The Effect of Calcium and Inhibitors on Corn Mitochondrial Respirationt

The effects of KCN, antimycin A, malonate, rotenone, and amytal on the oxidation of malate, succinate, and extramitochondrial reduced nicotinamide adenosine dinucleotide (NADH) by corn mitochondria were studied. Potassium cyanide and antimycin A inhibited the oxidation of all three substrates. Rotenone and amytal inhibited only the oxidation of malate, and malonate inhibited only the oxidation of succinate. Rotenone, amytal, and malonate did not inhibit the oxidation of extramitochondrial NADH. The calcium stimulation of the oxidation of extramitochondrial NADH was prevented by KCN and antimycin A but not by amytal, rotenone, or malonate. It is suggested that corn mitochondria possess a flavoprotein specific for extramitochondrial NADH and that this flavoprotein is sensitive to divalent cations. Divalent cations appear to affect the respiration of isolated corn mitochondria in two ways (10). First, divalent cations stimulate the oxidation of extramitochondrial NADH, a stimulation not found when corn mitochondria are oxidizing the Krebs cycle intermediates malate or succinate. The second divalent cation effect is the promotion of respiration in the presence of phosphate by Ca2+, Ba2+, and Sr2+ which is the result of phosphate transport and occurs when extramitochondrial NADH, malate, or succinate is being oxidized. The cause of the divalent cation stimulation of the oxidation of extramitochondrial NADH is not known. Hackett (6) has suggested that it may be due to increased membrane permeability or to the release of a rate-limiting step in the respiratory chain. Since the effect is not found with substrates other than NADH, the permeability effect is unlikely. If there is a flavoprotein for the oxidation of extramitochondrial NADH, as proposed by Bonner (1), and Storey (16), and if it is divalent cation-sensitive, it could account for the cation stimulation of the oxidation of extramitochondrial NADH. Such a divalent cation requirement has been suggested by Miller et al. (10). But the divalent cation effects could also be explained if an enzyme, such as the DT diaphorase (4, 11), was present to catalyze the oxidation of extramitochondrial NADH. Detailed spectrophotometric studies of the electron transport chain of animal mitochondria have been conducted. Similar 1 Supported in part by a grant from the Research Board, University of Illinois, and by funds from the Illinois Agriculture Experiment Station. studies have recently been performed with mung bean and skunk cabbage mitochondria (12-18), clarifying the characteristics of the cytochromes of the electron transport chain. Various inhibitors of electron transport have also been used to help elucidate the electron transport chain components of animal mitochondria (2, 5), and to a limited extent those of plant mitochondria (7, 18, 19). But inhibitors have not been used to study systematically the oxidation of extramitochondrial NADH by plant mitochondria. Three substrates-malate, succinate, and NADH-and the inhibitors KCN, antimycin A, malonate, rotenone, and amytal were used to characterize further the electron transport of corn mitochondria as it pertains specifically to extramitochondrial NADH oxidation. The experiments were not designed to study all of the effects of the inhibitors. The results of these experiments, plus the divalent cation effects, suggest that there is a specific flavoprotein for the oxidation of extramitochondrial NADH in corn mitochondria.