Purification of the receptor for 1 alpha,25-Dihydroxyvitamin D3 from chicken intestine

• Published in: Biochemistry (Easton) 1983-01, Vol.22 (10), p.2586-2594
• Main Authors: Simpson, R U, Hamstra, A, Kendrick, N C, DeLuca, H F
• Format: Article
• Language: English
• Subjects: Animals; Calcitriol - metabolism; Chickens; Duodenum - metabolism; Iodine Radioisotopes; Isomerism; Receptors, Calcitriol; Receptors, Steroid - isolation & purification; Space life sciences
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00279a041

Methods were investigated for use in the purification of the chicken intestinal receptor for 1 alpha,25-dihydroxyvitamin D3. The techniques investigated include column isoelectric focusing, gel exclusion, polyacrylamide gel electrophoresis, and DNA-cellulose, DEAE-cellulose, and hydroxylapatite chromatography. For the starting receptor preparation, a nuclear extract of chicken intestinal mucosa was found to be enriched above cytosol preparations and a plentiful source of receptor. A five-step purification scheme that resulted in the purification of the receptor protein by 5800-fold with 8% yield has been described. Analysis of the purified proteins on polyacrylamide gel electrophoresis containing sodium dodecyl sulfate suggests homogeneity. Analysis using two-dimensional polyacrylamide electrophoresis characterized the purified protein as having a molecular weight of approximately 63 000 and a pI of 6.0-6.2. Furthermore, assessment of protein purity by 125I iodination followed by sucrose gradient analysis revealed that approximately 90% of the iodinated macromolecules have the same sedimentation coefficient as the titrated 1 alpha,25-dihydroxyvitamin D3 receptor complex. The final purified receptor that bound tritiated 1 alpha,25-dihydroxyvitamin D3 retained affinity for DNA-cellulose and possesses a 3.7S sedimentation coefficient. The receptor has an estimated Stokes radius of 37 A.