The Effect of Respiratory Inhibitors on NADH, Succinate and Malate Oxidation in Corn Mitochondria

Exogenous NADH oxidation by cauliflower (Brassica oleracea L.) bud mitochondria was sensitive to antimycin A and gave ADP/O ratios of 1.4 to 1.9. In intact mitochondria, NADHcytochrome Previous studies with plant mitochondria (4,11,13,14) have indicated the presence of two NAD+-linked enzymes involved in the oxidation of malate; viz. malate dehydrogenase and "malic" enzyme. Plant mitochondria also appear to possess an alternative system for the oxidation of exogenous NADH.The oxidation involves an NADH dehydrogenase, apparently situated on the outside of the inner membrane (8,19), which is linked to the respiratory chain and coupled to two sites of phosphorylation (5,9,15,19,24 were carried out at about 2 C, using chilled solutions and apparatus.

“…3). These results support those of others (8,15,19,26) (Fig. 3A); Cyt c alone had no such effect on the inhibited malate oxidation (Fig.…”

Section: Methodssupporting

confidence: 82%

supporting

confidence: 77%

See 1 more Smart Citation

The Oxidation of Malate and Exogenous Reduced Nicotinamide Adenine Dinucleotide by Isolated Plant Mitochondria

Day¹,

Wiskich²

1974

“…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.…”

mentioning

confidence: 99%

Characteristics of External NADH Oxidation by Beetroot Mitochondria

Day¹,

Rayner²,

Wiskich³

1976

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 '

“…Table III shows that the promotion of ADP accumulation occurs with both AdN translocase inhibitors, making it unlikely that a side effect of C-ATR is responsible. In the course of other investigations we have found that rotenone will promote the phosphate swelling and 32Pi uptake energized by NADH oxidation (oxidation of exogenous NADH by corn mitochondria is not inhibited by rotenone, and can even be promoted slightly [21]). Rotenone also proves to promote ADP accumulation over the level of the oligomycin control (Table III) The rate of AMP and ATP exchange was partially inhibited by C-ATR, but not that of ADP (Fig.…”

Section: Methodsmentioning

confidence: 99%

Energy-linked Adenosine Diphosphate Accumulation by Corn Mitochondria

Abou‐Khalil

Hanson²

1979

There must exist a mechanism by which mitochondria gain the AdN2 which they contain, but this mechanism is still unknown. A recent report on AdN accumulation during the ontogeny of rat liver mitochondria indicates that the mechanism does not involve the well studied ADP/ATP exchanging translocator (13). However, Vignais (19) MATERIALS AND METHODSThe isolation of corn mitochondria was as previously described (1). Reactions were carried out at room temperature in 0.2 M sucrose, I mg/ml BSA, 20 mm Tes, 20 mm KCl, and 4 mm MgC12, adjusted to pH 7.5 with KOH. Additions to this standard medium (SBTKM) are given with the data.Millipore filtration procedures for determining uptake of labeled AdN were as described (1). It was important to presoak the filters in unlabeled 2 mm AdN to reduce the radioactivity absorbed by the filter from the labeled reaction solutions. Reaction solution blanks (no mitochondria) were used to determine the radioactivity retained by the filter.The adenine nucleotide content of mitochondria was determined by using the luciferine-luciferase enzyme system (1 1). For this purpose, mitochondria were incubated in the SBTKM medium in the presence of 5 mm Pi, NADH (2 ,umol/mg protein), 2 mM ADP, and in the presence or the absence of oligomycin or C-ATR. The silicone oil method (6) was used with some modification to separate the mitochondria from their media. Aliquots of mitochondria were carefully layered (in a 1.5-ml centrifuge tube) over 0.5 ml of silicone fluid, specific gravity 1.04, which in turn had been layered over 0.33 ml of 7% HC104. At 8 min the mitochondria were rapidly separated through the silicone fluid using an Eppendorf 5412 centrifuge. The supernatant and the oil were removed, and the HC104 extract was transferred to another tube with a Hamilton syringe. ATP content was determined with an Aminco Chem-Glow photometer. ADP and AMP were determined by converting these forms to ATP using pyruvate kinase and adenylate kinase and then measuring the ATP formed. Standard amounts of ATP, ADP, and AMP were treated in the same manner. Luciferine-luciferase solution was prepared from buffered firefly extract (Sigma). (Fig. 1) accumulation than oligomycin, and when both oligomycin and C-ATR are present, the level of accumulated ADP is the same as with C-ATR alone (Fig. 1). Without these inhibitors (i.e. state 3 conditions) there is uptake of about 8 nmol ADP/mg protein in 4 min. In the absence of NADH and in the presence of oligomycin to block ATPase activity there is a corresponding level of uptake.Endogenous respiration is scarcely detectable at pH 7.5 (8) but it must contribute to uptake under these conditions since addition of 1.25 ug antimycin A/mg protein reduces the uptake by 20 to 25% (data not shown). The bulk of the uptake in the absence of an exogenous energy supply is indicated to be by a passive mechanism. Succinate or malate plus pyruvate will effectively substitute for NADH in energy-linked [3HJADP uptake (data not shown), but organic acids were not routinely used as sub...