Bykova Natalia V., Møller Ian M.
Department of Plant Physiology, Lund University, Box 117, SE-221 00 Lund, Sweden; Umeå Plant Science Centre, Department of Plant Physiology, Umeå University, SE-901 87 Umeå, Sweden; Present address: Plant Biology and Biogeochemistry Department, Risø National Laboratory, Building 301, P.O. Box 49, DK-4000 Roskilde, Denmark.
Physiol Plant. 2001 Apr;111(4):448-456. doi: 10.1034/j.1399-3054.2001.1110404.x.
The involvement of the internal rotenone-insensitive NADPH dehydrogenase on the inner surface of the inner mitochondrial membrane [NDin(NADPH)] in the oxidation of strictly NAD+-linked substrates by pea (Pisum sativum L.) leaf mitochondria was measured. As estimated by the inhibition caused by 5 µM diphenyleneiodonium (DPI) in the presence of rotenone to inhibit complex I, the activity of NDin(NADPH) during glycine oxidation (measured both as O2 uptake and as CO2 release) was 40-50 nmol mg-1 protein min-1. No significant activity of NDin(NADPH) could be detected during the oxidation of 2-oxoglutarate, another strictly NAD+-linked substrate; this was possibly due to its relatively low oxidation rate. Control experiments showed that, even at 125 µM, DPI had no effect on the activity of glycine decarboxylase complex (GDC) and lipoamide dehydrogenase. The relative activity of complex I, NDin(NADPH), and NDin(NADH) during glycine oxidation, estimated using rotenone and DPI, differed depending on the pyridine nucleotide supply in the mitochondrial matrix. This was shown by loading the mitochondria with NAD+ and NADP+, both of which were taken up by the organelle. We conclude that the involvement of NADP turnover during glycine oxidation is not due to the direct production of NADPH by GDC but is an indirect result of this process. It probably occurs via the interconversion of NADH to NADPH by the two non-energy-linked transhydrogenase activities recently identified in plant mitochondria.
测定了豌豆（Pisum sativum L.）叶片线粒体中，线粒体内膜内表面对鱼藤酮不敏感的NADPH脱氢酶[NDin(NADPH)]参与严格依赖NAD⁺的底物氧化的情况。在鱼藤酮存在下，用5 μM二苯基碘鎓（DPI）抑制复合体I所引起的抑制作用来估算，甘氨酸氧化过程中（以O₂吸收和CO₂释放来衡量）NDin(NADPH)的活性为40 - 50 nmol mg⁻¹蛋白质 min⁻¹。在另一种严格依赖NAD⁺的底物2-氧代戊二酸氧化过程中，未检测到NDin(NADPH)的显著活性；这可能是由于其氧化速率相对较低。对照实验表明，即使在125 μM时，DPI对甘氨酸脱羧酶复合体（GDC）和硫辛酰胺脱氢酶的活性也没有影响。在甘氨酸氧化过程中，使用鱼藤酮和DPI估算的复合体I、NDin(NADPH)和NDin(NADH)的相对活性，因线粒体基质中吡啶核苷酸的供应情况而异。这通过向线粒体中加载NAD⁺和NADP⁺得以证明，二者均被细胞器摄取。我们得出结论，甘氨酸氧化过程中NADP周转的参与并非由于GDC直接产生NADPH，而是该过程的间接结果。它可能是通过植物线粒体中最近发现的两种非能量偶联的转氢酶活性，由NADH向NADPH的相互转化而发生的。
