Gorin Y, Leseney A M, Ohayon R, Dupuy C, Pommier J, Virion A, Dème D
INSERM Unité 96, Le Kremlin-Bicĕtre, France.
Biochem J. 1997 Jan 15;321 ( Pt 2)(Pt 2):383-8. doi: 10.1042/bj3210383.
Pig thyroid plasma membranes contain a Ca(2+)-dependent NADPH:O2 oxidoreductase, the thyroid NADPH-dependent H2O2 generator. This provided the H2O2 for the peroxidase-catalysed synthesis of thyroid hormones. The effect of the tervalent arsenical, phenylarsine oxide (PAO), on the NADPH oxidase was studied. PAO caused two directly related dose-dependent effects with similar half-effect concentrations of PAO (3 nmol of PAO/mg of protein): (i) partial inactivation of H2O2 formation by the Ca(2+)-stimulated enzyme, and (ii) desensitization of the enzyme activity to Ca2+. PAO had no effect on membranes that had been Ca(2+)-desensitized by alpha-chymotrypsin treatment. The NADPH oxidase in membranes treated with excess PAO had the same Vmax with and without Ca2+. This value was half the Vmax of the native enzyme. However, the K(m) for NADPH determined with Ca2+ (18 microM, identical with that of the native enzyme) was approx, one-third of the K(m) measured without Ca2+, showing the direct action of Ca2+ on the PAO-enzyme complex. PAO had the same effects, partial inactivation and Ca2+ desensitization, on the NADPH: ferricyanide oxidoreductase activity of the NADPH oxidase, suggesting that PAO acts on the flavodehydrogenase entity of the enzyme. Both partial inactivation and Ca2+ desensitization were completely and specifically reversed by 2.3-dimercaptopropanol, partly reversed by dithiothreitol and not reversed by 2-mercaptoethanol, indicating that PAO binds to vicinal thiol groups. These results suggest that thiol groups are involved in the control of thyroid NADPH oxidase by Ca2+; PAO bound to vicinal thiols might alter the structure of the enzyme so that electron transfer occurs without Ca2+ but more slowly.
猪甲状腺质膜含有一种钙依赖性NADPH：O2氧化还原酶，即甲状腺NADPH依赖性H2O2生成酶。它为过氧化物酶催化的甲状腺激素合成提供H2O2。研究了三价砷化合物苯砷氧化物（PAO）对NADPH氧化酶的影响。PAO产生了两种直接相关的剂量依赖性效应，其PAO的半数效应浓度相似（3 nmol PAO/mg蛋白质）：（i）钙刺激的酶形成H2O2的部分失活，以及（ii）酶活性对Ca2+的脱敏作用。PAO对经α-胰凝乳蛋白酶处理而对Ca2+脱敏的膜无影响。用过量PAO处理的膜中的NADPH氧化酶在有或没有Ca2+的情况下具有相同的Vmax。该值是天然酶Vmax的一半。然而，在有Ca2+的情况下测定的NADPH的K(m)（18 μM，与天然酶相同）约为在没有Ca2+的情况下测定的K(m)的三分之一，表明Ca2+对PAO-酶复合物有直接作用。PAO对NADPH氧化酶的NADPH：铁氰化物氧化还原酶活性具有相同的效应，即部分失活和Ca2+脱敏，这表明PAO作用于该酶的黄素脱氢酶部分。2,3-二巯基丙醇可完全且特异性地逆转部分失活和Ca2+脱敏，二硫苏糖醇可部分逆转，而2-巯基乙醇则不能逆转，这表明PAO与相邻的巯基结合。这些结果表明，巯基参与了Ca2+对甲状腺NADPH氧化酶的调控；与相邻巯基结合的PAO可能会改变酶的结构，从而使电子传递在没有Ca2+的情况下发生，但速度更慢。
