In Vitro biosynthesis of adipocyte proteins having the characteristics of glucocorticoid receptors.
A quantitative method for the measurement of putative glucocorticoid receptor biosynthesis in rat adipocytes is described. The method utilizes the incorporation of radioactive amino acids into newly synthesized putative receptor proteins and their subsequent separation from other labeled proteins by affinity chromatography. Dexamethasone and deoxycorticosterone-Sepharose are used as affinity adsorbants. Specific binding of radioactive putative receptors to these gels is time- and protein concentration-dependent, and is abolished by exposure of cells to cycloheximide, pretreatment of adipocyte cytosol preparations with unlabeled steroids or incubation of cytosols at 37C for 4 h. Specifically bound radioactivity, which represents about 10% of the radioactivity initially associated with affinity adsorbants can be quantitatively eluted under rigidly defined conditions including high ionic strength. Specifically eluted material, which comprises up to 50% of total eluted radioactivity sediments at 3.8 S in sucrose gradients containing 1 M KCl, and electrophoretically migrates on 0.1% SDS gels in single band with a molecular weight of about 50 000. The sedimentation coefficient is comparable to that of the native adipocyte cytosol receptor not subject to affinity chromatography (3.7 S). Under low ionic-strength conditions most of the native receptor sediments at 8 S. The molecular weight of 50 000 is in the range of those reported for glucocorticoid receptors of liver (45 000---66 000 for monomers). The properties of the protein or proteins measured in the present system are therefore consistent with the current state of knowledge regarding glucocorticoid receptors in adipocytes.
D000273:Adipose Tissue / Q000166:cytology; D000818:Animals; D002499:Centrifugation, Density Gradient; D002846:Chromatography, Affinity / Q000379:methods; D003600:Cytosol / Q000378:metabolism; D003907:Dexamethasone / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D066298:In Vitro Techniques; D008297:Male; D051381:Rats; D011965:Receptors, Glucocorticoid / Q000096:biosynthesis* / Q000302:isolation & purification; D011987:Receptors, Steroid / Q000096:biosynthesis*
D011965:Receptors, Glucocorticoid; D011987:Receptors, Steroid; D003907:Dexamethasone