Németi Balázs, Gregus Zoltán
Department of Pharmacology and Pharmacotherapy, Toxicology Section, University of Pécs, Medical School, Szigeti út 12, H-7624 Pécs, Hungary.
Toxicol Appl Pharmacol. 2009 Sep 1;239(2):154-61. doi: 10.1016/j.taap.2009.02.011. Epub 2009 Feb 25.
Three cytosolic phosphorolytic/arsenolytic enzymes, (purine nucleoside phosphorylase [PNP], glycogen phosphorylase, glyceraldehyde-3-phosphate dehydrogenase) have been shown to mediate reduction of arsenate (AsV) to the more toxic arsenite (AsIII) in a thiol-dependent manner. With unknown mechanism, hepatic mitochondria also reduce AsV. Mitochondria possess ornithine carbamoyl transferase (OCT), which catalyzes phosphorolytic or arsenolytic citrulline cleavage; therefore, we examined if mitochondrial OCT facilitated AsV reduction in presence of glutathione. Isolated rat liver mitochondria were incubated with AsV, and AsIII formed was quantified. Glutathione-supplemented permeabilized or solubilized mitochondria reduced AsV. Citrulline (substrate for OCT-catalyzed arsenolysis) increased AsV reduction. The citrulline-stimulated AsV reduction was abolished by ornithine (OCT substrate inhibiting citrulline cleavage), phosphate (OCT substrate competing with AsV), and the OCT inhibitor norvaline or PALO, indicating that AsV reduction is coupled to OCT-catalyzed arsenolysis of citrulline. Corroborating this conclusion, purified bacterial OCT mediated AsV reduction in presence of citrulline and glutathione with similar responsiveness to these agents. In contrast, AsIII formation by intact mitochondria was unaffected by PALO and slightly stimulated by citrulline, ornithine, and norvaline, suggesting minimal role for OCT in AsV reduction in intact mitochondria. In addition to OCT, mitochondrial PNP can also mediate AsIII formation; however, its role in AsV reduction appears severely limited by purine nucleoside supply. Collectively, mitochondrial and bacterial OCT promote glutathione-dependent AsV reduction with coupled arsenolysis of citrulline, supporting the hypothesis that AsV reduction is mediated by phosphorolytic/arsenolytic enzymes. Nevertheless, because citrulline cleavage is disfavored physiologically, OCT may have little role in AsV reduction in vivo.
三种胞质磷酸解/砷解酶（嘌呤核苷磷酸化酶[PNP]、糖原磷酸化酶、甘油醛-3-磷酸脱氢酶）已被证明以硫醇依赖的方式介导砷酸盐（AsV）还原为毒性更强的亚砷酸盐（AsIII）。肝脏线粒体以未知机制也能还原AsV。线粒体含有鸟氨酸氨甲酰基转移酶（OCT），该酶催化磷酸解或砷解瓜氨酸裂解；因此，我们研究了线粒体OCT在谷胱甘肽存在的情况下是否促进AsV还原。将分离的大鼠肝脏线粒体与AsV一起孵育，并对形成的AsIII进行定量。补充了谷胱甘肽的通透化或溶解的线粒体可还原AsV。瓜氨酸（OCT催化砷解的底物）增加了AsV的还原。鸟氨酸（抑制瓜氨酸裂解的OCT底物）、磷酸盐（与AsV竞争的OCT底物）以及OCT抑制剂正缬氨酸或Palo可消除瓜氨酸刺激的AsV还原，这表明AsV还原与OCT催化的瓜氨酸砷解偶联。支持这一结论的是，纯化的细菌OCT在瓜氨酸和谷胱甘肽存在的情况下介导AsV还原，对这些试剂具有相似的反应性。相比之下，完整线粒体形成AsIII不受Palo影响，而瓜氨酸、鸟氨酸和正缬氨酸对其有轻微刺激，这表明OCT在完整线粒体中AsV还原中的作用最小。除了OCT，线粒体PNP也可介导AsIII的形成；然而，其在AsV还原中的作用似乎受到嘌呤核苷供应的严重限制。总体而言，线粒体和细菌OCT通过瓜氨酸的偶联砷解促进谷胱甘肽依赖性AsV还原，支持AsV还原由磷酸解/砷解酶介导的假说。然而，由于瓜氨酸裂解在生理上不受青睐，OCT在体内AsV还原中可能作用不大。
