Sawada K, Hummel B C, Walfish P G
Biochem J. 1986 Mar 1;234(2):391-8. doi: 10.1042/bj2340391.
The effects of cytosol, NADPH and reduced glutathione (GSH) on the activity of 5'-deiodinase were studied by using washed hepatic microsomes from normal fed rats. Cytosol alone had little stimulatory effect on the activation of microsomal 5'-deiodinase. NADPH had no stimulatory effect on the microsomal 5'-deiodinase unless cytosol was added. 5'-deiodinase activity was greatly enhanced by the simultaneous addition of NADPH and cytosol (P less than 0.001); this was significantly higher than that with either NADPH or cytosol alone (P less than 0.001). GSH was active in stimulating the enzyme activity in the absence of cytosol, but the activity of 5'-deiodinase with 62 microM-NADPH in the presence of cytosol was significantly higher than that with 250 microM-GSH in the presence of the same concentration of cytosol (P less than 0.001). The properties of the cytosolic components essential for the NADPH-dependent activation of microsomal 5'-deiodinase independent of a glutathione/glutathione reductase system were further assessed using Sephadex G-50 column chromatography to yield three cytosolic fractions (A, B and C), wherein A represents pooled fractions near the void volume, B pooled fractions of intermediate Mr (approx. 13 000), and C of low Mr (approx. 300) containing glutathione. In the presence of NADPH (1 mM), the 5'-deiodination rate by hepatic washed microsomes is greatly increased if both A and B are added and is a function of the concentrations of A, B, washed microsomes and NADPH. A is heat-labile, whereas B is heat-stable and non-dialysable. These observations provide the first evidence of an NADPH-dependent cytosolic reductase system not involving glutathione which stimulates microsomal 5'-deiodinase of normal rat liver. The present data are consistent with a deiodination mechanism involving mediation by a reductase (other than glutathione reductase) in fraction A of an NADPH-dependent reduction of a hydrogen acceptor in fraction B, followed by reduction of oxidized microsomal deiodinase by the reduced acceptor (component in fraction B).
利用正常喂养大鼠的肝脏洗涤微粒体，研究了胞质溶胶、NADPH和还原型谷胱甘肽（GSH）对5'-脱碘酶活性的影响。单独的胞质溶胶对微粒体5'-脱碘酶的激活几乎没有刺激作用。除非添加胞质溶胶，否则NADPH对微粒体5'-脱碘酶没有刺激作用。同时添加NADPH和胞质溶胶可使5'-脱碘酶活性大大增强（P小于0.001）；这明显高于单独使用NADPH或胞质溶胶时的活性（P小于0.001）。在没有胞质溶胶的情况下，GSH能刺激酶活性，但在相同浓度胞质溶胶存在时，含62微摩尔NADPH的5'-脱碘酶活性明显高于含250微摩尔GSH的活性（P小于0.001）。使用Sephadex G-50柱色谱法进一步评估了微粒体5'-脱碘酶NADPH依赖性激活所必需的胞质成分的特性，得到三个胞质组分（A、B和C），其中A代表接近空体积的合并组分，B代表中等分子量（约13000）的合并组分，C代表低分子量（约300）且含谷胱甘肽的组分。在存在NADPH（1毫摩尔）的情况下，如果同时添加A和B，肝脏洗涤微粒体的5'-脱碘速率会大大增加，并且是A、B、洗涤微粒体和NADPH浓度的函数。A对热不稳定，而B对热稳定且不可透析。这些观察结果首次证明了一种不涉及谷胱甘肽的NADPH依赖性胞质还原酶系统，该系统可刺激正常大鼠肝脏的微粒体5'-脱碘酶。目前的数据与一种脱碘机制一致，该机制涉及由A组分中的一种还原酶（而非谷胱甘肽还原酶）介导NADPH依赖性还原B组分中的氢受体，随后由还原的受体（B组分中的成分）还原氧化的微粒体脱碘酶。
