Involvement of matrix NADP turnover in the oxidation of NAD(+)-linked substrates by pea leaf mitochondria

The involvement of the internal rotenone-insensitive NADPH dehydrogenase on the inner surface of the inner mitochondrial membrane INDin(NADPH)I in the oxidation of strictly NAD(+)-linked substrates by pea (Pisum sativum L,) l eaf mitochondria was measured. As estimated by the inhibition caused by 5 m uM diphenyieneiodonium (DPI) in the presence of rotenone to inhibit complex I, the activity of NDin(NAI)PH) during glycine oxidation (measured both as O-2 uptake and as CO2 release) was 40-50 nmol mg(-1) protein min(-1). No s ignificant activity of NDin(NADPH) could be detected during the oxidation o f 2-oxoglutarate, another strictly NAD(+)- Linked substrate; this was possi bly due to its relatively Low oxidation rate. Control experiments showed th at, even at 125 muM, DPI had no effect on the activity of glycine decarboxy lase complex (GDC) and lipoamide dehydrogenase, The relative activity of co mplex I, NDin(NADPH), and NDin(NADH) during glycine oxidation. estimated us ing rotenone and DPI, differed depending on the pyridine nucleotide supply in the mitochondrial matrix. This was shown by loading the mitochondria wit h NAD(+) and NADP(+), both of which were taken up by the organelle, We conc lude 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 t his process. It probably occurs via the interconversion of NADH to NADPH by the two non-energy-linked transhydrogenase activities recently identified in plant mitochondria.