Radiation-inactivation size of transformed and non-transformed androgen receptors.
The nucleic acid sequence of the androgen receptor (AR) gene predicts that the protein structure possesses DNA- and steroid-binding domains that show high degrees of sequence similarity with those of other steroid receptors. Since the steroid-binding domain of the AR corresponds to a 30 kDa portion of the protein, and the AR structure may be monomeric or hetero-oligomeric depending on its transformation state, we have herein determined the AR radiation-inactivation size (RIS) in relation to the molecular structure whose binding activity toward methyltrienolone (R1881) is abolished by a radiation 'hit'. Soluble fractions from whole canine prostatic tissue were used as a source of non-transformed AR. The AR transformation was induced by the addition of 0.6 M-KCl, and these preparations were used together with high-salt nuclear extracts as a source of transformed AR. To maximize the binding activity, molybdate and dithiothreitol were included during AR extraction. Receptor transformation was verified by modifications of both the sedimentation coefficients (from 7.5 S to 4.1 S on sucrose gradients) and molecular masses (from 260 kDa to 115 kDa by gel filtration). The RIS values of the non-transformed and transformed ARs were not statistically different: 92 +/- 19 kDa and 110 +/- 25 kDa respectively. In addition, the inactivation of AR binding activity by radiation was attributed to a loss of binding sites, with no significant change in the Kd. When benzoic acid, a free-electron scavenger, was added together with dithiothreitol before and after irradiation, no change in the RIS value was observed. Thus, in the canine prostate, the RIS value of the AR represents the monomeric protein, independently of its association with other proteins, and this value corresponds to that predicted by cloning studies and photoaffinity-labelling of AR.
Turcotte
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G
Research Center of *Maisonneuve, Rosemont Hospital, University of Montréal, Québec, Canada.
Beauregard
G
G
Potier
M
M
Chevalier
S
S
D000818:Animals; D001665:Binding Sites / Q000528:radiation effects; D002467:Cell Nucleus / Q000737:chemistry; D002499:Centrifugation, Density Gradient; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002850:Chromatography, Gel; D004229:Dithiothreitol / Q000494:pharmacology; D004285:Dogs; D004355:Drug Stability; D008297:Male; D015741:Metribolone / Q000378:metabolism; D008970:Molecular Weight; D008982:Molybdenum / Q000494:pharmacology; D011189:Potassium Chloride / Q000494:pharmacology; D011467:Prostate / Q000737:chemistry / Q000648:ultrastructure; D011944:Receptors, Androgen / Q000737:chemistry / Q000378:metabolism / Q000528:radiation effects*
D011944:Receptors, Androgen; C044659:molybdate; D015741:Metribolone; D011189:Potassium Chloride; D008982:Molybdenum; D004229:Dithiothreitol