Oxidation of External NAD(P)H by Purified Mitochondria from Fresh and Aged Red Beetroots (Beta vulgaris L.)

Abstract

Mitochondria were isolated from fresh beetroots (Beta vulgaris L. cvs Rubria and Nina) by differential centrifugation followed by Percoll gradient centrifugation. These purified mitochondria oxidized external NADH, although relatively slowly (20-40 versus 100-120 nanomoles oxygen per minute times milligram protein for NADH and succinate oxidation, respectively), with respiratory control ratios of two to three and ADP/O ratios of 1.2 to 1.6. NADPH was also oxidized, but even more slowly and with little or no coupling. The optimum for both NADH and NADPH oxidation by fresh beetroot mitochondria was pH 6. The rate of external NADH oxidation by isolated mitochondria was enhanced threefold during storage of the intact tubers at 10 degrees C for 12 weeks. The optimum of the induced NADH oxidation was approximately pH 6.8. Succinate and malate oxidation only increased by 30% during the same period and NADPH oxidation was constant. This is strong evidence that NADH and NADPH oxidation are catalyzed by different enzymes at least in beetroots. Activity staining of nondenaturing polyacrylamide gels with NADH and Nitro Blue Tetrazolium did not show differences in banding pattern between mitochondria isolated from fresh and stored beetroots. The induction is discussed in relation to physiological aging processes.