Characteristics of External NADH Oxidation by Beetroot Mitochondria

Abstract

Mitochondria isolated from fresh red beetroot (Beta vulgaris L.) tissue do not oxidize external NADH with 02 as the electron acceptor. These mitochondria have a rotenone-and antimycin-insensitive pathway of NADH oxidation associated with the outer membrane and are capable of reducing cytochrome c or potassium ferricyanide. They are also capable of oxidizing internal NADH via the inner membrane electron transport chain with normal rotenone and antimycin sensitivity and ADP/O ratios. They differ from other plant mitochondria in the apparent lack of the NADH dehydrogenase located on the outer surface of the inner membrane. It is shown that this activity develops during the aging of red beetroot slices in aerated dilute CaSO4 solutions, and is present in the mitochondria isolated from aged tissue.Mitochondria isolated from fresh red beetroot tissue are capable of oxidizing external NADH via a malate-oxaloacetate shuttle system. It is suggested that these mitochondria possess a rapid oxaloacetate-transporting system. Isolated plant mitochondria appear able to oxidize NADH via three distinct pathways (5, 8). Thesc arc: (a) an clectron transport chain on the outer membrane (7. 16). consisting of a flavoprotein and Cyt b555 which operates when an external electron acceptor is present. This pathway is inscnsitive to respiratory inhibitors (5. 8. 16). (b) A dehydrogenase located on the outside of the inner membrane which can transfer electrons from exogenous NADH to the respiratorv chain. This pathway is inhibited by antimycin A but is insensitive to rotenone (5. 7. 1 0. 17). (c) A dehydrogenase situated on the matrix side of the inner membrane (5. 7). NADH oxidation via this enzyme is inhibited by both rotenone and antimycin A. and coupled to three sites of ATP formation (10,17). Pathways (a) and (b) above are available to exogenous NADH, while pathwvay (c) is accessible only to NADH generated within the matrix, i.e. by oxidation of NADlinked substrates.On the other hand, mitochondria from mammalian tissues, such as rat liver, do not oxidize exogenous NADH directly via the inner membrane respiratory chain unless the inner membrane is disrupted (13). In this studyv. we report observations which indicate that mitochondria isolated from fresh beetroot (Beta vulgaris L.) also lack pathway (b) above for the oxidation '

Oxidation of External NAD(P)H by Mitochondria from Taproots and Tissue Cultures of Sugar Beet (Beta vulgaris)

“…However, no substantia1 differences in terms of electron transport rates, sensitivities to inhibitors, and energy coupling were observed between the two genotypes (data not shown). The induction of the NADPH dehydrogenase activity (4.7 times) during in vitro growth of B. vulgaris cells might be analogous to induction by the mechanism previously observed in red beetroots upon wounding (Day et al, 1976;Arron and Edwards, 1979) or cold storage (Fredlund et al, 1991), with the significant difference that in these latter cases, NADH, but not NADPH, oxidation was enhanced. The immediate conclusion derived from the present results is that, at least in mitochondria of B. vulgaris, the oxidation of cytosolic NADH and NADPH (when present) are catalyzed by separate, specific enzymes.…”

“…It is apparent that results in (a) are different from those of Day et al (1976), Soole et al (1990b) with fresh red beetroot mitochondria (cvs Red Globe and Derwent Globe), in which no NADH oxidation . was found; our data are also qualitatively and quantitatively discordant with those of Fredlund et al (1991) with two different cultivars of red beetroots (Rubria and Nina), in which both NADH and NADPH were oxidized, although relatively slowly (12.5 and 18 nmol min-' mg-' protein, respectively).…”

“…The NADH dehydrogenase catalyzing this activity is insensitive to rotenone, is greatly stimulated by Ca2+, and is not protonmotive (Mdler and Lin, 1986). At variance with this general behavior, Day et al (1976), Soole et al (1990b) found no NADH oxidation by mitochondria isolated from fresh red beetroots (Beta vulgaris cvs Red Globe and Derwent Globe). Conversely, mitochondria isolated from beetroot that had been sliced into discs and 'aged" by bubbling in a CaSO, solution were able to oxidize exogenous NADH (Day et al, 1976).…”

“…At variance with this general behavior, Day et al (1976), Soole et al (1990b) found no NADH oxidation by mitochondria isolated from fresh red beetroots (Beta vulgaris cvs Red Globe and Derwent Globe). Conversely, mitochondria isolated from beetroot that had been sliced into discs and 'aged" by bubbling in a CaSO, solution were able to oxidize exogenous NADH (Day et al, 1976). Recently, Fredlund et al (1991) have shown that mitochondria purified from fresh red beetroots of two different cultivars (Rubria and Nina) can oxidize extemal NADH, although relatively slowly (25 nmol O2 min-' mg-' protein, state 3).…”

“…Whereas the specific induction of an exogenous NADH dehydrogenase in cold-stored and aged beetroot tissues is a well-recognized phenomenon (Day et al, 1976;Arron and Edwards, 1979;Rayner and Wiskich, 1983;Fredlund et al, 1991), the physiological role of the extemal NAD(P)H dehydrogenase in fresh tissues remains to be established (Arron and Edwards, 1979;Rayner and Wiskick, 1983;Soole et al, 1990aSoole et al, , 1990bFredlund et al, 1991;Luethy et al, 1991).…”