Overexpression of isocitrate lyase—glyoxylate bypass influence on metabolism in Aspergillus niger

In order to improve the production of succinate and malate by the filamentous fungus Aspergillus niger the activity of the glyoxylate bypass pathway was increased by over-expression of the isocitrate lyase (icl) gene. The hypothesis was that when isocitrate lyase was up-regulated the flux towards glyoxylate would increase, leading to excess formation of malate and succinate compared to the wild-type. However, metabolic network analysis showed that an increased icl expression did not result in an increased glyoxylate bypass flux. The analysis did show a global response with respect to gene expression, leading to an increased flux through the oxidative part of the TCA cycle. Instead of an increased production of succinate and malate, a major increase in fumarate production was observed.The effect of malonate, a competitive inhibitor of succinate dehydrogenase (SDH), on the physiological behaviour of the cells was investigated. Inhibition of SDH was expected to lead to succinate production, but this was not observed. There was an increase in citrate and oxalate production in the wild-type strain. Furthermore, in the strain with over-expression of icl the organic acid production shifted from fumarate towards malate production when malonate was added to the cultivation medium.Overall, the icl over-expression and malonate addition had a significant impact on metabolism and on organic acid production profiles. Although the expected succinate and malate formation was not observed, a distinct and interesting production of fumarate and malate was found.     

Organic acid utilization,succinate excretion,encystation and oscillating nitrogenase activity inAzospirillum brasilense under microaerobic conditions

Oscillating nitrogenase activity in long lasting batch cultures ofAzospirillum brasilense ATCC 29145 is independent of the carbon source malate. With fumarate, succinate or pyruvate as sole carbon source nitrogenase activity is also oscillating. Cultivation in a medium with 20-fold the buffer concentration also results in oscillating nitrogenase activity. Nitrogen-fixing cultures ofAzospirillum brasilense ATCC 29145 excrete ammonia into the culture medium varying between 0.02 and 0.04 mM concentrations. This is not sufficient to cause a drop of nitrogenase activity inAzospirillum brasilense after the first maximum. During growth under nitrogen-fixing conditions with malate as carbon source, the cells excrete significant quantities of succinate into the culture medium. Cultures with only 0.05% malate reutilized the excreted succinate as soon as malate disappeared from the medium. Azospirillum brasilense ATCC 29145 is shown to have the capability of encystation. Encysted cells are different from vegetative cells in their resistance to desiccation, by the spherical shape and by immotility. The results indicate that oscillating nitrogenase activity in long lasting cultures reflects the development from vegetative cells to cysts and again to vegetative cells under microaerobic conditions.     

Aerotaxis and chemotaxis ofAzospirillum brasilense: A note

Azospirillum brasilense was attracted to capillaries containing either phosphate buffer, distilled water, or saline. The number of bacteria in these capillaries was 3–4×10⁴, after 1 h of incubation. In the presence of phosphate buffer + attractants, the number of cells accumulated in the capillary increased only to 5×10⁴–1.1×10⁵ cells. It was not possible, therefore, to measure chemotaxis inA. brasilense as distinct from aerotaxis by the capillary method. Chemotaxis was observed in semi-solid agar plates and was determined by a growth band oriented towards the attractant. Positive chemotactic response was obtained with peptone, tryptone, yeast extract, amino acids, organic acids, arabinose and galactose.     

Formation of l(-)malate by Saccharomyces cerevisiae during fermentation

Summary When grown in a synthetic medium most of the 51 strains of the genera Saccharomyces, Saccharomycodes, Zygosaccharomyces and Schizosaccharomyces investigated formed l-malate during fermentation. The quantity varied between 0.1 and 2.6 g malate per liter. Two strains of Saccharomyces cerevisiae synthesized malate at a rate of about 1.5 g/l. Malate was liberated during the growth phase and not metabolized during the stationary phase. Optimum malate formation was observed at a sugar concentration of about 20% (w/v), at pH 5 and at suboptimal nitrogen concentrations of less than 300 mg N/liter. Of the amino acids aspartate and glutamate were most favourable. If ammonium salts were used as the nitrogen source, significant amounts of malate were formed when the pH was kept constant by buffering. Trace metals had no or only little influence on malate synthesis. Biotin and pantothenate were essential for growth. Added ¹⁴CO₂ led to the formation of approximately equal quantities of labelled malate and succinate by S. cerevisiae strain 52, whereas about ten times more malate than succinate was formed by Saccharomyces uvarum. Avidin strongly inhibited the formation of malate while the inhibiton of succinate synthesis and of growth was comparatively much less. Malate is obviously formed by reduction of oxalacetate, the synthesis of which is catalysed by a biotin-dependent pyruvate carboxylase.     

Oxidation of organic compounds to CO2 with sulfur or thiosulfate as electron acceptor in the anaerobic hyperthermophilic archaea Thermoproteus tenax and Pyrobaculum islandicum proceeds via the citric acid cycle

The oxidation of organic compounds with elemental sulfur or thiosulfate as electron acceptor was studied in the anaerobic hyperthermophilic archaea Thermoproteus tenax and Pyrobaculum islandicum. T. tenax was grown on either glucose or casamino acids and sulfur; P. islandicum on peptone and either elemental sulfur or thiosulfate as electron acceptor. During exponential growth only CO₂ and H₂S rather than acetate, alanine, lactate, and succinate were detected as fermentation products of both organisms; the ratio of CO₂/H₂S formed was 1:2 with elemental sulfur and 1:1 with thiosulfate as electron acceptor. Cell extracts of T. tenax and P. islandicum contained all enzymes of the citric acid cycle in catabolic activities: citrate synthase, aconitase, isocitrate dehydrogenase (NADP⁺-reducing), oxoglutarate: benzylviologen oxidoreductase, succinyl-CoA synthetase, succinate dehydrogenase, fumarase and malate dehydrogenase (NAD⁺-reducing). Carbon monoxide dehydrogenase activity was not detected. We conclude that in T. tenax and P. islandicum organic compounds are completely oxidized to CO₂ with sulfur or thiosulfate as electron acceptor and that acetyl-CoA oxidation to CO₂ proceeds via the citric acid cycle.     

Effect of temperature on Pseudomonas fluorescens chemotaxis.

The effects of temperature and attractants on chemotaxis in psychrotrophic Pseudomonas fluorescens were examined using the Adler capillary assay technique. Several organic acids, amino acids, and uronic acids were shown to be attractants, whereas glucose and its oxidation products, gluconate and 2-ketogluconate, elicited no detectable response. Chemotaxis toward many attractants was dependent on prior growth of the microorganism with these compounds. However, the organic acids, malate and succinate, caused strong chemotactic responses regardless of the carbon source used for growth of the bacteria. The temperature at which the cells were grown (30 or 5 degrees C) had no significant detectable effect on chemotaxis to the above attractants. The temperature at which the cells were assayed appeared to affect the rate but the extent of the chemotactic response, nor the concentration response curves. The ratios of the rate of accumulation of cells to the attractant malate were approximately 2, 4, and 1 at 30, 17, and 5 degrees C, respectively. Strong chemotactic responses were observed with cells assayed at temperatures approaching 0 degree C and appeared to be functional over a broad temperature range of 3 to 35 degrees C.     

Kinetics of malate transport and decomposition in acid soils and isolated bacterial populations: The effect of microorganisms on root exudation of malate under Al stress

The kinetics and characteristics of malate degradation were studied in four acid soils ranging in both pH (4.30 to 5.00) and vegetation type. The breakdown of malate was rapid in all soils with a half life of approximately 1.7 h, K_m of 1.7 mM and V_max of 70 nmol g^–1 soil h^–1. No relationship was observed between malate decomposition rate and pH. Co-metabolism studies with other C and N substrates (glucose, glycine, glutamate, citrate and succinate) indicated that the microorganisms were not N limited and competitive inhibition of malate breakdown was only observed in the presence of succinate. Studies with isolated mixed bacterial cultures indicated that the bacterial malate uptake was mediated by an energy dependent, dicarboxylate transporter which can be inhibited by succinate and is independent of pH between pH 5.0 and 7.0. The K_m and V_max parameters ranged from 279–955 M and 0.1–17 mol mg^–1 protein h^–1 for the mixed bacterial cultures depending on the bacteria's previous C source. The results indicate that in acid topsoils where microbial populations are high, the microbes may provide a considerable sink for organic acids. If organic acids are being released by roots in response to an environmental stress (e.g. Al toxicity, P deficiency) it can be expected that the efficiency of these root mediated metal resistance mechanisms will be markedly reduced by rapid microbial degradation.     

Oxydation of substrates in organic acids utilization negative mutants and the wild typeRhizobium meliloti strain S'14

Two mutants defective in succinate utilization were isolated by NTG mutagenesis of the effective wild typeRhizobium meliloti strain S₁₄. The mutants used carbon sources in a fashion similar to strain S₁₄, but they were not able to grow on succinate, fumarate or malate. The mutants nodulated alfalfa plants but did not exhibit any nitrogenase activity. The mutants oxidized glucose and fructose, but were not able to oxidize organic acids. Cultured free-living bacteria of strain S₁₄ appeared to have an inducible C₄-dicarboxylic acid uptake system and a constitutive glucose uptake system. When S₁₄ cells were grown on glucose in the presence of 5mM or more succinate or malate, the rate of glucose-dependent O₂ consumption significantly decreased suggesting the presence of a catabolite repression like phenomenom. Contribution no. 301, Station de Recherches, Agriculture Canada.     

Surface iron polymers and hydroxy acids. A model of iron supply in sideramine-free fungi

Biochemical and electron microscopic evidence is presented that sideramine-free fungi form iron hydroxide polymer layers on the cell surface when grown in an iron containing medium.Iron hydroxide polymer formation on the cell surface is completely prevented in sideramine producing strains of Neurospora crassa. After feeding a sideramine-free mutant of Neurospora crassa with ornithine in order to restore the sideramine synthesis the iron hydroxide coat is gradually dissolved.The addition of excess citrate and malate to the incubation medium also prevents iron polymer adsorption, suggesting that hydroxy acids may be involved in iron supply, when sideramine-free organisms are grown in iron containing media.In order to study the interaction between iron hydroxide polymer deposition upon the cell surface and iron chelating acids in Neurospora crassa, the amount and the proportion of excreted acids was studied under various experimental conditions. Gas chromatographic analysis of the acids produced under iron deficient conditions revealed that succinate, malate and citrate were present within the cells in the early growth phase. The acids were sequentially excreted into the medium in the order succinate, malate and citrate. The amount of succinate decreased after 2 days of cultivation, whereas the amount of malate and citrate continually increased. Although citrate was present within the cells from the 1st day, excretion occurred very late, generally after the 3rd day.It is suggested that sideramine-free fungi first adsorb iron as a hydroxide polymer on the cell surface, and that it is gradually solubilized by excreted hydroxy acids such as citrate or malate. Thus high local concentrations of iron chelated by hydroxy acids provide sideramine-free fungi with a continuous iron supply.Abbreviations BSTFA N,O-Bis(trimethylsilyl)-trifluoracetamide - GC Gaschromatography - EGTA Ethylenglykol-bis(2-aminoethylether) N,N-tetraacetic acid - TMS Trimethylsilyl     

NMR metabolomics of berry quality in sea buckthorn (Hippophae L.)

Sea buckthorn (Hippophae L.) is a woody perennial shrub or small tree whose berries are rich in bioactive compounds with powerful nutritional and medicinal properties. Untargeted ¹H nuclear magnetic resonance (NMR) spectroscopy and principal component analysis (PCA) were used to characterize the metabolic profile of berry quality. There was strong separation in the NMR signal intensity of bioactive compounds between pulp and seeds, such as amino acids [gamma-aminobutyric acid (GABA), aspartate, glutamate, theanine, and proline], organic acids (citrate, succinate, malate, acetate, quinate, and heriguard), and carbohydrates (sucrose, fructose, glucose, and melibiose). Sea buckthorn cultivars could be clearly separated into two groups using principal component analysis (PCA) based on NMR spectroscopy of bioactive compounds in the pulp and seeds. Several metabolic compounds such as sugar, organic acids, and amino acids could serve as biomarkers for prediction of berry quality and for classification of germplasm collections. This dataset provides potential information concerning the mechanisms of berry quality formation and contributes to increasing the breeding efficiency of sea buckthorn quality improvement.     

Net efflux of citrate in Penicillium simplicissimum is mediated by a transport protein

Penicillium simplicissimum excreted citrate, isocitrate, and succinate when grown in a strongly buffered medium [1 M Mes (pH 6) or 1 M Hepes (pH 7.3)]. Growth in a weakly buffered medium did not lead to citrate excretion despite a similar intracellular citrate concentration. When nongrowing, citrate-excreting hyphae were aerated in a glucose solution, the following steady-state intracellular concentrations of organic acids were measured: succinate (25 mM); citrate, isocitrate, malate, and fumarate (all less than 5 mM). After 2 h of incubation, the extracellular concentrations of these acids were [μmol (g dry wt.)^–1]: isocitrate [100], citrate [60], succinate [30], and malate, fumarate, and α-ketoglutarate [<5]. The excretion of citrate was due neither to an unspecific change in the permeability of the plasma membrane nor to simple diffusion of undissociated citric acid. The involvement of a transport protein in citrate excretion was indicated because N-ethylmaleimide and sodium azide inhibited citrate excretion strongly despite an unchanged outward-directed citrate gradient. Arguments are given why efflux via a citrate uptake carrier is not considered probable. These results indicate that citrate is excreted by P. simplicissimum via a transport protein that probably specifically mediates the efflux of citrate. Received: 28 July 1997 / Accepted: 19 November 1997     

Photosystem I-dependent oxidation of organic acids in blue-green alga, Anabaena variabilis

Photosynthetic lamellar fragments isolated from photoautotrophicallygrown cells of Anabaena variabilis were tested for their methylviologen photoreduction activity by coupled oxygen uptake inthe presence of CMU. Four kinds of organic acids, glycolate,malate, succinate and isocitrate, were found to serve as electrondonors of photosystem I, bypassing photosystem II, as in thecase of reduced DCPIP. Spectrophotometric measurement revealedthat the dark reduction of photooxidized cytochrome f and P700was markedly accelerated if one of the four organic acids waspresent. (Received April 18, 1975; )     

Changes induced by aluminum stress in the organic acid content of maize seedlings: The crucial role of exogenous citrate in enhancing seedling growth

We have studied the effect of Al on growth and morphology of maize seedlings (Zea mays L.), the changes in organic acid content as well as the role of application of exogenous citrate in enhancing the Al tolerance. Al treatment induced inhibition of root growth, causing morphological symptoms of Al toxicity. Al decreased significantly the malate content in roots compared to control plants. However, the citrate and total organic acids did not show any change, indicating that one mechanism underlying plant defense may involve the maintenance a normal levels of organic acids in roots. The succinate content increased in roots at 1000 μmol L⁻¹ Al, while that of lactate decreased. However, 500 and 1000 μmol L⁻¹ Al significantly increased the total organic acid in shoots, due to an increase in the succinate and malate contents. By contrast, the citrate and lactate levels decreased at 250 and 500 μmol L⁻¹ Al. To investigate the role of citrate in enhancing the plant growth, citrate was supplied to nutrient medium containing 500 μmol L⁻¹ Al at different Al:Citrate ratios (1:1, 1:2 and 1:3). The addition of citrate in the nutrient solution resulted in an alleviation of Al toxicity, with the maximal effect obtained at Al:Citrate ratio of 1:2. These data provide evidence that in maize, the organic acids, mainly citrate play an important role in enabling the plant to tolerate elevated exposure to Al concentration.     

Specificity and regulation of the dicarboxylate carrier on the peribacteroid membrane of soybean nodules

Malate and succinate were taken up rapidly by isolated, intact peribacteroid units (PBUs) from soybean (Glycine max (L.) Merr.) root nodules and inhibited each other in a competitive manner. Malonate uptake was slower and was severely inhibited by equimolar malate in the reaction medium. The apparent K_m for malonate uptake was higher than that for malate and succinate uptake. Malate uptake by PBUs was inhibited by (in diminishing order of severity) oxaloacetate, fumarate, succinate, phthalonate and oxoglutarate. Malonate and butylmalonate inhibited only slightly and pyruvate,isocitrate and glutamate not at all. Of these compounds, only oxaloacetate, fumarate and succinate inhibited malate uptake by free bacteroids. Malate uptake by PBUs was inhibited severely by the uncoupler carbonylcyanidem-chlorophenyl hydrazone and the respiratory poison KCN, and was stimulated by ATP. We conclude that the peribacteroid membrane contains a dicarboxylate transport system which is distinct from that on the bacteroid membrane and other plant membranes. This system can catalyse the rapid uptake of a range of dicarboxylates into PBUs, with malate and succinate preferred substrates, and is likely to play an important role in symbiotic nitrogen fixation. Energization of both the bacteroid and peribacteroid membranes controls the rate of dicarboxylate transport into peribacteroid units.     

Changes of Some Mitochondrial Enzyme Activities of Cucumber Leaves during Ammonium Toxicity

The following enzyme activities were determined in the mitochondria of cucumber leaves (Cucumis sativus L. cv. Suisei No. 2) during ammonium toxicity: malate dehydrogenase, succinate dehydrogenase, glutamate dehydrogenase, cytochrome c oxidase, NADH diaphorase, NADH oxidase, succinate: cytochrome c oxidoreductase, NADH: cytochrome c oxidoreductase and adenosine triphosphatase. The activities of all enzymes except ATPase increased more or less during ammonium toxicity. Generally speaking the marked increase was found at 7 days treatment with 200 mg/1 NH₃-N. The adenosine triphosphatase activity of injured plants was lower than that of normal plants through treatment. The addition of various organic acids (15 mM) to the culture solution contaning 200 mg/1 NH₃-N (14.3 mM NH₄Cl) suppressed the ammonium toxicity. The accumulation of free ammonia in the leaves was also repressed by the addition of organic acids. The results of present and previous reports suggest that the increase of respiratory metabolism due to ammonium toxicity is required for the supply of organic acids, specially δ-ketoglutaric acid, to counteract ammonia. Uncoupling in mitochondria resulting in the increase of respiration does not seem to occur during ammonium toxicity.     

Nutrition of Acetobacter rancens S3 and F11 Isolated from Tanks for Vinegar Production

The strains S3 and F11 which were isolated respectively from static and submerged tanks for vinegar production were identified as Acetobacter rancens. Neither strain grew in an ammonium defined medium containing ethanol, glucose, glycerol or organic acids as the sole carbon source. When casamino acids were added, they grew luxuriantly with lactate, ethanol or glycerol as the carbon source and less well with acetate or glucose. They grew, forming much acetic acid, in defined ethanol medium when alanine was supplied in place of casamino acids, but strain S3 showed a longer lag time than strain Fl1. This lag time could be shortened by addition of aspartate and glutamate. These amino acids could be replaced by succinate, fumarate, malate, lactate, pyruvate or propionate but not by glucose. Both strains required lactate or pyruvate in defined glucose medium but many other organic acids, which were effective in defined ethanol medium, were ineffective or slightly effective in glucose medium.     

Effects of organic compounds on growth of chemostat cultures of Thiomicrospira pelophila,Thiobacillus thioparus and Thiobacillus neapolitanus

Summary Cultures of Thiomicrospira pelophila, Thiobacillus thioparus and Thiobacillus neapolitanus were grown in thiosulfate-limited chemostats in a mineralsthiosulfate medium with and without organic supplements. Acetate, succinate and mixtures of amino acids increased the dry weight by 12–24% and the protein by 11–38%. Addition of both acetate and succinate had a cumulative effect. Saccharose, glucose, fructose, ribose, glycerol, glycerate, pyruvate, lactate or malate were without effect. The increase in dry weight of T. neapolitanus by ¹⁴C-acetate was directly related to the relative contribution of this compound to the total cell carbon.In CO₂-limited cultures of T. neapolitanus the effects of acetate on dry weight and protein were similar to those found in thiosulfate-limited cultures. In CO₂-limited cultures of T. pelophila a combination of acetate and succinate caused an increase in dry weight of 27% and of 50% in protein, the increase in protein being twice as high as in thiosulfate-limited cultures.There were no measurable differences in the activities of ribulosediphosphate carboxylase (RudPcase) in cell free extracts obtained from thiosulfate- or CO₂-limited cultures of T. pelophila or T. neapolitanus grown in the presence or absence of organic compounds. In T. pelophila the RudPcase activity was almost constant at all growth rates tested, and independent of the type of growth-limitation. For T. neapolitanus the specific RudPcase activity varied slightly with the growth rate. In CO₂-limited cultures the activity was three times that found in thiosulfate-limited cultures, thus showing that the RudPcase activity can be influenced by nutritional conditions.     

The nature of the interaction Azospirillum-Arabidopsis determine the molecular and morphological changes in root and plant growth promotion

Plant growth promoting rhizobacteria influence host functional and adaptive traits via complex mechanisms that are just started to be clarified. Azospirillum brasilense acts as a probiotic bacterium, but detailed information about its molecular mechanisms of phytostimulation is scarce. Three interaction systems were established to analyze the impact of A. brasilense Sp245 on the phenotype of Arabidopsis seedlings, and underlying molecular responses were assessed under the following growth conditions: (1) direct contact of roots with the bacterium, (2) chemical communication via diffusible compounds produced by the bacterium, (3) signaling via volatiles. A. brasilense Sp245 improved shoot and root biomass and lateral root production in the three interaction systems assayed. Cell division, quiescent center, and differentiation protein reporters pCYCB1;1::GUS, WOX5::GFP, and pAtEXP7::GUS had a variable expression in roots depending of the nature of interaction. pCYCB1;1::GUS and WOX5::GFP increased with volatile compounds, whereas pAtEXP7::GUS expression was enhanced towards the root tip in plants with direct contact with the bacterium. The auxin reporter DR5::GUS was highly expressed with diffusible and volatile compounds, and accordingly, auxin signaling mutants pin3, slr1, arf7arf19, and tir1afb2afb3 showed differential phytostimulant responses when compared with the wild type. By contrast, ethylene signaling was not determinant to mediate root changes in response to the different interactions, as observed using the ethylene-related mutants etr1, ein2, and ein3. Our data highlight the diverse effects by which A. brasilense Sp245 improves plant growth and root architectural traits and define a critical role of auxin but not ethylene in mediating root response to bacterization.

     

Blue LED and succinic acid enhance the growth of Rhodobacter sphaeroides

Rhodobacter sphaeroides is a purple non-sulfur photosynthetic bacteria that participates in the anoxic cycling of carbon both as the primary producer and as the light-stimulated consumers of the reduced organic compounds. In this study, six different organic acids, i.e. acetate, lactate, oxaloacetate, malate, succinate, and citrate, were selected and used to analyze the relationships between the organic acid source and the cell growth. The C₄ compound exhibited an enhanced cell growth compared to the other organic acids, and the growth rate of R. sphaeroides that was grown with 0.03 M succinic acid was significantly 3.2-fold faster than the C₆ compound of 0.03 M citrate. Additionally, the cell growth of R. sphaeroides was enhanced with increasing light intensity, and the growth rate and the dry cell weight of R. sphaeroides that were grown under the light conditions of 15 W/m² were 2.0- and 1.2-fold higher than R. sphaeroides at 3 W/m². Therefore, the high light intensity probably affected the growth of R. sphaeroides. Moreover, the blue-colored light emitting diode (LED) exhibited a highest growth rate and cell concentration of R. sphaeroides among the various types of LEDs, and the enhanced cell growth phenomenon under the blue LED conditions was dramatically stimulated at low concentrations of succinic acid, which was compensatory for succinic acid. Therefore, a high light intensity and a blue LED as the light source were necessary for the enhanced cell growth for the C₄ organic acid, i.e. succinic acid.     

The anaerobic formation of propionic acid in the mitochondria of the lugwormArenicola marina

Summary The anaerobic transformation of malate and succinate into propionate was demonstrated in homogenates and mitochondria isolated from the body wall musculature ofArenicola marina, a facultative anaerobic polychaete. Synthesis of propionate from succinate was enhanced by the addition of malate and ADP. In the presence of malate, acetate was formed in addition to propionate. Maximal quantities of both fatty acids were produced by mitochondria incubated with malate, succinate, and ADP. Since the rate of propionate production in this case was about the same as in homogenates when related to fresh weight, it is concluded that the enzymatic system involved is localized exclusively in the mitochondria. The rate of propionate production is correlated with the concentration of succinate, saturation being reached at about 5 mM. In tracer experiments using (methyl-¹⁴C)-malonyl-CoA, 2,3-¹⁴C-succinate, and 1-¹⁴C-propionate as precursors, the pathway of the transformation of succinate into propionate was examined. The results indicate that methylmalonyl-CoA is an intermediary product. It was shown that the synthesis of propionate from succinate is coupled to the formation of ATP. The ratio ATP/propionate was 0.76. Dinitrophenol had only a slight effect on this ratio, although the utilization of succinate was inhibited considerably. It is concluded that in vivo substrate level phosphorylation occurs equimolar to the formation of propionate from succinate.Abbreviations Ap ₅ A P₁,P₅-di(adenosine-5-)pentaphosphate - DNP 2,4-dinitrophenol - mma methylmalonic acid - mm-CoA methylmalonyl-CoA Enzymes EC 6.2.1.1 Acetate thiokinase (AMP) - EC 3.6.1.3 actomyosin ATPase - EC 2.7.4.3 adenylate kinase - EC 2.8.3.1 CoA transferase - EC 2.7.1.1 hexokinase - EC 2.1.3.1 methylmalonyl-CoA carboxyltransferase - EC 5.4.99.1 methylmalonyl-CoA isomerase - EC 5.1.99.1 methylmalonyl-CoA racemase - EC 6.4.1.3 propionyl-CoA carboxylase - EC 1.2.4.1 pyruvate dehydrogenase Supported by Deutsche Forschungsgemeinschaft Gr 456/6