Regulation of Malate Oxidation in Isolated Mung Bean Mitochondria

Abstract

Effects of ADP and ATP on products of malate oxidation in the presence or absence of respiratory inhibitors and an uncoupler were investigated in mitochondria isolated from mung bean (Phaseolus aureus var. Jumbo) hypocotyls. Changes in levels of products from malate oxidation generally correlated directly with changes in oxygen uptake. Effects of ADP and ATP were indistinguishable from each other when respiratory chain activity was limited. We concluded that adenylates indirectly act on malate oxidation via the oxidation-reduction status of the pyridine nudeotides which are linked to the respiratory chain. The possibility of allosteric action of ADP and ATP on malate dehydrogenase activity was examined in both intact mitochondria and a partially purified enzyme preparation. Although small inhibition, 16% with 500 ,UM ATP and 8% with 500 jAM ADP, was observed at pH 9.5, this effect was abolished by the addition of magnesium ions or by lowering the pH to 7.2. We concluded that these adenylate effects are probably not a significant factor in regulation under physiological conditions. Furthermore, the equilibrium constant of malate dehydrogenase (to 1.5 x 10-5) in both mitochondria and the partially purified enzyme calculated from the steady state level of NADH formed suggested that the enzyme functions in an equilibrium manner in intact mitochondria.Studies of citric acid cycle activity in mitochondria isolated from mung bean hypocotyls led to the conclusion that malate oxidation plays an important role in cycle activity (6). Adenylates are implicated in the control of malate oxidation by the following observations in the literature: (a) ADP and ATP drastically altered the levels of OAA5 produced from oxidation of malate (6) purified from mung bean cotyledon mitochondria (1). On the other hand, some investigators contend that control of oxidation of cycle intermediates does not reside in the cycle itself but is confined to interaction of adenine nucleotides with the respiratory chain (25,27). The concept of respiratory control proposed by Chance and Williams (7) can also be viewed as supporting the latter possibility.This publichation is aimed at resolving how adenylates act in the regulation of malate oxidation in isolated mung bean mitochondria, whether they act directly as effectors of malate dehydrogenase, indirectly via their action on the oxidative-phosphorylation system, or both. MATERIALS AND METHODSPreparation and Incubation of Mitochondria. Mitochondria were prepared as described previously (6) from mung bean hypocotyls by the method of Ikuma (13). The standard reaction medium contained 500 mm mannitol, 10 mm KCI, 5 mM MgCl2, and 10 mm K phosphate buffer, pH 7.2. In studies aimed at evaluating the effect of adenylates on malate oxidation products, mitochondria were incubated with 10 mm malate as substrate, and 02 consumption was followed to a steady state level. During the period of linear 02 uptake, ADP or ATP was added, and the oxidation products were analyzed at two to three timed intervals ...

Effects of Rotenoids on Isolated Plant Mitochondria

“…MDH activity is reversible and, in the mitochondrial matrix, the characteristics of malate oxidation strongly support the evidence that the regulation in vivo of MDH activity can be readily accounted for by equilibrium effect alone (2,20). MDH (2,20).…”

Adenine Nucleotide Regulation of Malate Oxidation in Isolated Mung Bean Hypocotyl Mitochondria

“…In the present study, we observed no stimulation in the rate of malate oxidation when ADP was added after uncoupling the mitochondria. We therefore conclude that the most straightforward explanation for the increase in oxaloacetate and pyruvate production brought about by ADP in coupled mitochondria is that it is primarily due to increased electron transport activity (2).…”

“…Since ADP increases the rate of electron transport and oxygen uptake in coupled mitochondria (3), higher rates of NADH oxidation will take place so that intramitochondrial NADH levels decrease and NAD+ increases (16). The equilibrium position of the malate dehydrogenase reaction is such that oxaloacetate production would be favored when NADH levels are low in the mitochondrial matrix (2,16). Therefore, in coupled mitochondria, ADP can control malate oxidation by regulation ofelectron transport activity.…”

“…Bowman and Ikuma (2) concluded that the increase in pyruvate production and the decreased production of oxaloacetate brought about by ATP in coupled mitochondria could, like the effects of ADP, be due principally to sg/ml oligomycin. The experimental procedure was the same as in Figure 3 except that 0.8 jM FCCP was omitted.…”

“…The results suggest that, whereas the ADP effects may be exerted primarily through the control of electron transport (cf. 2), ATP can strongly regulate malate oxidation by affecting directly the activity of one or both of the enzymes responsible for malate oxidation.…”