The role of oxygen limitation in the formation of poly-β-hydroxybutyrate during batch and continuous culture of Azotobacter beijerinckii

Azotobacter beijerinckii was grown in ammonia-free glucose-mineral salts media in batch culture and in chemostat cultures limited by the supply of glucose, oxygen or molecular nitrogen. In batch culture poly-beta-hydroxybutyrate was formed towards the end of exponential growth and accumulated to about 74% of the cell dry weight. In chemostat cultures little poly-beta-hydroxybutyrate accumulated in organisms that were nitrogen-limited, but when oxygen limited a much increased yield of cells per mol of glucose was observed, and the organisms contained up to 50% of their dry weight of poly-beta-hydroxybutyrate. In carbon-limited cultures (D, the dilution rate,=0.035-0.240h(-1)), the growth yield ranged from 13.1 to 19.8g/mol of glucose and the poly-beta-hydroxybutyrate content did not exceed 3.0% of the dry weight. In oxygen-limited cultures (D=0.049-0.252h(-1)) the growth yield ranged from 48.4 to 70.1g/mol of glucose and the poly-beta-hydroxybutyrate content was between 19.6 and 44.6% of dry weight. In nitrogen-limited cultures (D=0.053-0.255h(-1)) the growth yield ranged from 7.45 to 19.9g/mol of glucose and the poly-beta-hydroxybutyrate content was less than 1.5% of dry weight. The sudden imposition of oxygen limitation on a nitrogen-limited chemostat culture produced a rapid increase in poly-beta-hydroxybutyrate content and cell yield. Determinations on chemostat cultures revealed that during oxygen-limited steady states (D=0.1h(-1)) the oxygen uptake decreased to 100mul h(-1) per mg dry wt. compared with 675 for a glucose-limited culture (D=0.1h(-1)). Nitrogen-limited cultures had CO(2) production values in situ ranging from 660 to 1055mul h(-1) per mg dry wt. at growth rates of 0.053-0.234h(-1) and carbon-limited cultures exhibited a variation of CO(2) production between 185 and 1328mul h(-1) per mg dry wt. at growth rates between 0.035 and 0.240h(-1). These findings are discussed in relation to poly-beta-hydroxybutyrate formation, growth efficiency and growth yield during growth on glucose. We suggest that poly-beta-hydroxybutyrate is produced in response to oxygen limitation and represents not only a store of carbon and energy but also an electron sink into which excess of reducing power can be channelled.     

The regulation of poly-β-hydroxybutyrate metabolism in Azotobacter beijerinckii

1. The enzymes beta-ketothiolase, acetoacetyl-CoA reductase, acetoacetate-succinate CoA-transferase (;thiophorase') and d(-)-3-hydroxybutyrate dehydrogenase have been partially purified from crude extracts of glucose-grown nitrogen-fixing batch cultures of Azotobacter beijerinckii. The condensation of acetyl-CoA to acetoacetyl-CoA catalysed by beta-ketothiolase is inhibited by CoASH, and the reverse reaction is inhibited by acetoacetyl-CoA. Acetoacetyl-CoA reductase has K(m) for acetoacetyl-CoA of 1.8mum and is inhibited by acetoacetyl-CoA above 10mum. The enzyme utilizes either NADH or NADPH as electron donor. The second enzyme of poly-beta-hydroxybutyrate degradation, d(-)-3-hydroxybutyrate dehydrogenase, is NAD(+)-specific and is inhibited by NADH, pyruvate and alpha-oxoglutarate. CoA transferase is inhibited by acetoacetate, the product of hydroxybutyrate oxidation. In continuous cultures poly-beta-hydroxybutyrate biosynthesis ceased on relaxation of oxygen-limitation and the rates in situ of oxygen consumption and carbon dioxide evolution of such cultures increased without a concomitant increase in glucose uptake. 2. On the basis of these and other findings a cyclic mechanism for the biosynthesis and degradation of poly-beta-hydroxybutyrate is proposed, together with a regulatory scheme suggesting that poly-beta-hydroxybutyrate metabolism is controlled by the redox state of the cell and the availability of CoASH, pyruvate and alpha-oxoglutarate. beta-Ketothiolase plays a key role in the regulatory process. Similarities to the pathways of poly-beta-hydroxybutyrate biosynthesis and degradation in Hydrogenomonas are discussed.     

Poly-β-hydroxybutyrate biosynthesis and the regulation of glucose metabolism in Azotobacter beijerinckii

Azotobacter beijerinckii possesses the enzymes of both the Entner-Doudoroff and the oxidative pentose phosphate cycle pathways of glucose catabolism and both pathways are subject to feedback inhibition by products of glucose oxidation. The allosteric glucose 6-phosphate dehydrogenase utilizes both NADP(+) and NAD(+) as electron acceptors and is inhibited by ATP, ADP, NADH and NADPH. 6-Phosphogluconate dehydrogenase (NADP-specific) is unaffected by adenosine nucleotides but is strongly inhibited by NADH and NADPH. The formation of pyruvate and glyceraldehyde 3-phosphate from 6-phosphogluconate by the action of the Entner-Doudoroff enzymes is inhibited by ATP, citrate, isocitrate and cis-aconitate. Glyceraldehyde 3-phosphate dehydrogenase is unaffected by adenosine and nicotinamide nucleotides but the enzyme is non-specific with respect to NADP and NAD. Citrate synthase is strongly inhibited by NADH and the inhibition is reversed by the addition of AMP. Isocitrate dehydrogenase, a highly active NADP-specific enzyme, is inhibited by NADPH, NADH, ATP and by high concentrations of NADP(+). These findings are discussed in relation to the massive synthesis of poly-beta-hydroxybutyrate that occurs under certain nutritional conditions. We propose that synthesis of this reserve material, to the extent of 70% of the dry weight of the organism, serves as an electron and carbon ;sink' when conditions prevail that would otherwise inhibit nitrogen fixation and growth.     

Perfusion cultures require optimum respiratory ATP supply to maximize cell-specific and volumetric productivities

Perfusion processes are an emerging alternative to common fed-batch processes in the growing biopharmaceutical industry. However, the challenge of maintaining high cell-specific productivities remains. In this study, glucose limitation was applied to two perfusion steady states and compared with a third steady state without any detectable limitation. The metabolic phenotype was enhanced under glucose limitation with a decrease of 30% in glucose uptake and 75% in lactate formation. Cell-specific productivities were substantially improved by 50%. Remarkably, the productivities showed a strong correlation to respiratory adenosine triphosphate (ATP) supply. As less reduced nicotinamide adenine dinucleotide (NADH) remained in the cytosol, the ATP generation from oxidative phosphorylation was increased by almost 30%. Consequently, the efficiency of carbon metabolism and the resulting respiratory ATP supply was crucial for maintaining the highly productive cellular state. This study highlights that glucose limitation can be used for process intensification in perfusion cultures as ATP generation via respiration is significantly increased, leading to elevated productivities.     

Oxygen Response of the Wine Yeast Saccharomyces cerevisiae EC1118 Grown under Carbon-Sufficient,Nitrogen-Limited Enological Conditions

Discrete additions of oxygen play a critical role in alcoholic fermentation. However, few studies have quantitated the fate of dissolved oxygen and its impact on wine yeast cell physiology under enological conditions. We simulated the range of dissolved oxygen concentrations that occur after a pump-over during the winemaking process by sparging nitrogen-limited continuous cultures with oxygen-nitrogen gaseous mixtures. When the dissolved oxygen concentration increased from 1.2 to 2.7 μM, yeast cells changed from a fully fermentative to a mixed respirofermentative metabolism. This transition is characterized by a switch in the operation of the tricarboxylic acid cycle (TCA) and an activation of NADH shuttling from the cytosol to mitochondria. Nevertheless, fermentative ethanol production remained the major cytosolic NADH sink under all oxygen conditions, suggesting that the limitation of mitochondrial NADH reoxidation is the major cause of the Crabtree effect. This is reinforced by the induction of several key respiratory genes by oxygen, despite the high sugar concentration, indicating that oxygen overrides glucose repression. Genes associated with other processes, such as proline uptake, cell wall remodeling, and oxidative stress, were also significantly affected by oxygen. The results of this study indicate that respiration is responsible for a substantial part of the oxygen response in yeast cells during alcoholic fermentation. This information will facilitate the development of temporal oxygen addition strategies to optimize yeast performance in industrial fermentations.     

Competence regulation by oxygen availability and by Nox is not related to specific adjustment of central metabolism in Streptococcus pneumoniae

In Streptococcus pneumoniae oxygen availability is a major determinant for competence development in exponentially growing cultures. NADH oxidase activity is required for optimal competence in cultures grown aerobically. The implication of oxidative metabolism and more specifically of Nox on central metabolism has been examined. Glycolytic flux throughout exponential growth revealed homolactic fermentation with a lactate production/glucose utilization ratio close to 2, whatever the aerobiosis level of the culture. Loss-of-function mutations in nox, which encodes NADH oxidase, did not change this trait. Consistently, mRNA levels of glyceraldehyde-3-phosphate dehydrogenase, L-lactate dehydrogenase, pyruvate oxidase, and NADH oxidase remained comparable to wild-type levels, as did the specific activities of key enzymes which control central metabolism. Competence regulation by oxygen involving the NADH oxidase activity is not due to significant modification of carbon flux through glycolysis. Failure to obtain loss-of-function mutation in L-ldh, which encodes the L-lactate dehydrogenase, indicates its essential role in pneumococci whatever their growth status.     

Stimulation by light of nitrogenase synthesis in cells of Rhodopseudomonas capsulata growing in N-limited continuous cultures

In cells of Rhodopseudomonas capsulata growing in nitrogen-limited continuous culture the nitrogenase-specific activity was found to be closely dependent on the light intensity. As light intensity, measured with a photodiode immersed in the culture, was varied stepwise from 1000 to 7000 lux, the nitrogenase activity, measured at steady state, increased gradually up to 5-fold. Shifting light intensity from 1200 to 7000 lux resulted in a sharp rise in nitrogenase activity which doubled within the first two hours. The determination by immunoassays of the intracellular levels of each nitrogenase component revealed that the light-dependent stimulation of nitrogenase activity was correlated with the accumulation of the nitrogenase enzyme inside the cells. Under high illumination, nitrogenase represented up to 40% of the cytoplasmic proteins. The specific activities of each component in intact cells, calculated on the basis of their relative concentration in the cells and on in vivo nitrogenase assays, appeared roughly constant and hardly affected by changes of light intensity. The specific activity of the Fe protein was about 7-fold higher in intact cells than in the purified state. The ratio of the two nitrogenase components remained fairly constant and close to one, irrespective of the light intensity to which cells were exposed. These results demonstrate that in nitrogen-limited grown cells of Rps. capsulata light brings about an induction or a derepression of nitrogenase synthesis the extent of which is dependent on light intensity.     

Activity of nitrogenase and glutamine synthetase in relation to availability of oxygen in continuous cultures of a strain of cowpea Rhizobium sp. supplied with excess ammonium.

In samples from nitrogen-fixing continuous cultures of strain CB756 of the cowpea type rhizobia (Rhizobium sp.), newly fixed NH+4 is in equiblibrium with the medium, from where it is assimilated by the glutamine synthetase/glutamate synthase pathway. In samples from steady state cultures with different degrees of oxygen-limitation, nitrogenase activity was positively correlated with the biosynthetic of glutamine synthetase in cell free extracts. Also, activities in biosynthetic assays were positively correlated with activities in gamma-glutamyl transferase assays containing 60 mM Mg2+. Relative adenylylation of glutamine synthetase was conveniently measured in cell free extracts as the ratio of gamma-glutamyl transferase activities without and with addition of 60 mM Mg2+. Automatic control of oxygen supply was used to facilitate the study of transitions between steady-state continuous cultures with high and low nitrogenase activities. Adenylylation of glutamine synthetase and repression of nitrogenase activity in the presence of excess NH+4, were masked when oxygen strongly limited culture yield. Partial relief of the limitation in cultures supplied with 10 mM NH+4 produced early decline in nitrogenase activity and increase in relative adenylylation of glutamine synthetase. Decreased oxygen supply produced a rapid decline in relative adenylylation, followed by increased nitrogenase activity, supporting the concept that control of nitrogenase synthesis is modulated by glutamine synthetase adenylylation in these bacteria.     

Coordinate reciprocal trends in glycolytic and mitochondrial transcript accumulations during the in vitro differentiation of human myoblasts

Changes in the mRNA levels during mammalian myogenesis were compared for seven polypeptides of mitochondrial respiration (the mitochondrial DNA-encoded cytochrome oxidase subunit III, ATP synthase subunit 6, NADH dehydrogenase subunits 1 and 2, and 16S ribosomal RNA; the nuclear encoded ATP synthase beta subunit and the adenine nucleotide translocase) and three polypeptides of glycolysis (glyceraldehyde-3-phosphate dehydrogenase, pyruvate kinase, and triose-phosphate isomerase). Progressive changes during the conversion from myoblasts to myotubes were monitored under both atmospheric oxygen (normoxic) and hypoxic environments. Northern analyses revealed coordinate, biphasic, and reciprocal expression of the respiratory and glycolytic mRNAs during myogenesis. In normoxic cells the mitochondrial respiratory enzymes were highest in myoblasts, declined 3- to 5-fold during commitment and exist from the cell cycle, and increased progressively as the myotubes matured. By contrast, the glycolytic enzyme mRNAs rose 3- to 6-fold on commitment and then progressively declined. When partially differentiated myotubes were switched to hypoxic conditions, the glycolytic enzyme mRNAs increased and the respiratory mRNAs declined. Hence, the developmental regulation of muscle bioenergetic metabolism appears to be regulated at the pretranslational level and is modulated by oxygen tension.     

Oxygen- and glucose-dependent regulation of central carbon metabolism in Pichia anomala

We investigated the regulation of the central aerobic and hypoxic metabolism of the biocontrol and non-Saccharomyces wine yeast Pichia anomala. In aerobic batch culture, P. anomala grows in the respiratory mode with a high biomass yield (0.59 g [dry weight] of cells g of glucose(-1)) and marginal ethanol, glycerol, acetate, and ethyl acetate production. Oxygen limitation, but not glucose pulse, induced fermentation with substantial ethanol production and 10-fold-increased ethyl acetate production. Despite low or absent ethanol formation, the activities of pyruvate decarboxylase and alcohol dehydrogenase were high during aerobic growth on glucose or succinate. No activation of these enzyme activities was observed after a glucose pulse. However, after the shift to oxygen limitation, both enzymes were activated threefold. Metabolic flux analysis revealed that the tricarboxylic acid pathway operates as a cycle during aerobic batch culture and as a two-branched pathway under oxygen limitation. Glucose catabolism through the pentose phosphate pathway was lower during oxygen limitation than under aerobic growth. Overall, our results demonstrate that P. anomala exhibits a Pasteur effect and not a Crabtree effect, i.e., oxygen availability, but not glucose concentration, is the main stimulus for the regulation of the central carbon metabolism.     

Effect of Oxygen on Several Enzymes Involved in the Aerobic and Anaerobic Utilization of Glucose in Escherichia coli

By using the continuous culture technique, the transition from aerobiosis to anaerobiosis and its effect on a number of enzymes has been investigated in Escherichia coli K-12. A decrease in the oxygen partial pressure below 28.0 mm of Hg resulted firstly in an increase of the respiratory enzymes (reduced nicotinamide adenine dinucleotide [NADH] oxidase, 2.53-fold; succinic dehydrogenase, 1.4-fold; cytochrome b(1), 3.91-fold; and cytochrome a(2), 2.45-fold) before the electron transport system gradually collapsed as cytochrome a(2), followed by cytochrome b(1), succinic dehydrogenase, and finally NADH oxidase decreased in activity. The change from respiration to fermentation was initiated well before the oxygen tension reached zero by the increase in levels of fructose diphosphate-aldolase, glucose 6-phosphate, and 6-phosphogluconate dehydrogenases and a decrease in 2-oxoglutarate dehydrogenase. Whem the dissolved oxygen tension reached zero, dry weight and CO(2) formation together with isocitrate dehydrogenase decreased, whereas acid production and phosphofructokinase synthesis started to increase. Enzymatic investigations revealed that the kinetics of the enzyme phosphofructokinase from strict aerobic cultures (6.9 ppm oxygen in solution) was adenosine triphosphate (ATP)-insensitive, whereas the same enzyme from anaerobic cultures was ATP-sensitive. A mechanism is proposed for the change from aerobiosis to anaerobiosis together with the occurring change in glucose regulation.     

Activity of nitrogenase and glutamine synthetase in relation to availability of oxygen in continuous cultures of a strain of cowpea Rhizobium sp. supplied with excess ammonium

In samples from nitrogen-fixing continuous cultures of strain CB756 of the cowpea type rhizobia (Rhizobium sp.), newly fixed NH₄⁺ is in equilibrium with the medium, from where it is assimilated by the glutamine synthetase/glutamate synthase pathway. In samples from steady state cultures with different degrees of oxygen-limitation, nitrogenase activity was positively correlated with the biosynthetic activity of glutamine synthetase in cell free extracts. Also, activities in biosynthetic assays were positively correlated with activities in γ-glutamyl transferase assays containing 60 mM Mg²⁺. Relative adenylylation of glutamine synthetase was conveniently measured in cell free extracts as the ratio of γ-glutamyl transferase activities without and with addition of 60 mM Mg²⁺.Automatic control of oxygen supply was used to facilitate the study of transitions between steady-state continuous cultures with high and low nitrogenase activities. Adenylylation of glutamine synthetase and repression of nitrogenase activity in the presence of excess NH₄⁺, were masked when oxygen strongly limited culture yield. Partial relief of the limitation in cultures supplied with 10 mM NH₄⁺ produced early decline in nitrogenase activity and increase in relative adenylylation of glutamine synthetase. Decreased oxygen supply produced a rapid decline in relative adenylylation, followed by increased nitrogenase activity, supporting the concept that control of nitrogenase synthesis is modulated by glutamine synthetase adenylylation in these bacteria.     

Physiological Studies of Oxygen Protection Mechanisms in the Heterocysts of Anabaena cylindrica

The mechanism of O₂ protection of nitrogenase in the heterocysts of Anabaena cylindrica was studied in vivo. Resistance to O₂ inhibition of nitrogenase activity correlated with the O₂ tension of the medium in which heterocyst formation was induced. O₂ resistance also correlated with the apparent K_m for acetylene, indicating that O₂ tension may influence the development of a gas diffusion barrier in the heterocysts. The role of respiratory activity in protecting nitrogenase from O₂ that diffuses into the heterocyst was studied using inhibitors of carbon metabolism. Reductant limitation induced by 3-(3,4-dichlorophenyl)-1, 1-dimethylurea increased the O₂ sensitivity of in vivo acetylene reduction. Azide, at concentrations (30 mM) sufficient to completely inhibit dark nitrogenase activity (a process dependent on oxidative phosphorylation for its ATP supply), severely inhibited short-term light-dependent acetylene reduction in the presence of O₂ but not in its absence. After 3 h of aerobic incubation in the presence of 20 mM azide, 75% of cross-reactive component I (Fe-Mo protein) in nitrogenase was lost; less than 35% was lost under microaerophilic conditions. Sodium malonate and monofluoroacetate, inhibitors of Krebs cycle activity, had only small inhibitory effects on nitrogenase activity in the light and on cross-reactive material. The results suggest that oxygen protection is dependent on both an O₂ diffusion barrier and active respiration by the heterocyst.     

Nitrogenase activity and regeneration of the cellular ATP pool in Azotobacter vinelandii adapted to different oxygen concentrations.

The in vivo activity of nitrogenase under aerobiosis was studied with diazotrophic chemostat cultures of Azotobacter vinelandii grown under glucose- or phosphate-limited conditions at different dilution rates (Ds, representing the growth rate mu) and different dissolved oxygen concentrations. Under steady-state conditions, the concentration as well as the cellular level of ATP increased in glucose-limited cultures when D was increased. Irrespective of the type of growth limitation or the dissolved oxygen concentration, the steady-state concentrations of ATP and of dinitrogen fixed by nitrogenase increased in direct proportion to each other. Specific rates of dinitrogen fixation as well as of the regeneration of the cellular ATP pool were compared with specific rates of cellular respiration. With glucose-limited cultures, the rate of regeneration of the ATP pool and the rate of respiration varied in direct proportion to each other. This relationship, however, was dependent on the dissolved oxygen concentration. As compared to the phosphate-sufficient control, phosphate-limited cultures exhibited the same nitrogenase activity but significantly increased respiratory activities. Rates of ATP regeneration and of cellular respiration of phosphate-limited cultures did not fit into the relationship characteristic of glucose-limited cultures. However, a linear relationship between the rates of dinitrogen fixation and ATP regeneration was identified irrespective of the type of growth limitation and the dissolved oxygen concentration. The results suggest that the ATP supply rather than cellular oxygen consumption is of primary importance in keeping nitrogenase activity in aerobic cultures of A. vinelandii.     

Nitrogen fixation is synchronized with carbon metabolism in Lotus japonicus and Medicago truncatula nodules under salt stress

Abstract

In the present work, the response to NaCl applied at the vegetative stage to Medicago truncatula and Lotus japonicus has been evaluated in order to ascertain whether the effect of salt stress on nitrogen fixation is due to a limitation on nodular carbon metabolism. Results show maximum sucrose synthase (SS) and alkaline invertase (AI) activities were obtained at the vegetative stage, when maximum nitrogenase activity was detected in both species. SS activity decreased with the salt treatment, providing evidence of the regulatory role of this enzyme for the carbon supply to the bacteroids. Phosphoenolpyruvate carboxylase (PEPC) and malate dehydrogenase (MDH) activities could account for higher nitrogen fixation efficiency detected in L. japonicus nodules and isocitrate dehydrogenase (ICDH) activity compensated for the carbon limitations that occur under salt stress. These results support that nitrogenase inhibition in nodules experiencing salt stress is doubt to a carbon flux shortage, as result of carbon metabolism enzymes activities down-regulation.     

Are respiratory enzymes the primary sources of intracellular hydrogen peroxide?

Endogenous H2O2 is believed to be a source of chronic damage in aerobic organisms. To quantify H2O2 formation, we have generated strains of Escherichia coli that lack intracellular scavenging enzymes. The H2O2 that is formed within these mutants diffuses out into the medium, where it can be measured. We sought to test the prevailing hypothesis that this H2O2 is primarily generated by the autoxidation of redox enzymes within the respiratory chain. The rate of H2O2 production increased when oxygen levels were raised, confirming that H2O2 is formed by an adventitious chemical process. However, mutants that lacked NADH dehydrogenase II and fumarate reductase, the most oxidizable components of the respiratory chain in vitro, continued to form H2O2 at normal rates. NADH dehydrogenase II did generate substantial H2O2 when it was when overproduced or quinones were absent, forcing electrons to accumulate on the enzyme. Mutants that lacked both NADH dehydrogenases respired very slowly, as expected; however, these mutants showed no diminution of H2O2 excretion, suggesting that H2O2 is primarily formed by a source outside the respiratory chain. That source has not yet been identified. In respiring cells the rate of H2O2 production was approximately 0.5% the rate of total oxygen consumption, with only modest changes when cells used different carbon sources.     

Investigations into the pathway of electron transport to the nitrogenase from nodule bacteroids

Summary A series of investigations were conducted with the objective of elucidating natural pathways of electron transport from respiratory processes to the site of N₂ fixation in nodule bacteroids. A survey of dehydrogenase activities in a crude extract of soybean nodule bacteroids revealed relatively high activities of NAD-specific β-hydroxybutyrate and glyceraldehyde-3-phosphate dehydrogenases. Moderate activities of NADP-specific isocitrate and glucose-6-phosphate dehydrogenases were observed. By use of the ATP-dependent acetylene reduction reaction catalyzed by soybean bacteroid nitrogenase, and enzymes and cofactors from bacteroids and other sources, the following sequences of electron transport to bacteroid nitrogenase were demonstrated: (1) H₂ to bacteroid nitrogenase in presence of a nitrogenase-free extract ofC. pasteurianum; (2) β-hydroxybutyrate to bacteroid nitrogenase in a reaction containing β-hydroxybutyrate dehydrogenase, NADH dehydrogenase, NAD and benzyl viologen; (3) β-hydroxybutyrate dehydrogenase, to nitrogenase in reaction containing NADH dehydrogenase, NAD and either FMN or FAD; (4) light-dependent transfer of electrons from ascorbate to bacteroid nitrogenase in a reaction containing photosystem I from spinach chloroplasts, 2,6-dichlorophenolindophenol, and either azotoflavin from Azotobacter or non-heme iron protein from bacteroids; (5) glucose-6-phosphate to bacteroid nitrogenase in a system that included glucose-6-phosphate dehydrogenase, NADP, NADP-ferredoxin reductase from spinach, azotoflavin from Azotobacter and bacteroid non-heme iron protein. The electron transport factors, azotoflavin and bacteroid non-heme iron protein, failed to function in the transfer of electrons from an NADH-generating system to bacteroid nitrogenase. When FMN or FAD were added to systems containing azotoflavin and bacteroid non-heme iron protein, electrons apparently were transferred to the flavin-nucleotides and then nitrogenase without involvement of azotoflavin and bacteroid non-heme iron protein. Evidence is available indicating that nodule bacteroids contain flavoproteins analogous to Azotobacter, azotoflavin, and spinach ferredoxin-NADP reductase. It is concluded that physiologically important systems involved in transport of electrons from dehydrogenases to nitrogenase in bacteroids very likely will include relatively specific electron transport proteins such as bacteroid non-heme iron protein and a flavoprotein from bacteroids that is analogous to azotoflavin.     

Studies on rat liver mitochondria: 4. Enzyme activities in mitochondria preserved at 0-4 degrees C

Rat liver mitochondria, stored with the energy-linked functions preserved or in aging conditions, were used to assay the activity of various enzymes during five days. The preservation of energy-linked functions was monitored by the respiratory control coefficient. ATPase, cytochrome oxidase and NADH dehydrogenase showed increased activity when the energy-linked functions were preserved. In aging conditions, cytochrome oxidase, NADH dehydrogenase and ATPase showed decreased activity. The ATPase activity increased only when mitochondria were stored in the presence of inhibitors of the electron transport chain. The activity of NADH oxidase did not change, and succinate oxidase and succinate dehydrogenase showed a small decrease in their activity. The enzymes of the matrix, alpha-ketoglutarate dehydrogenase, malate dehydrogenase and aspartate aminotransferase showed little decrease in activity under either of the conditions of storage. The total protein content decreased slightly under both conditions of storage. These results show that the activity of the enzymes analysed was maintained at reasonable levels, when the energy-linked functions of isolated mitochondria were preserved.