Activation of glucocorticoid-type II receptor complexes in brain cytosol leads to an increase in surface hydrophobicity as determined by hydrophobic interaction chromatography.
Hydrophobic interaction chromatography has been used to demonstrate an increase in the surface hydrophobicity of [3H]triamcinolone acetonide ([3H]TA)-labeled type II receptors in mouse brain cytosol following transformation of these receptor complexes to the activated DNA-binding form. After removing unbound [3H]TA and molybdate (which prevents activation) by gel filtration, [3H]TA-type II receptors were activated by incubation at 22 degrees C for 20 min. Gel filtration was then used to remove newly dissociated steroid and to readjust the molybdate and/or KCl concentration. Unactivated and activated receptors were then added to propyl, butyl, pentyl, hexyl, octyl, decyl, and dodecyl alkyl agarose, phenyl agarose, or unmodified agarose columns equilibrated and eluted with buffers of various molybdate and KCl concentrations and/or other additions, including glycerol, ethylene glycol, and urea. Under high-salt conditions, activated receptors were retained longer than unactivated receptors run on butyl, pentyl, hexyl, and phenyl agaroses. With the longer alkyl chain columns, essentially none of the [3H]TA was eluted in association with receptor macromolecules. Removal of the remaining steroid required receptor denaturation with urea. Under low-salt conditions, both receptor forms were retained more avidly on all alkyl agarose columns; however, on phenyl agarose only activated receptors displayed this increased retention. Further studies revealed that optimal separation and subsequent recovery of unactivated and activated [3H]TA-type II receptor complexes were achieved on pentyl agarose columns equilibrated and eluted with buffers containing 50 mM molybdate and 600-1,200 mM KCl.(ABSTRACT TRUNCATED AT 250 WORDS)
Densmore
C L
CL
Department of Neuroscience, University of Florida, College of Medicine, Gainesville 32610.
Chou
Y C
YC
Luttge
W G
WG
D000818:Animals; D001921:Brain / Q000378:metabolism*; D002021:Buffers; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002845:Chromatography; D002850:Chromatography, Gel; D003600:Cytosol / Q000378:metabolism; D005260:Female; D051379:Mice; D008982:Molybdenum; D009994:Osmolar Concentration; D011189:Potassium Chloride; D011965:Receptors, Glucocorticoid / Q000302:isolation & purification / Q000378:metabolism*; D012685:Sepharose; D014222:Triamcinolone Acetonide / Q000378:metabolism
D002021:Buffers; D011965:Receptors, Glucocorticoid; C044659:molybdate; D011189:Potassium Chloride; D008982:Molybdenum; D012685:Sepharose; D014222:Triamcinolone Acetonide