Sulfhydryl group content of chicken progesterone receptor: effect of oxidation on DNA binding activity.
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.
Peleg
S
S
Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030.
Schrader
W T
WT
O'Malley
B W
BW
D000818:Animals; D002645:Chickens; D004247:DNA / Q000378:metabolism*; D004229:Dithiothreitol / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D005260:Female; D007700:Kinetics; D010057:Oviducts / Q000378:metabolism*; D010084:Oxidation-Reduction; D011485:Protein Binding; D011980:Receptors, Progesterone / Q000302:isolation & purification / Q000378:metabolism*; D013438:Sulfhydryl Compounds / Q000378:metabolism
D011980:Receptors, Progesterone; D013438:Sulfhydryl Compounds; D004247:DNA; D005033:Ethylmaleimide; D004229:Dithiothreitol