Biochemical characterization of the protein affinity labeled by dihydrotestosterone 17 beta-bromoacetate: comparison with the human androgen receptor.
Dihydrotestosterone 17 beta-bromoacetate covalently binds to a single protein of 58,000 mol wt from human genital skin fibroblast cytosol. Previous experiments have suggested that this protein is related to the human androgen receptor and may be a proteolytic fragment of the intact protein. In the present study the biochemical properties of the covalently radiolabeled protein were compared to those of the classically defined human androgen receptor radiolabeled noncovalently with [3H]dihydrotestosterone. The radiolabeled proteins were indistinguishable by gel filtration chromatography, sucrose density gradient centrifugation analysis, chromatofocusing, and hydrophobic interaction chromatography. Both ligands labeled a protein with an apparent Stokes radius of 4.4 nm under high salt conditions. Analysis on sucrose density gradients showed peaks of 4.6S and 9.2S with either ligand. The protein radiolabeled with either ligand chromatofocused as two isoforms, a predominant form with a pI of about 5.4 and a minor isoform with a pI of about 4.5. Both radiolabeled proteins were found to have a high degree of hydrophobicity and eluted identically from a phenyl-Sepharose column. While the radiolabeled proteins were qualitatively indistinguishable, significantly more radiolabeled protein was quantitated using the affinity ligand. These data suggest that the affinity ligand may recognize precursor or degraded forms of the receptor that do not bind the natural ligand or that assays based on the use of noncovalent ligands could underestimate the receptor content of target cells.
Kovacs
W J
WJ
Department of Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee 37232.
Turney
M K
MK
Skinner
M K
MK
D000345:Affinity Labels; D002499:Centrifugation, Density Gradient; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D003600:Cytosol / Q000378:metabolism; D013196:Dihydrotestosterone / Q000031:analogs & derivatives* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005347:Fibroblasts / Q000378:metabolism; D005837:Genitalia, Male / Q000378:metabolism; D007526:Isoelectric Point; D008297:Male; D008970:Molecular Weight; D011498:Protein Precursors / Q000378:metabolism; D011506:Proteins / Q000378:metabolism*; D011944:Receptors, Androgen / Q000378:metabolism*; D012867:Skin / Q000378:metabolism
D000345:Affinity Labels; D011498:Protein Precursors; D011506:Proteins; D011944:Receptors, Androgen; D013196:Dihydrotestosterone; C023562:dihydrotestosterone 17-bromoacetate