Tashima Y, Terui M, Itoh H, Mizunuma H, Kobayashi R, Marumo F
Department of Biochemistry, School of Medicine, Akita University.
J Biochem. 1990 Aug;108(2):271-7. doi: 10.1093/oxfordjournals.jbchem.a123193.
We purified glucocorticoid receptors quickly but very partially using DEAE-resin. [3H]-Triamcinolone acetonide-labeled and non-activated receptors in the quickly purified fraction were found to be separated into two fractions (P-2 and P-3) by hydroxyapatite column chromatography. The P-2 receptor was the main component, and the ratio of P-2/P-3 was around 2. The molecular weights of the two receptors were calculated to be the same, 242,000: Rs = 6.2 nm and s20,w = 9.0. Treatment of the receptor with catalytic subunits of phosphoprotein phosphatase 2A1 reduced the P-2/P-3 ratio from 2 to 0.5, while treatment with catalytic subunits of cAMP-dependent protein kinase and ATP increased it to 2.5. The isolated P-3 receptor could be converted into the P-2 type by the kinase treatment. Tungstate, a phosphatase inhibitor, stabilized the P-2 receptor, and the P-2/P-3 ratio was larger than 3 when the DEAE-fraction was prepared in the presence of tungstate. However, the tungstate effect was not very strong, and the P-2 type tended to change into the P-3. [3H]-Triamcinolone acetonide-labeled and non-activated receptors were purified very highly by using an affinity gel; the procedure required more than 10 h. Only the P-3 form was observed in the preparation of highly purified receptors. Hormone-free receptors were affected by neither the phosphatase nor the kinase. The results indicate that the hormone binding makes the receptor sensitive to phosphatase. The reversibly dephosphorylated receptor is more stable than the non-dephosphorylated one, and can be activated.
我们使用DEAE树脂快速但仅部分纯化了糖皮质激素受体。在快速纯化的组分中，[3H] - 曲安奈德标记且未活化的受体通过羟基磷灰石柱色谱法被分离为两个组分（P - 2和P - 3）。P - 2受体是主要成分，P - 2 / P - 3的比例约为2。两种受体的分子量经计算相同，为242,000：Rs = 6.2 nm且s20,w = 9.0。用蛋白磷酸酶2A1的催化亚基处理受体可使P - 2 / P - 3的比例从2降至0.5，而用cAMP依赖性蛋白激酶的催化亚基和ATP处理则使其升至2.5。分离出的P - 3受体经激酶处理可转化为P - 2型。磷酸酶抑制剂钨酸盐可稳定P - 2受体，当在钨酸盐存在下制备DEAE组分时，P - 2 / P - 3的比例大于3。然而，钨酸盐的作用不是很强，P - 2型倾向于转变为P - 3型。使用亲和凝胶可非常高效地纯化[3H] - 曲安奈德标记且未活化的受体；该过程需要超过10小时。在高度纯化的受体制剂中仅观察到P - 3形式。无激素受体不受磷酸酶或激酶的影响。结果表明，激素结合使受体对磷酸酶敏感。可逆去磷酸化的受体比未去磷酸化的受体更稳定，并且可以被激活。
