Zava D T, Goldhirsch A
Eur J Cancer Clin Oncol. 1983 Aug;19(8):1151-9. doi: 10.1016/0277-5379(83)90041-x.
A model system is presented to explain how tyrosinase, an enzyme unique to pigmented cells such as normal and malignant melanocytes can oxidize [3H]-estradiol to radiolabeled products which closely resemble the tight binding of [3H]-estradiol to estrogen receptor. In the model system studied, tyrosinase oxidized [2,4,6,7-3H]-estradiol to [3H]-water and [3H]-estradiol metabolites, the latter of which formed ring-substituted conjugates with nucleophiles like monothioglycerol and BSA. Radiolabeled estradiol without tritium in the C-2 position (i.e. [6,7-3H]-estradiol) failed to liberate [3H]-water when exposed to tyrosinase but, nevertheless, did form ring-substituted [3H]-estradiol adducts with nucleophiles. The [3H]-water and the ring-substituted radiolabeled products possessed several characteristics of a genuine estrogen receptor protein in that they were resistant to dextran-coated charcoal (DCC) adsorption and their enzymatic formation was inhibited with non-steroidal estrogens like diethylstilbestrol. Other natural (estradiol) and synthetic (hydroxytamoxifen) estrogens which contain the phenol grouping also inhibited the enzymatic oxidation of [3H]-estradiol. Although it was difficult to differentiate estrogen receptor from tyrosinase using the conventional DCC assay system, several differences in these two proteins permitted a distinction to be made between them. First, tyrosinase oxidation of [3H]-estradiol was markedly inhibited by sulfhydryl reducing agents (monothioglycerol) that stabilize [3H]-estradiol binding to estrogen receptor. Second, estrogen receptor adsorbed by hydroxylapatite whereas tyrosinase did not, thus permitting the separation of these two proteins prior to incubation with [3H]-estradiol. We conclude that the [3H]-estradiol binding components in melanoma previously reported to be estrogen receptor probably represent instead the radiolabeled products of the tyrosinase-catalyzed oxidation of [3H]-estradiol.
本文提出了一个模型系统，用以解释酪氨酸酶（一种正常和恶性黑素细胞等色素细胞特有的酶）如何将[3H]-雌二醇氧化为放射性标记产物，这些产物与[3H]-雌二醇与雌激素受体的紧密结合非常相似。在所研究的模型系统中，酪氨酸酶将[2,4,6,7-3H]-雌二醇氧化为[3H]-水和[3H]-雌二醇代谢物，后者与亲核试剂如单硫甘油和牛血清白蛋白形成环取代共轭物。在C-2位没有氚的放射性标记雌二醇（即[6,7-3H]-雌二醇）在暴露于酪氨酸酶时未能释放[3H]-水，但仍与亲核试剂形成环取代的[3H]-雌二醇加合物。[3H]-水和环取代的放射性标记产物具有真正雌激素受体蛋白的几个特征，即它们对葡聚糖包被活性炭（DCC）吸附具有抗性，并且它们的酶促形成受到己烯雌酚等非甾体雌激素的抑制。其他含有酚基团的天然（雌二醇）和合成（羟基他莫昔芬）雌激素也抑制[3H]-雌二醇的酶促氧化。尽管使用传统的DCC分析系统很难区分雌激素受体和酪氨酸酶，但这两种蛋白质的一些差异使它们得以区分。首先，[3H]-雌二醇的酪氨酸酶氧化受到巯基还原剂（单硫甘油）的显著抑制，而巯基还原剂可稳定[3H]-雌二醇与雌激素受体的结合。其次，雌激素受体可被羟基磷灰石吸附，而酪氨酸酶则不能，因此在与[3H]-雌二醇孵育之前可以分离这两种蛋白质。我们得出结论，先前报道的黑色素瘤中[3H]-雌二醇结合成分可能不是雌激素受体，而是酪氨酸酶催化氧化[3H]-雌二醇的放射性标记产物。
