Pyridine Nucleotides in Plant Mitochondria. Amounts, metabolism and contribution to electron transport

Abstract: HPLC analysis was used for direct quantification of amounts and redox levels of pyridine nucleotides in plant mitochondria. The amount of NAD(H) and NADP+ varied, 1.0-3.7 and 0-0.5 nmol (mg protein)-1, respectively, depending on the species from which nucleotides were extracted. NADPH was never detected. Submitochondrial localisation of NAD+-degrading enzymes indicated that matrix NAD+ diffuses intact across the inner mitochondrial membrane and is metabolised by NAD+-catabolising enzymes in the outer membrane. NAD pyrophosphatase activity was identified as being involved in the pyridine nucleotide degradation. During malate oxidation by isolated Solanum tuberosum mitochondria, the highest levels of NAD(H) reduction were obtained when malic enzyme was active. The lowest NAD(H) reduction levels were seen when conditions favoured malate dehydrogenase activity. Rotenone increased the reduction level of intramitochondrial NADH. Diphenylene iodonium (DPI) inhibited malate oxidation in the presence of rotenone in isolated mitochondria. This result indicated participation of matrix NADPH and the internal rotenone-insensitive, DPI-sensitive, NADPH dehydrogenase during malate oxidation. Antisera raised against peptides deduced from two S. tuberosum genes (nda1 and ndb1), homologous to the yeast genes for the internal and external NADH:ubiquinone dehydrogenases specifically cross-reacted with 48 and 61 kDa polypeptides in potato tuber mitochondria, respectively. Native NDA1 and NDB1 occurred as higher molecular mass forms, and show different association to the inner mitochondrial membrane. The physiological significance of pyridine nucleotide turnover and occurrence of multiple NAD(P)H dehydrogenases in the inner mitochondrial membrane is discussed.