Application of the rat thymus receptor in a specific, sensitive and simplified assay of 1,25-dihydroxyvitamin D in blood serum.
A simple method has been employed to prepare crude nuclear extract from rat thymus, using hypertonic buffer after previous treatment with hypotonic buffer. The preparation is free from serum vitamin D-binding protein and contains a 3.7 S receptor molecule, which specifically binds 1,25-dihydroxyvitamin D-3 (1,25-(OH)2D3). The receptor is of high affinity (KD = 0.85 X 10(-11) M at O degrees C) and low capacity (260-460 fmol/g tissue). The Scatchard analysis of ligand binding results in a concave downward curve. The Hill analysis of the same data gives good linear fitting (r = +0.971) with the Hill coefficient nH = 1.63. These facts indicate positive cooperativity between two ligand binding sites of the rat thymus 1,25-(OH)2D3 receptor. The preparation was used in a competitive protein binding assay of 1,25-(OH)2D in serum extracts, purified on Sep-Pak C18 followed by silica Sep-Pak cartridges. The method was sensitive to 0.5 pg/tube (2.0 ng/l) when 1 ml of serum was extracted. Intra- and interassay coefficients of variation were 9% and 14%, respectively. The serum 1,25-(OH)2D concentration estimated in 33 children (mean age 6.5 +/- 3 years) was 46.6 +/- 18.4 ng/l (mean +/- S.D.).
Gradzka
I
I
Department of Biochemistry and Experimental Medicine, Child's Health Center, Warsaw, Poland.
Lukaszkiewicz
J
J
Lorenc
R
R
D000818:Animals; D002117:Calcitriol / Q000097:blood*; D002467:Cell Nucleus / Q000032:analysis; D002648:Child; D002675:Child, Preschool; D004247:DNA / Q000378:metabolism; D005260:Female; D006801:Humans; D007700:Kinetics; D008297:Male; D009994:Osmolar Concentration; D011869:Radioligand Assay; D051381:Rats; D011919:Rats, Inbred Strains; D018167:Receptors, Calcitriol; D011987:Receptors, Steroid / Q000302:isolation & purification* / Q000378:metabolism; D012016:Reference Values; D013696:Temperature; D013950:Thymus Gland / Q000032:analysis*
D018167:Receptors, Calcitriol; D011987:Receptors, Steroid; D004247:DNA; D002117:Calcitriol