Resolution of the effects of sulfhydryl-blocking reagents on hormone-and DNA-binding activities of the chick oviduct progesterone receptor

1988

DNA binding activity of chicken progesterone receptor B form (PRB) and A form (PRA) has been examined. This activity is strongly dependent upon the presence of thiols in the buffer. Stability studies showed that PRB was more sensitive to oxidation than was PRA. Receptor preparations were fractionated by DNA-cellulose chromatography to DNA-positive and DNA-negative subpopulations, and sulfhydryl groups were quantified on immunopurified receptor by labeling with [3H]-N-ethylmaleimide. Labeling of DNA-negative receptors with [3H]-N-ethylmaleimide showed 21-23 sulfhydryl groups on either PRA or PRB form when the proteins were reduced and denatured. A similar number was seen without reduction if denatured DNA-positive receptor species were tested. In contrast, the DNA-negative PRB had only 10-12 sulfhydryl groups detectable without reduction. A similar number (12-13 sulfhydryl groups) was found for PRA species that lost DNA binding activity after exposure to a nonreducing environment in vitro. We concluded that the naturally occurring receptor forms unable to bind to DNA, as well as receptor forms that have lost DNA binding activity due to exposure to a nonreducing environment in vitro, contain 10-12 oxidized cysteine residues, likely present as disulfide bonds. Since we were unable to reduce the disulfide bonds when the native DNA-negative receptor proteins were treated with dithiothreitol (DTT), we speculate that irreversible loss of DNA binding activity of receptor in vitro is due to oxidation of cysteine residues that are not accessible to DTT in the native state.

Section: Discussionsupporting

confidence: 81%

mentioning

confidence: 97%

Sulfhydryl group content of chicken progesterone receptor: effect of oxidation on DNA binding activity

Peleg¹,

Schrader²,

1988

“…First, the relatively low molecular weight (about 40000) is smaller than that reported for the estrogen receptor in either its multimeric or monomeric forms [about 300000 and 60 000-70 000 respectively (Lubahn et al, 1985;Parmar et al, 1988)]. The sensitivity of the type II site to sulfhydryl reducing reagents is contrary to the findings for essentially all of the intracellular high-affinity steroid receptors, including those for estrogen (Jensen et al, 1967), progesterone (Coty et al, 1983), glucocorticoids (Rees & Bell, 1975), mineralocorticoids (Emadian et al, 1986), and androgens (Wilson et al, 1986), which have generally been shown to be stabilized by the presence of sulfhydryl reagents.…”

Section: Discussionmentioning

confidence: 76%

Characterization and partial purification of an estrogen type II binding site in chick oviduct cytosol

Densmore¹,

Markaverich²,

et al. 1989

An estrogen binding site of moderate affinity (Kd approximately 10 nM) and high capacity (approximately 25-70 pmol/g of tissue) was measured in DES-stimulated chick oviduct cytosol. Saturation analysis by [3H]estradiol exchange demonstrated that this binding site displayed sigmoidal binding characteristics suggesting a cooperative binding mechanism. Competition analysis with a number of compounds demonstrated that the bioflavonoid luteolin was a better competitor for binding to type II sites in chick than either estradiol or DES. Steroid specificity was demonstrated by the inability of 17 alpha-estradiol, progesterone, testosterone, corticosterone, and the triphenylethylene antiestrogen nafoxidine (U-1100A) to compete for [3H]-17 beta-estradiol binding to chick oviduct cytosol preparations. In addition, the binding site appeared to be sensitive to sulfhydryl reducing reagents as evidenced by a 75% reduction in binding activity in the presence of dithiothreitol. Both prelabeling and postlabeling procedures used in conjunction with Sephacryl S-300 chromatography resulted in a single major peak of type II binding activity representing a molecular weight in the 40,000 range. Type II binding activity was recoverable after precipitation with ammonium sulfate, and this material was subjected to a variety of column chromatography procedures in order to achieve further purification of the type II site. Significant purification of the site was achieved with a bioflavonoid-Sepharose (quercetin-Sepharose) affinity matrix. The purified type II sites eluted from quercetin-Sepharose displayed the same sigmoidal binding curves characteristic of native cytosol.

“…The reagents /V-ethylmalcimide and iodoacetamide have been described previously as poor inhibitors of DNA binding of chicken PR (Coty et al" 1983). We modified the conditions previously described and prolonged the incubation period of the reagents with receptor.…”

Section: Resultsmentioning

confidence: 99%

Differential sensitivity of chicken progesterone receptor forms to sulfhydryl reactive reagents

Peleg

Schrader²,

1989

DNA binding of chick progesterone receptor B form (PRB) has been examined and compared to that of the A form (PRA). We found that the elution profiles of the two receptors overlap on DNA-cellulose columns. Both PRA or PRB could bind to plasmid DNA equivalently as assayed by sedimentation velocity studies. However, DNA-binding activity of the two receptor forms showed differential sensitivity to reducing agents and to sulfhydryl (SH) reactive reagents. Reducing agents stabilized DNA-binding activity of PRA more efficiently than they stabilized PRB. Moreover, removal of reducing agents from receptor preparations caused preferential loss of DNA binding by PRB compared to the PRA. DNA-binding activity of PRA was readily destroyed by sulfhydryl modifying reagents such as N-ethylmaleimide and iodoacetamide while PRB was 3-4 times less sensitive to these reagents. We conclude the DNA-binding activity of PRB is less stable due to altered accessibility of SH groups despite the amino acid sequence identity of the DNA-binding domains of PRA and PRB.