Affinity labeling of the androgen receptor in rat prostate cytosol with 17.beta.-[(bromoacetyl)oxy]-5.alpha.-androstan-3-one

Abstract

An androgen affinity label, 17 beta-[(bromoacetyl)-oxy]-5 alpha-androstan-3-one, has been synthesized in both radioactive and nonradioactive forms. The affinity label (170 Ci/mmol) was characterized and found to have a high degree of purity. Affinity labeling of the androgen receptor from rat ventral prostate was androgen specific and appeared to be directed at the steroid binding site of the protein. Covalent binding was achieved at 0 degrees C; however, heat treatment at 23 degrees C for 30 min enhanced covalent binding by 31%. The covalently bound steroid was resistant to extraction with organic solvents and precipitation with trichloracetate. The Stokes radius (4.2 nm) and sedimentation coefficient (4.5 S) were identical with those found for receptor bound noncovalently to dihydrotestosterone. Gel electrophoresis of the affinity-labeled receptor under denaturing conditions revealed a molecular weight of 86000. The same molecular weight was observed for the receptor from rat seminal vesicle. This affinity label will be useful in future studies on the structure and function of androgen receptors.

High efficiency covalent radiolabeling of the human androgen receptor. Studies in cultured fibroblasts using dihydrotestosterone 17 beta-bromoacetate.

“…The bromoacetate methylene singlet was at 6 = 3.8251 while the Cl7a-hydrogen produced a triplet with peaks at a = 4.5906, 4.66, and 4.767. This spectrum is identical to the published spectrum of the compound (13).…”

“…The commercial availability of high specific activity radiolabeled dihydrotestosterone has now obviated those difficulties. Chang et al (13) [1,2,4,5,6,7,16,17-3H]dihydrotestosterone and used it to covalently label highly purified rat prostate cytosolic androgen receptors (8,13,14), as well as purified androgen receptors from bovine seminal vesicle (7). We report here the first application of such methodology to human cells, and the crucial demonstration that the protein labeled by dihydrotestosterone 1 7f3-bromoacetate is absent from receptor negative cell strains.…”

“…The results have been uniformly disappointing either because of the very low efficiency ofcovalently labeling, because ofextensive purification of receptors necessary prior to introduction of the affinity label, or because of lability ofthe receptor protein itself under conditions of photolysis. Recently Chang et al (7,8,13,14) have reported evidence that the electrophilic affinity label 173-bromoacetyl oxy-5a androstane-3-one (dihydrotestosterone 173-bromoacetate)' originally synthesized by Le Gaillard and Dautreavaux (15) and tested by Mainwaring (16), binds covalently to purified androgen receptors from rat prostate, from a transplantable prostatic tumor and from bovine seminal vesicle.…”

Toward the Characterization and Identification of γ‐Secretases Using Transition‐state Analogue Inhibitors

“…To identify the target of these γ‐secretase inhibitors, we have designed derivatives of these peptide analogues that may bind covalently and irreversibly. The N‐terminal Boc group of 1b was replaced with a bromoacetyl group, a functionality susceptible to nucleophilic attack that has been extensively employed as a means of creating affinity labels 32–37. Thus, the difluoro ketone transition‐state mimic should interact with the two active site aspartates of β‐secretase,38 while the bromoacetamide should react with any proximal nucleophilic residues (e.g., Ser, Cys, Thr) to form a stable covalent bond and irreversibly inactivate the enzyme.…”

“…The N-terminal Boc group of 1b was replaced with a bromoacetyl group, a functionality susceptible to nucleophilic attack that has been extensively employed as a means of creating affinity labels. [32][33][34][35][36][37] Thus, the difluoro ketone transition-state mimic should interact with the two active site aspartates of γ-secretase, 38 while the bromoacetamide should react with any proximal nucleophilic residues (e.g., Ser, Cys, Thr) to form a stable covalent bond and irreversibly inactivate the enzyme. As expected, we found that the Boc-containing 1b reversibly inhibits γ-secretase: Upon replacement with inhibitorfree media, Aβ production returned to control levels (FIG.…”