A procedure for the biosynthesis and purification of 1α,24(R),25-trihydroxy[26,27-methyl-3H]-vitamin D3 (1,24,25-(OH)3[3H]D3) is reported. A kidney homogenate from chicks receiving a high calcium diet (3%) and oral supplements of 1α,25-dihydroxy vitamin d3 (1,25-(OH)2D3) was used for C-24-hydroxylation of 1α,25-dihydroxy[26,27-methyl-3H]-vitamin F3 (1,25-(OH)2[3H]D3), in vitro. Extraction and purification of the homogenate lipid fraction by Sephadex LH-20 and high performance liquid chromatography yielded radiochemically pure 1,24,25-(OH)3[3H]D3 with a specific radioactivity equivalent to the initial substrate (166Ci/mmol). The authenticity of the generated metabolite was assessed by comigration with synthetic 1,24,25-(OH)3D3 on high performance liquid chromatography and by equimolar competition with authentic radioinert 1,24,25-(OH)3D3 for binding to a purified receptor protein from rat kidney. Binding studies indicate the trihydroxylated metabolite competes 40-50% as effectively as 1,25-(OH)2D3 for hormone binding sites. Further analysis of l,24,25-(OH)3D3-receptor interaction reveals a high-affinity, saturable binding with an apparent Kd of 2.2 × 10-9 M. These studies demonstrate that although slightly less active than 1,25-(OH)2D3, 1,24,25-(OH)3D3 is capable of hormone-like interactions, in vitro. The availability of this high specific radioactivity sterol should allow for clarification of its potential physiologic significance.
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