Biosynthesis, purification and receptor binding properties of high specific radioactivity 1 alpha, 24(R),25-trihydroxy-[26,27-methyl-3H]-vitamin D3.
A procedure for the biosynthesis and purification of 1 alpha, 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 1 alpha, 25-dihydroxyvitamin D3 (1,25-(OH)2D3) was used for C-24-hydroxylation of 1 alpha, 25-dihydroxy[26,27-methyl-3H]-vitamin D3 (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 (166 Ci/mmol). The authenticity of the generated metabolite was assessed by co-migration 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 1,24,25-(OH)3D3-receptor interaction reveals a high-affinity, saturable binding with an apparent K4 of 2.2 x 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.
D000818:Animals; D002117:Calcitriol / Q000494:pharmacology; D002645:Chickens; D003577:Cytochrome P-450 Enzyme System; D004790:Enzyme Induction / Q000187:drug effects; D006887:Hydroxycholecalciferols / Q000096:biosynthesis* / Q000302:isolation & purification; D066298:In Vitro Techniques; D007668:Kidney / Q000378:metabolism; D007700:Kinetics; D008297:Male; D051381:Rats; D018167:Receptors, Calcitriol; D011987:Receptors, Steroid / Q000378:metabolism*; D013250:Steroid Hydroxylases / Q000096:biosynthesis; D013997:Time Factors; D065668:Vitamin D3 24-Hydroxylase
D006887:Hydroxycholecalciferols; D018167:Receptors, Calcitriol; D011987:Receptors, Steroid; C013721:1,24,25-trihydroxyvitamin D3; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D065668:Vitamin D3 24-Hydroxylase; D002117:Calcitriol