NAD(P) turnover in plant mitochondria

An analytical procedure based on alkaline extraction and HPLC analysis was adapted for quantification of pyridine nucleotides in plant mitochondria. The amounts of NAD and NADP extracted from seven different species varied from 1.0 to 3.7 and 0 to 0.5 nmol (mg protein)(-1), respectively. Although NADP was found in four species, its reduced form was in all cases below the detection limit of 0.1 nmol (mg protein)(-1). The NAD pool was mainly oxidized in the absence of substrates. However, oxidation of substrates followed by anaerobiosis caused 50-92% NAD pool reduction, indicating that the majority of the NAD(+) was metabolically active. The NAD reduction level in potato tuber mitochondria oxidizing malate varied with assay conditions. The highest level of reduction (> 80%) was reached at anaerobiosis, at pH 6.5 and 7.2, conditions favouring malic enzyme (ME), whereas the lowest reduction level (0%) was observed at pH 7.5, conditions favouring malate dehydrogenase (MDH). Mitochondria incubated at 0 degreesC without respiratory substrate showed a loss of endogenous NAD(+) which correlated with a decline in the rate of oxidation of NAD(+)-linked substrates. The lost NAD(+) was mainly recovered as breakdown products in both the surrounding medium and the mitochondria. When submitochondrial fractions were incubated with NAD(+) or NADP(+), the highest rate of NAD(P)(+) metabolism was detected in the outer membrane fraction. The metabolites detected, adenosine monophosphate (AMP), nicotinamide mononucleotide (NMN) and adenosine, imply that several enzymes involved in pyridine nucleotide degradation, including an NAD pyrophosphatase, are localized to the outer membrane.