 1.0-2.5 pg of 1,25(OH)2D3 reproducibly displaced the tracer sterol from the binding protein. Analyses of human plasma 1,25(OH)2D3 content yielded values consistent with published results. The vitamin D-replete rabbit provides a convenient, plentiful, and inexpensive source of binding protein for 1,25(OH)2D3 assays.