Biosynthesis, purification and receptor binding properties of high specific radioactivity 1α,24(R),25-trihydroxy-[26,27-methyl-3H]-vitamin D3

John S. Chandler, J. Wesley PikO, Mark R Haussler

Research output: Contribution to journalArticle

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Abstract

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.

Original languageEnglish (US)
Pages (from-to)303-310
Number of pages8
JournalJournal of Steroid Biochemistry
Volume16
Issue number2
DOIs
StatePublished - 1982

Fingerprint

Cholecalciferol
Biosynthesis
Radioactivity
High performance liquid chromatography
Metabolites
Purification
High Pressure Liquid Chromatography
Hormones
Kidney
Hydroxylation
Sterols
Nutrition
Vitamins
Rats
Binding Sites
Availability
Diet
Calcium
Lipids
Substrates

ASJC Scopus subject areas

  • Biochemistry
  • Endocrinology

Cite this

Biosynthesis, purification and receptor binding properties of high specific radioactivity 1α,24(R),25-trihydroxy-[26,27-methyl-3H]-vitamin D3. / Chandler, John S.; PikO, J. Wesley; Haussler, Mark R.

In: Journal of Steroid Biochemistry, Vol. 16, No. 2, 1982, p. 303-310.

Research output: Contribution to journalArticle

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abstract = "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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