Preparation and some properties of submitochondrial particles from tightly coupled mung bean mitochondria

Abstract

Osmotic shock was found to be better than freezing and thawing, a French press, or sonic oscillation for the preparation of submitochondrial particles from mung bean (Phaseolus aureus) hypocotyl mitochondria. Particles prepared by osmotic shock rapidly oxidize reduced nicotinamide adenine dinucleotide and succinate, but they oxidize malate slowly. NADH oxidation was slightly stimulated by cytochrome c, ATP, and ADP; succinate oxidation was markedly increased by ATP, slightly by ADP and cytochrome c; and malate oxidation required the addition of NAD(+) NADH oxidation is inhibited weakly by amytal, completely by antimycin A and KCN, but not by rotenone. Chlorsuccinate, malonate, antimycin A, and KCN inhibit succinate oxidation. The action of antimycin A and KCN is incomplete, while chlorsuccinate and malonate were competitive inhibitors. Antimycin A combined stoichiometrically with particle protein in the ratio of 0.23 millimicromole per milligram of protein.Oligomycin and bis(hexafluoroacetonitryl) acetone, a potent uncoupler of oxidative phosphorylation, were without effect on oxygen uptake but did influence the ATP-stimulated onset of respiration when succinate was substrate. Fresh particles were markedly inhibited by oxtylguanidine, indicating energy conservation, but this inhibition decreased on storage of the particles.Spectra show the presence of cytochrome components the same as those of the intact mung bean mitochondrion, but present at higher concentrations. The molar concentrations of the particle cytochromes were two to three times those of the intact mitochondrion and the molar ratios were calculated as 0.9:1.0:1.0:2.8 for cytochromes a:b:c:flavoprotein, respectively.