Comparative study of some parameters of energy metabolism in two strains of Saccharomyces cerevisiae

A comparative study of energy metabolism in two strains Saccharomyces cerevisiae (the initial strain N 73 and laser-irradiated mutant strain Y-503) was performed. In all growth phases, the rates of oxygen consumption by cells of Y-503 were higher than in the initial strain. The maximum (threefold) increase in the rate of oxygen consumption was observed in the linear phase. The effects of respiratory chain inhibitors rotenone, antimycin A, and cyanide on cellular and mitochondrial respiration were identical. There are two sites of energy coupling in the respiratory chain of mitochondria in S. cerevisiae N 73 and Y-503, and electron flow mainly is mainly mediated by cytochrome oxidase. The data suggest that a higher respiratory activity of S. cerevisiae Y-503 cells in comparison with N 73 is associated with greater amounts of mitochondria and total surface area of coupling mitochondrial membranes, which appears to be a factor contributing to a high physiological and biochemical activity of this strain.     

Effects of uncoupling of mitochondrial energy conservation on the ultradian clock-driven oscillations in Saccharomyces cerevisiae continuous culture

Protonophores have several different perturbative effects on dissolved O2 concentrations in continuous cultures of Saccharomyces cerevisiae. As well as uncoupling energy conservation from mitochondrial electron transport in vivo, they reset ultradian clock-driven respiratory oscillations and produce cell cycle effects. Thus, additions at low concentration (1.25 microM) of either m-chlorocarbonyl-cyanide phenylhydrazone (CCCP) or 5-chloro-3-t-butyl-2-chloro-4(1)-nitrosalicylanilide (S13) led to phase resetting of the 48 min ultradian clock-driven respiratory oscillations. At 2.5 microM CCCP or 4 microM S13, transient inhibition of oscillatory respiration (for 5 h) preceded synchronisation of the cell division cycle seen as a slow (9 h period) wave that enveloped the 48 min oscillation. At still higher concentrations of CCCP (5 microM), the cell division cycle was prolonged by about 7 h, and during this phase, the respiratory oscillation became undetectable. The significance of these observations with respect to the time-keeping functions of the ultradian clock is discussed.     

Some Specific Features of the Respiration of Hydrocarbon-utilizable Candida Yeasts

The respiration of both glucose-grown and hydrocarbon-grown cells of Candida tropicalis pK 233 harvested in the stationary phases was not inhibited by cyanide when glucose was used as oxidation substrate, but the former was rather stimulated in the presence of cyanide. When n-alkanes were used as oxidation substrate, cyanide lowered the respiratory activities of both cells to about 50%. With respect to the susceptibility to cyanide, the younger cells growing on n-alkanes were less sensitive in hydrocarbon oxidizing ability than the older cells, whereas the older cells growing on glucose or n-alkanes were more resistant in glucose oxidizing ability than the younger cells. Acetate was oxidized by both glucose-grown and hydrocarbon-grown cells of the yeast. Laurate was oxidized by hydrocarbon-grown cells, but not by glucose-grown cells. The respiration on laurate was inhibited completely by 3.3 mM of cyanide. In general, hydrocarbon-grown cells of Candida tropicalis pK 233 were more sensitive to various respiratory inhibitors than glucose-grown cells, although the oxidation substrates had a significant effect.

The respiration of both glucose-grown and hydrocarbon-grown cells of C. albicans, C. guilliermondii and C. lipolytica harvested in the stationary phases was also resistant to cyanide when glucose was used as oxidation substrate. But the respiration on n-alkanes of these cells was inhibited significantly by 3.3 mM of cyanide except for C. albicans.     

The enzymic interconversion of acetate and acetyl-coenzyme A in Escherichia coli.

Mutants of Escherichia coli K12 have been isolated that grow on media containing pyruvate of proline as sole carbon sources despite the presence of 10 or 50 mM-sodium fluoroacetate. Such mutants lack either acetate kinase [ATP: acetate phosphotransferase; EC 2.7.2.1] or phosphotransacetylase [acetyl-CoA: orthophosphate acetyltransferase; EC 2.3.1.8] activity. Unlike wild-type E. coli, phosphotransacetylase mutants do not excrete acetate when growing aerobically or anaerobically on glucose; their anaerobic growth on this sugar is slow. The genes that specify acetate kinase (ack) and phosphotransacetylase (pta) activities are cotransducible with each other and with purF and are thus located at about min 50 on the E. coli linkage map. Although Pta- and Ack- mutants are greatly impaired in their growth on acetate, they incorporate [2-14C]acetate added to cultures growing on glycerol, but not on glucose. An inducible acetyl-CoA synthetase [acetate: CoA ligase (AMP-forming); EC 6.2.1.1] effects this uptake of acetate.     

Growth and respiration of Petunia hybrida cells in chemostat cultures: A comparison of glucose-limited and nitrate-limited cultures

Nitrate-limited and glucose-limited chemostat cultures of Petunia hybrida cells were compared at a specific biomass (+extracellular product) formation rate of 0.0042 C.mol/C.mol h. The composition of the biomass differed considerably in both culture types. The N/C (mol/mol) ratio in the biomass was almost four times lower in the nitrate-limited than in the glucose-limited cultures. On a dry weight basis (g/g DW) the biomass in the nitrate-limited cultures contained about 2.5 times less ions and protein N and about 2.5 times more carbohydrates than the biomass in the glucose-limited cultures. On a fresh weight basis (mmol/g FW) the biomass in nitrate-limited and glucose-limited cultures differed mainly in carbohydrate content. The yields of biomass on glucose and oxygen were generally higher in the nitrate-limited than in the glucose-limited cultures. Average values for these parameters were 0.27 C . mol biomass/C . mol glucose and 0.42 C . mol biomass/mol O(2) in the glucose-limited cultures and 0.34 C . mol biomass/C . mol glucose and 0.55 C . mol biomass/mol O(2) in the nitrate-limited cultures. On a C . mol basis the total respiration was about 25% and the maximally attainable cytochrome pathway activity (measured in the presence of hydroxamate) about 30% higher in the glucose-limited than in the nitrate-limited cultures. The maximally attainable activity of the alternative pathway (measured in the presence of KCN) was significantly lower in the glucose-limited cultures. On an organic N ( approximately protein) basis all respiratory parameters were significantly higher in the nitrate-limited cultures. In the presence of the respiratory uncoupler carbonyl cyanide p-trifluoromethoxy phenylhydrazone (FCCP) and excess glucose, cellular respiratory activity shows its maximal activity; under these conditions the total respiration increased more than 150% in the glucose-limited and only 30% in the nitrate-limited cultures. It is suggested that glucose-limited cultures are able to react more flexibly to changes in the environmental conditions than nitrate-limited cultures. (c) 1996 John Wiley & Sons, Inc.     

The effect of phosphate deficiency on mitochondrial activity and adenylate levels in bean roots

Bean plants ( Phaseolus vulgaris ) were grown for 16–20 days with or without phosphate in Knop nutrient medium. It was found in previous experiments that for roots grown on a P_i-deficient medium respiration is mainly carried out by the cyanide-insensitive pathway. Mitochondria isolated from—P_i, roots had poor respiratory control and their respiration exhibited 62% inhibition by cyanide and was inhibited (30%) by salicylhydroxamic acid (SHAM). In contrast, mitochondria obtained with control (+P_i) roots had respiratory control and ADP/O ratios typical for succinate as the substrate; their respiration was inhibited to 95% by cyanide and insensitive to SHAM. The integrity of mitochondrial membranes was similar in both types of mitochondria. Cytochrome oxidase activity, however, was about 20% lower in -P_i mitochondria, but the cytochrome composition was the same in both types of mitochondria. The cytochrorae pathway was not operating at full capacity in mitochondria isolated from—P_i roots but the alternative oxidation pathway participated in a great part in mitochondrial respiration, similar to in vivo whole roots. The participation of the non-phosphorylating., alternative pathway decreased the respiratory control ratio in mitochondria and had an effect on the total adenine nucleotide pool and energy charge values which were lower (16 and 13% respectively) in -P_i roots. About 50% lower ADP and 20% lower ATP levels were observed whereas AMP levels were several times higher.     

Pathways of glucose catabolism in procyclic Trypanosoma congolense

Studies of respiration on glucose in procyclic Trypanosoma congolense in the presence of rotenone, antimycin, cyanide, salicylhydroxamic acid and malonate have indicated the presence of NADH dehydrogenase, cytochrome b-c1, cytochrome aa3, trypanosome alternate oxidase and NADH fumarate reductase/succinate dehydrogenase pathway that contributes electrons to coenzyme Q of the respiratory chain. The rotenone sensitive NADH dehydrogenase, the trypanosome alternate oxidase, and cytochrome aa3 accounted for 24.5 +/- 6.5, 36.2 +/- 4.2 and 54.1 +/- 5.5% respectively of the total respiration. Activities of lactate dehydrogenase, NAD(+)-linked malic enzyme and pyruvate kinase were less than 6 nanomoles/min/mg protein suggesting that they play a minor role in energy metabolism of the parasite. Phosphoenolpyruvate carboxykinase, pyruvate dehydrogenase, succinate dehydrogenase, NADP(+)-linked malic enzyme, NADH fumarate reductase, malate dehydrogenase, and alpha-ketoglutarate dehydrogenase and glycerol kinase on the other hand had specific activities greater than 60 nanomoles/min/mg protein. These enzyme activities could account for the production of pyruvate, acetate, succinate and glycerol. The results further show that the amount of glycerol produced was 35-48% of the combined total of pyruvate, acetate and succinate produced. It is apparent that some of the glycerol 3-phosphate produced in glycolysis in the presence of salicylhydroxamic acid is dephosphorylated to form glycerol while the rest is oxidised via cytochrome aa3 to form acetate, succinate and pyruvate.     

The respiratory chain of Paramecium tetraurelia in wild type and the mutant Cl1. II. Cyanide-insensitive respiration. Function and regulation.

1. The cyanide-insensitive respiration in Paramecium tetraurelia was found to be located in mitochondria. 2. Sensitivity of the mitochondrial respiration to cyanide depended on growth conditions. Under standard conditions of growth, 15--20% of respiration was insensitive to 1 mM cyanide. Full resistance to 1 mM cyanide was observed by growing cells in the presence of erythromycin (100--400 microgram/ml) 0.2 mM cyanide. The mitochondrial respiration of the mutant Cl1 harvested during the exponential phase of growth was largely insensitive to cyanide (more than 80%). 3. Pyruvate was oxidized at the same rate by wild type mitochondria and mitochondria of the mutant Cl1. In contrast, succinate oxidation was 2--3 times faster in mitochondria of the mutant Cl1 than in wild type mitochondria. 4. The cyanide-insensitive respiration was inhibited by 1 mM salicylhydroxamic acid to nearly 100%. Other efficient respiratory inhibitors included amytal and heptylhydroxyquinoline. Antimycin was not inhibitory even at concentrations as high as 5 microgram/mg protein, a finding consistent with the lack of antimycin binding sites.     

Role of growth phase and ethanol in freeze-thaw stress resistance of Saccharomyces cerevisiae.

The freeze-thaw tolerance of Saccharomyces cerevisiae was examined throughout growth in aerobic batch culture. Minimum tolerance to rapid freezing (immersion in liquid nitrogen; cooling rate, approximately 200 degrees C min-1) was associated with respirofermentative (exponential) growth on glucose. However, maximum tolerance occurred not during the stationary phase but during active respiratory growth on ethanol accumulated during respirofermentative growth on glucose. The peak in tolerance occurred several hours after entry into the respiratory growth phase and did not correspond to a transient accumulation of trehalose which occurred at the point of glucose exhaustion. Substitution of ethanol with other carbon sources which permit high levels of respiration (acetate and galactose) also induced high freeze-thaw tolerance, and the peak did not occur in cells shifted directly from fermentative growth to starvation conditions or in two respiratorily incompetent mutants. These results imply a direct link with respiration, rather than exhaustion of glucose. The role of ethanol as a cryoprotectant per se was also investigated, and under conditions of rapid freezing (cooling rate, approximately 200 degrees C min-1), ethanol demonstrated a significant cryoprotective effect. Under the same freezing conditions, glycerol had little effect at high concentrations and acted as a cryosensitizer at low concentrations. Conversely, under slow-freezing conditions (step freezing at -20, -70, and then -196 degrees C; initial cooling rate, approximately 3 degrees C min-1), glycerol acted as a cryoprotectant while ethanol lost this ability. Ethanol may thus have two effects on the cryotolerance of baker's yeast, as a respirable carbon source and as a cryoprotectant under rapid-freezing conditions.     

Respiratory metabolism of a "petite negative"yeast Schizosaccharomyces pombe 972h-

The respiratory metabolism of Schizosaccharomyces pombe 972h(-), a fission, haplontic, "petite negative" yeast, was studied. Glucose and glycerol are good growth substrates and are oxidized under appropriate conditions. l-Lactate, ethanol, malate, and succinate are oxidized but are poor substrates for growth. d-Lactate and pyruvate are neither oxidized nor used for growth. Limited growth was observed under anaerobic conditions. The addition of 0.3% KNO(3) to a rich medium relieves the oxygen requirement. A continuous increase of cell respiration during growth on repressive concentration of glucose was observed, suggesting the presence of glucose repression of respiration. Reduced nicotinamide adenine dinucleotide (NADH), succinate, alpha-glycerophosphate, and ascorbate plus tetramethyl-p-phenylenediamine are oxidized by a mitochondrial fraction. NADH and succinate oxidations are inhibited by antimycin A and NaCN but not by rotenone, suggesting the absence of the phosphorylation site I and the presence of sites II and III. The effects of several mitochondrial inhibitors on growth and respiration indicate that the requirement of an oxidant for growth is related neither to the functioning of the respiratory electron transport chain nor to the formation of respiratory energy. The previously suggested correlations between the nonviability of vegetative "petites" mutants, the absence of repression of respiration by glucose, and the incapacity to grow under anaerobic conditions are thus not strictly valid for S. pombe.     

Studies on the respiratory system of Aspergillus oryzae. V. Some properties of the respiratory system of mitochondria from mycelia grown in the presence of chloramphenicol.

Presence of chloramphenicol in the growth medium for mycelia of Aspergillus oryzae was without effect on the oxidative activity, respiratory control, or P/O ratio of isolated mitochondria. The mitochondria oxidized Krebs cycle intermediates even in the presence of cyanide at the concentration markedly inhibiting the normal mitochondrial oxidation. However, the P/O ratio during the mitochondrial oxidation decreased by about 1.0 on addition of cyanide. The c-type cytochromes, shown to occur in large amounts than in normal mitochondria (Wakiyama and Ogura, 1972), were suggested to act as electron carriers in this cyanide-resistant oxidation. A novel pigment, demonstrated only in the mitochondria prepared from chloramphenicol-treated mycelia by a CO-difference spectrum, was presumed to be the terminal oxidase of the respiration in the presence of cyanide.     

Uptake and respiration of organic compounds and heterotrophic growth in Pediastrum duplex (Meyen)*

SUMMARY. Axenic cultures of Pediastrum duplex, a green alga prominent in Lake Kinneret, Israel, assimilated and respired amino acids, acetate, glucose, glycollate and glycerol under conditions of light or darkness. Increased rates of uptake and respiration were observed in nutrient (P and N) depleted medium. Although ineffective in moderate light (30–100 μ Einstein m^?2 s^?1) glycerol, glucose and leucine, but not glycollate, stimulated growth and yields under faint light (～ 2 μ Einstein m^?2 s^?1). In the dark, glycerol (and sometimes leucine) permitted growth. Kinetic studies with leucine indicated an active uptake mechanism effective at substrate concentrations from 0.5 to 47 μg 1^?1.     

Selective Inhibition of Enzyme Synthesis Under Conditions of Respiratory Inhibition

When Neurospora mycelium is transferred from a medium containing sucrose to one containing acetate as sole source of carbon, a preferential synthesis of many Krebs cycle, glyoxylate cycle, and associated enzymes occurs. Respiration was inhibited during preferential enzyme synthesis in the following ways. (i) The amount of aeration (shaking) was reduced, (ii) cyanide was added to the culture, (iii) the carbon source, acetate, was removed, (iv) a mutant strain was starved of its Krebs cycle intermediates, and (v) respiration was inhibited by mutation. The effect of this respiratory inhibition on the synthesis of a number of enzymes was measured. It was found that the synthesis of nicotinamide adenine dinucleotide (NAD)-linked glutamate dehydrogenase and phosphoenolpyruvate carboxykinase was significantly less inhibited under conditions of respiratory inhibition than was the synthesis of Krebs cycle, glyoxylate cycle, and most other cell proteins synthesized during the adaptation period. This differential inhibition of enzyme synthesis was almost certainly not due to differential repression by regulatory metabolic end product effectors. Inhibition of mitochondrial respiration under these conditions most likely results in a limitation of the energy supply of the cell. Thus, it is suggested that the inhibition of synthesis of most proteins after inhibition of mitochondrial respiration results from a lack of energy in a utilizable form. Possible reasons to account for the relative insensitivity of NAD-linked glutamate dehydrogenase and phosphoenolpyruvate carboxykinase to inhibition under these conditions are discussed.     

The regulation of carbohydrate metabolism in Klebsiella aerogenes NCTC 418 organisms,growing in chemostat culture

Klebsiella aerogenes NCTC 418 was grown in chemostat cultures (D=0.17 hr^-1; pH 6.8; 35° C) that were, successively, carbon-, sulphate-, ammonia-, and phosphate-limited, and which contained as the sole carbon-substrate first glucose, then glycerol, mannitol and lactate. Quantitative analyses of carbon-substrate used and products formed allowed carbon balances to be constructed and direct comparisons to be made of the effciency of substrate utilization. With all sixteen cultures, carbon recoveries of better than 90% were obtained.Optimum utilization of the carbon substrate was invariably found with the carbon-limited cultures, the sole products being organisms and carbon dioxide. But the extent to which excess substrate was over-utilized varied markedly with both the nature of the growth-limitation and the identity of the carbon-substrate. In general, sulphate-, ammonia-, and phosphate-limited cultures utilized glycerol more efficiently than mannitol, mannitol better than lactate, and glucose least efficiently. Glucose-containing cultures also synthesized some extracellular polysaccharide.When the carbon source was in excess, a range of acidic compounds generally were excreted. Sulphate-limited cultures, growing on glucose, excreted much pyruvate and acetate, whereas similarly-limited cultures growing on glycerol, mannitol or lactate produced only acetate. Ammonialimited cultures invariably excreted 2-oxoglutarate and acetate, whereas phosphate-limited cultures produced gluconic acid, 2-ketogluconic acid and acetate, when growing on glucose, but only acetate when growing on mannitol or lactate.From the rates of substrate and oxygen consumption, and the rates of cell synthesis, yield values for both substrate and oxygen were calculated. These showed different trends, but were similar in being highest under carbon-limitation and substantially lower under all other limitations.The physiological significance of these findings, and the probable nature of the regulatory mechanisms underlying overflow metabolism are discussed.     

Respiratory Shifts in Developing Petals of Saxifraga cernua

Changes in the oxygen uptake of petal slices by the cytochrome and alternative respiratory pathways were monitored during petal development in the arctic herb Saxifraga cernua. As the petals developed, rates of total respiration increased to a maximum rate during petal unfolding (day 4.5), and thereafter declined. Respiration in petals of all ages was at least partially resistant to cyanide, indicating the capacity for the alternative pathway. In all, except day 1 and senescing day 8 petals, respiration was inhibited by salicylhydroxamic acid, indicating engagement of the alternative pathway. In general, temporal changes in the respiratory activity along each pathway were similar and in parallel with changes in total respiration, although maximum rates along each pathway occurred at different times. Maximum cytochrome pathway activity occurred during petal expansion (day 4) whereas the alternative pathway peaked during petal unfolding at day 4.5. The control of respiration was also investigated. In the presence of salicylhydroxamic acid, the addition of the uncoupler carbonyl cyanide m-chlorophenylhydrazone was never stimulatory, suggesting that the cytochrome pathway was not restricted by adenylate levels. The addition of sucrose stimulated respiration only in day 1 petals, suggesting substrate limitation at this developmental stage. Since the rate of alternative pathway respiration peaked during petal unfolding, a time of high energy requirement, we suggest that the alternative pathway may have been used as an inefficient energy source during petal development.     

The influence of organic substrates and inhibitors on the induction of a respiratory oscillation in the cyanobacterium Anacystis nidulans

The rate of respiratory oxygen uptake of the cyanobacterium (blue-green alga) Anacystis nidulans oscillated under certain physiological conditions after light pulses or after addition of sodium acetate. The oscillation started either by the photosynthetic inhibition of respiration or by the stimulation of oxygen uptake caused by sodium acetate. The photosynthetic inhibition of respiration decreased the rate of oxygen uptake to about 20% of the rate in the dark.Starved cells (48 h dark) had lost the inducibility of the oscillation. In starved as well as in non-starved cells oscillations were inducible in the presence of fructose or glucose. Well developed oscillations were not promoted further. All other substances tested as substrates did not restore the inducibility of the oscillation in starved cells. The induction of the oscillation was inhibited by iodoacetamide (0.1 mM), p-hydroxymercuribenzoate (0.1 mM) and sodium fluoride (100 mM). It is suggested that a flow from glyceraldehyde-3-phosphate to the incomplete tricarboxylic acid cycle is a prerequisite for the oscillation.     

Physiological and genetic engineering of cytosolic redox metabolism in Saccharomyces cerevisiae for improved glycerol production

Previous metabolic engineering strategies for improving glycerol production by Saccharomyces cerevisiae were constrained to a maximum theoretical glycerol yield of 1 mol.(molglucose)(-1) due to the introduction of rigid carbon, ATP or redox stoichiometries. In the present study, we sought to circumvent these constraints by (i) maintaining flexibility at fructose-1,6-bisphosphatase and triosephosphate isomerase, while (ii) eliminating reactions that compete with glycerol formation for cytosolic NADH and (iii) enabling oxidative catabolism within the mitochondrial matrix. In aerobic, glucose-grown batch cultures a S. cerevisiae strain, in which the pyruvate decarboxylases the external NADH dehydrogenases and the respiratory chain-linked glycerol-3-phosphate dehydrogenase were deleted for this purpose, produced glycerol at a yield of 0.90 mol.(molglucose)(-1). In aerobic glucose-limited chemostat cultures, the glycerol yield was ca. 25% lower, suggesting the involvement of an alternative glucose-sensitive mechanism for oxidation of cytosolic NADH. Nevertheless, in vivo generation of additional cytosolic NADH by co-feeding of formate to aerobic, glucose-limited chemostat cultures increased the glycerol yield on glucose to 1.08 mol mol(-1). To our knowledge, this is the highest glycerol yield reported for S. cerevisiae.     

Inhibition of aerobic respiration and dissimilatory perchlorate reduction using cyanide

The effect of low concentrations of cyanide on dissimilatory perchlorate and chlorate reduction and aerobic respiration was examined using pure cultures of Azospira sp. KJ. Cyanide at a concentration of 38 microM inhibited cell growth on perchlorate, chlorate and molecular oxygen, but it did not inhibit the activity of chlorite dismutase. When oxygen accumulation was prevented by adding an oxygen scavenger (Oxyrase or L-cysteine), however, cells completely reduced perchlorate in the presence of cyanide. It was concluded that the inhibition of dissimilative perchlorate reduction by cyanide at this concentration was a consequence of oxygen accumulation, not inhibition of the enzymes used for perchlorate reduction. This finding on the effect of cyanide on respiratory enzymes provides a new method to control and study respiratory enzymes used for perchlorate reduction.     

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

1. Mitochondria isolated from cultures of Acanthamoeba castellanii exhibit respiratory control and oxidize alpha-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 (K(m)[unk]15mum), 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 MgCl(2). 7. Addition of rotenone to mitochondria oxidizing alpha-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.