Peleg S, Schrader W T, O'Malley B W
Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030.
Biochemistry. 1988 Jan 12;27(1):358-67. doi: 10.1021/bi00401a054.
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.
已对鸡孕酮受体B型（PRB）和A型（PRA）的DNA结合活性进行了检测。这种活性强烈依赖于缓冲液中硫醇的存在。稳定性研究表明，PRB比PRA对氧化更敏感。通过DNA纤维素色谱法将受体制剂分离为DNA阳性和DNA阴性亚群，并用[3H]-N-乙基马来酰亚胺标记对免疫纯化的受体上的巯基进行定量。当蛋白质被还原和变性时，用[3H]-N-乙基马来酰亚胺标记DNA阴性受体显示PRA或PRB形式上有21 - 23个巯基。如果测试变性的DNA阳性受体种类，在未还原的情况下也能看到类似数量的巯基。相比之下，未还原时DNA阴性PRB仅能检测到10 - 12个巯基。对于在体外暴露于非还原环境后失去DNA结合活性的PRA种类，也发现了类似数量（12 - 13个巯基）。我们得出结论，天然存在的无法与DNA结合的受体形式，以及由于在体外暴露于非还原环境而失去DNA结合活性的受体形式，含有10 - 12个氧化的半胱氨酸残基，可能以二硫键形式存在。由于当用二硫苏糖醇（DTT）处理天然DNA阴性受体蛋白时我们无法还原二硫键，我们推测受体在体外DNA结合活性的不可逆丧失是由于天然状态下DTT无法接触到的半胱氨酸残基的氧化。
