Glucocorticoid receptor from lactating goat mammary tissue comparison of native and activated forms in a cell free system.
Physicochemical properties of native and activated (DNA-binding) forms of the glucocorticoid receptor in cytosol prepared from lactating goat mammary tissue have been examined. Under hypotonic conditions the cytosolic receptor sediments at 8.4 S or 9.9 S in the absence or presence of 10 mM molybdate, respectively. The receptor in cytosol, either with or without molybdate elutes from DEAE-cellulose at approximately 200 mM potassium phosphate concentration. Isoelectric focusing reveals that this form of the receptor focuses at pH 5.5. Further, the cytosolic form of the receptor exhibits minimal binding affinity for polyanions such as DNA-cellulose. Its Stokes radius is 77 A and the mol. wt is approximately 331,000. Following exposure to in vitro activating conditions (including elevated ionic strength or temperature), the liganded receptor exhibits much lower affinity for DEAE-cellulose (elution at 35-55 mM potassium phosphate concentration). Other alterations in properties of the activated receptor, after partial purification, include sedimentation at 3.9 S in hypotonic sucrose gradients, binding to polyanions (DNA-cellulose), and an isoelectric point at pH 7.2. This receptor has a Stokes radius of 58 A and a mol wt of 98,000. A degraded form, with a mol. wt of approximately 57,000 and high affinity for polyanions, was the major form of the receptor obtained if appropriate precautions to prevent or remove proteolytic activity were not observed during purification and/or characterization of the activated receptor.
Hutchens
T W
TW
Department of Biochemistry, University of Southern California, School of Medicine, Los Angeles 90033.
Hawkins
E F
EF
Markland
F S
FS
D000818:Animals; D002474:Cell-Free System; D002499:Centrifugation, Density Gradient; D002853:Chromatography, Liquid; D003600:Cytosol / Q000032:analysis; D003907:Dexamethasone / Q000378:metabolism; D005260:Female; D006041:Goats; D006358:Hot Temperature; D007525:Isoelectric Focusing; D007774:Lactation / Q000378:metabolism; D008321:Mammary Glands, Animal / Q000032:analysis*; D008970:Molecular Weight; D011189:Potassium Chloride; D011247:Pregnancy; D011869:Radioligand Assay; D011965:Receptors, Glucocorticoid / Q000032:analysis* / Q000378:metabolism
D011965:Receptors, Glucocorticoid; D011189:Potassium Chloride; D003907:Dexamethasone