scholarly journals Properties of mitochondria isolated from cyanide-sensitive and cyanide-stimulated cultures of Acanthamoeba castellanii

1978 ◽  
Vol 174 (1) ◽  
pp. 203-211 ◽  
Author(s):  
Steven W. Edwards ◽  
David Lloyd
Keyword(s):  
Electron Transport ◽  
Respiratory Control ◽  
Acanthamoeba Castellanii ◽  
Type A ◽  
Antimycin A ◽  
Type B ◽  
Transport Pathway ◽  
Salicylhydroxamic Acid ◽  
Stages Of Growth ◽  
Electron Transport Pathway

1. Mitochondria isolated from cultures of Acanthamoeba castellanii exhibit respiratory control and oxidize α-oxoglutarate, succinate and NADH with ADP:O ratios of about 2.4, 1.4 and 1.25 respectively. 2. Mitochondria from cultures of which the respiration was stimulated up to 50% by 1mm-cyanide (type-A mitochondria) and from cyanide-sensitive cultures (type-B mitochondria) had similar respiratory-control ratios and ADP:O ratios. 3. State-3 rates of respiration were generally more cyanide-sensitive than State-4 rates, and the respiration of type-A mitochondria was more cyanide-resistant than that of type-B mitochondria. 4. Salicylhydroxamic acid alone had little effect on respiratory activities of either type of mitochondria, but when added together with cyanide, irrespective of the order of addition, inhibition was almost complete. 5. Oxidation of externally added NADH by type-A mitochondria was mainly via an oxidase with a low affinity for oxygen (Km[unk]15μm), which was largely cyanide-sensitive and partially antimycin A-sensitive; this electron-transport pathway was inhibited by ADP. 6. Cyanide-insensitive but salicylhydroxamic acid-sensitive respiration was stimulated by AMP and ADP, and by ATP after incubation in the presence of MgCl2. 7. Addition of rotenone to mitochondria oxidizing α-oxoglutarate lowered the ADP:O ratios by about one-third and rendered inhibition by cyanide more complete. 8. The results suggest that mitochondria of A. castellanii possess branched pathways of electron transport which terminate in three separate oxidases; the proportions of electron fluxes via these pathways vary at different stages of growth.

scholarly journals Hydrogen peroxide production in uncoupled mitochondria of the parasitic nematode worm Nippostrongylus brasiliensis

1987 ◽  
Vol 243 (2) ◽  
pp. 589-595 ◽  
Author(s):  
T A Paget ◽  
M Fry ◽  
D Lloyd
Keyword(s):  
Electron Transport ◽  
Parasitic Nematode ◽  
H2o2 Production ◽  
Nippostrongylus Brasiliensis ◽  
Antimycin A ◽  
Transport Pathway ◽  
Salicylhydroxamic Acid ◽  
Low Concentrations ◽  
Carbonyl Cyanide ◽  
Nematode Worm

1. Mitochondria from the parasitic nematode worm Nippostrongylus brasiliensis produce H2O2 in the energized state; higher rates of H2O2 production were observed in the presence of the uncoupler carbonyl cyanide m-chlorophenylhydrazone. 2. Antimycin A inhibits respiration and H2O2 production by 70 and 65% respectively; the residual activities can be attributed to alternative electron-transport pathway(s). 3. o-Hydroxydiphenyl and 1,3,5-trihydroxybenzene, inhibitors of alternative electron transport, inhibit respiration by 37% and H2O2 production by 26%. 4. Another inhibitor of alternative electron transport, salicylhydroxamic acid, shows a complex mode of action; low concentrations (less than 0.5 mM) stimulate respiration and H2O2 production, whereas 2 mM-salicylhydroxamic acid inhibited respiration by 35% and stopped H2O2 production completely. 5. O2 thresholds were observed for the inhibition of respiration at O2 concentrations greater than 57.7 microM and inhibition of H2O2 production (greater than 20.5 microM-O2); apparent Km values for oxygen were 5.5 microM and 3.0 microM respectively. 6. In the presence of antimycin A the O2-inhibition thresholds and apparent Km values for O2 of respiration and H2O2 production matched closely, suggesting that the alternative oxidase is a likely site of H2O2 production. 7. These results are discussed in relation to O2 toxicity to N. brasiliensis.

scholarly journals Pyruvate metabolism in castor-bean mitochondria

1986 ◽  
Vol 239 (2) ◽  
pp. 355-361 ◽  
Author(s):  
M A Brailsford ◽  
A G Thompson ◽  
N Kaderbhai ◽  
R B Beechey
Keyword(s):  
Electron Transport ◽  
Castor Bean ◽  
Silicone Oil ◽  
Ricinus Communis ◽  
Mitochondrial Protein ◽  
Respiratory Control ◽  
O2 Uptake ◽  
Salicylhydroxamic Acid ◽  
Oxidation Rates ◽  
Pyruvate Oxidation

We report the isolation of mitochondria from the endosperm of castor beans (Ricinus communis). These mitochondria oxidized succinate, external NADH, malate and pyruvate with respiratory-control and ADP/O ratios consistent with those found previously with mitochondria from other plant sources. The mitochondria exhibited considerable sensitivity to the electron-transport-chain inhibitors antimycin A and cyanide when oxidizing succinate and external NADH. Pyruvate-dependent O2 uptake was relatively insensitive to these inhibitors, although the residual O2 uptake could be inhibited by salicylhydroxamic acid. We conclude that a cyanide-insensitive alternative terminal oxidase is functional in these mitochondria. However, electrons from the succinate dehydrogenase or external NADH dehydrogenase seem to have no access to this pathway. There is little interconnection between the salicylhydroxamic acid-sensitive and cyanide-sensitive pathways of electron transport. alpha-Cyanocinnamate and its analogues, compound UK5099 [alpha-cyano-beta-(1-phenylindol-3-yl)acrylate] and alpha-cyano-4-hydroxycinnamate, were all found to be potent non-competitive inhibitors of pyruvate oxidation in castor-bean mitochondria. The accumulation of pyruvate by castor-bean mitochondria was determined by using a silicone-oil-centrifugation technique. The accumulation was shown to observe Michaelis-Menten kinetics, with a Km for pyruvate of 0.10 mM and a Vmax. of 0.95 nmol/min per mg of mitochondrial protein. However, the observed rates of pyruvate accumulation were insufficient to account for the pyruvate oxidation rates found in the oxygen-electrode studies. We were able to demonstrate that this is due to the immediate export of the accumulated radiolabel in the form of malate and citrate. Compound UK5099 inhibited the accumulation of [2-14C]pyruvate by castor-bean mitochondria at concentrations similar to those required to inhibit pyruvate oxidation.

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