Purification of the receptor for 1 alpha,25-dihydroxyvitamin D3 from chicken intestine.
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.
D000818:Animals; D002117:Calcitriol / Q000378:metabolism*; D002645:Chickens; D004386:Duodenum / Q000378:metabolism*; D007457:Iodine Radioisotopes; D007536:Isomerism; D018167:Receptors, Calcitriol; D011987:Receptors, Steroid / Q000302:isolation & purification*
D007457:Iodine Radioisotopes; D018167:Receptors, Calcitriol; D011987:Receptors, Steroid; C025169:1,25-dihydroxy-previtamin D(3); D002117:Calcitriol