1,25-Dihydroxyvitamin D3 receptor from Japanese quail intestine; kinetic studies

The cytosolic receptor from Japanese quail intestine has been partially purified and selected kinetic properties of the receptor preparation have been characterized. The receptor--chromatin complex technique developed by Haussler and his associates for separation of the bound from free 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] was used with modifications. Examination of the conditions for incubation of the receptor--chromatin complex with tritiated 1,25-(OH)2D3 led to the selection of 25 degrees for 25 min at pH 7.4. Under these conditions the following kinetic characteristics prevailed. Specific binding was greater than 90% and the affinity of the receptor for 1,25-(OH)2D3 was 100 times greater than that for 25-(OH)D3 and 24,25-(OH)2D3 and 1.0 x 10(5) times greater than that for vitamin D3. Scatchard analysis revealed an equilibrium dissociation constant of 1.1 nM. Determination of rate constants yielded an association rate constant (ka) of 4.2 x 10(7) M-1 min-1 and a dissociation rate constant (kd) of 4.1 x 10(-2) min-1. The equilibrium dissociation constant, calculated from the ratio kd/ka, is 1.0 nM. When used as a radioreceptor assay the preparation has a sensitivity of 5 pg of 1,25-(OH)2D3 per tube and a working range of 5 to 80 pg per tube.