Crassulacean acid metabolism (CAM) in Kalanchoë daigremontiana: Temperature response of phosphoenolpyruvate (PEP)-carboxylase in relation to allosteric effectors

Net CO₂ dark fixation of Kalanchoë daigremontiana varies with night temperature. We found an optimum of fixation at about 15° C; with increasing night temperature fixation decreased. We studied the temperature dependence of the activity of phosphoenolpyruvate (PEP)-carboxylase, the key enzyme for CO₂ dark fixation. We varied the pH, the substrate concentration (PEP), and the L-malate and glucose-6-phosphate (G-6-P) concentration in the assay. Generally, lowering the pH and reducing the amount of substrate resulted in an increase in activation by G-6-P and in an increase in malate inhibition of the enzyme. Furthermore, malate inhibition and G-6-P activation increased with increasing temperature. Activity measurements between 10° C and 45°C at a given concentration of the effectors revealed that the temperature optimum and maximum activities at that optimum varied with the effector applied. Under the influence of 5 mol m^-3 L-malate the temperature optimum and maximum activity dropped drastically, especially when the substrate level was low (at 0.5 mol m^-3 PEP from 32° C to 20° C). G-6-P raised the temperature optimum and maximum activity when the substrate level was low. If both malate and G-6-P were present, intermediate values were measured. We suggest that changes in metabolite levels in K. daigremontiana leaves can alter the temperature features of PEP-carboxylase so that the observed in vivo CO₂ dark fixation can be explained on the basis of PEP-carboxylase activity.Abbreviations PEP-c phosphoenolpyruvate carboxylase - CAM crassulacean acid metabolism - PEP phosphoenolpyruvate - G-6-P glucose-6-phosphate     

The Effect of Sodium Chloride on the Balance between the C3- and C4-Carbon Fixation Pathways

Young leaves of salt-depleted Aeluropus litoralis Parl. plants show CO₂ fixation by the C₃-carbon fixation pathway. No detectable activity of phosphoenol pyruvate (PEP) carboxylase was found. When A. litoralis plants were exposed to a NaCl solution, the leaves showed a high activity of PEP carboxylase as well as a significant CO₂ fixation by the C₄-pathway. — Also in Zea mays L. and Chloris gayana Kunth., the presence of NaCl in the medium influences the balance between phosphoenol pyruvate carboxylase and ribulose-1,5-diphosphate carboxylase.     

Wide range of metabolic adaptations to the acquisition of the Calvin cycle revealed by comparison of microbial genomes

Knowledge of the genetic basis for autotrophic metabolism is valuable since it relates to both the emergence of life and to the metabolic engineering challenge of incorporating CO₂ as a potential substrate for biorefining. The most common CO₂ fixation pathway is the Calvin cycle, which utilizes Rubisco and phosphoribulokinase enzymes. We searched thousands of microbial genomes and found that 6.0% contained the Calvin cycle. We then contrasted the genomes of Calvin cycle-positive, non-cyanobacterial microbes and their closest relatives by enrichment analysis, ancestral character estimation, and random forest machine learning, to explore genetic adaptations associated with acquisition of the Calvin cycle. The Calvin cycle overlaps with the pentose phosphate pathway and glycolysis, and we could confirm positive associations with fructose-1,6-bisphosphatase, aldolase, and transketolase, constituting a conserved operon, as well as ribulose-phosphate 3-epimerase, ribose-5-phosphate isomerase, and phosphoglycerate kinase. Additionally, carbohydrate storage enzymes, carboxysome proteins (that raise CO₂ concentration around Rubisco), and Rubisco activases CbbQ and CbbX accompanied the Calvin cycle. Photorespiration did not appear to be adapted specifically for the Calvin cycle in the non-cyanobacterial microbes under study. Our results suggest that chemoautotrophy in Calvin cycle-positive organisms was commonly enabled by hydrogenase, and less commonly ammonia monooxygenase (nitrification). The enrichment of specific DNA-binding domains indicated Calvin-cycle associated genetic regulation. Metabolic regulatory adaptations were illustrated by negative correlation to AraC and the enzyme arabinose-5-phosphate isomerase, which suggests a downregulation of the metabolite arabinose-5-phosphate, which may interfere with the Calvin cycle through enzyme inhibition and substrate competition. Certain domains of unknown function that were found to be important in the analysis may indicate yet unknown regulatory mechanisms in Calvin cycle-utilizing microbes. Our gene ranking provides targets for experiments seeking to improve CO₂ fixation, or engineer novel CO₂-fixing organisms.     

In vitro enzyme activities and products of 14CO2 assimilation in flag leaf and ear parts of wheat (Triticum aestivum L.)

Activities of key enzymes of Calvin cycle and C₄ metabolism, rate of ¹⁴CO₂ fixation in light and dark and the initial products of photosynthetic ¹⁴CO₂ fixation were determined in flag leaf and different ear parts of wheat viz. pericarp, awn and glumes. Compared to the activities of RuBP carboxylase and other Calvin cycle enzymes viz. NADP-glyceraldehyde-3-phosphate dehydrogenase, NAD-glyceraldehyde-3-phosphate dehydrogenase and ribulose-5-phosphate kinase, the levels of PEP carboxylase and other enzymes of C₄ metabolism viz. NADP-malate dehydrogenase, NAD-malate dehydrogenase, NADP-malic enzyme, NAD-malic enzyme, glutamate oxaloacetate transaminase genase, NADP-malic enzyme, NAD-malic enzyme, glutamate oxaloacetate transaminase and glutamate pyruvate transaminase, were generally greater in ear parts than in the flag leaf. In contrast to CO₂ fixation in light, the various ear parts incorporated CO₂ in darkness at much higher rates than flag leaf. In short term assimilation of ¹⁴CO₂ by illuminated ear parts, most of the ¹⁴C was in malate with less in 3-phosphoglyceric acid, whereas flag leaves incorporated most into 3-phosphoglyceric acid. It seems likely that ear parts have the capability of assimilating CO₂ by the C₄ pathway of photosynthesis and utilise PEP carboxylase for recapturing the respired CO₂.     

Diurnal modulation of phosphoenolpyruvate carboxylation in pea leaves and roots as related to tissue malate concentrations and to the nitrogen source

Phosphoenolpyruvate (PEP) carboxylation was measured as dark ¹⁴CO₂ fixation in leaves and roots (in vivo) or as PEP carboxylase (PEPCase) activity in desalted leaf and roof extracts (in vitro) from Pisum sativum L. cv. Kleine Rheinländerin. Its relation to the malate content and to the nitrogen source (nitrate or ammonium) was investigated. In tissue from nitrate-grown plants, PEP carboxylation varied diurnally, showing an increase upon illumination and a decrease upon darkening. Diurnal variations in roots were much lower than in leaves. Fixation rates in leaves remained constantly low in continuous darkness or high in continuous light. Dark CO₂ fixation of leaf slices also decreased when leaves were preilluminated for 1 h in CO₂-free air, suggesting that the modulation of dark CO2 fixation was related to assimilate availability in leaves and roots. Phosphoenolpyruvate carboxylase activity was also measured in vitro. However, no difference in maximum enzyme activity was found in extracts from illuminated or darkened leaves, and the response to substrate and effectors (PEP, malate, glucose-6-phosphate, pH) was also identical. The serine/threonine protein kinase inhibitors K252b, H7 and staurosporine, and the protein phosphatase 2A inhibitors okadaic acid and cantharidin, fed through the leaf petiole, did not have the effects on dark CO₂ fixation predicted by a regulatory system in which PEPCase is modulated via reversible protein phosphorylation. Therefore, it is suggested that the diurnal modulation of PEP carboxylation in vivo in leaves and roots of pea is not caused by protein phosphorylation, but rather by direct allosteric effects. Upon transfer of plants to ammonium-N or to an N-free nutrient solution, mean daily malate levels in leaves decreased drastically within 4–5 d. At that time, the diurnal oscillations of PEP carboxylation in vivo disappeared and rates remained at the high light-level. The coincidence of the two events suggests that PEPCase was de-regulated because malate levels became very low. The drastic decrease of leaf malate contents upon transfer of plants from nitrate to ammonium nutrition was apparently not caused by increased amino acid or protein synthesis, but probably by higher decarboxylation rates.Abbreviations CAM crassulacean acid metabolism - PEP Phosphoenolpyruvate - PEPCase phosphoenolpyruvate carboxylase - PP protein phosphatase - PK protein kinase This work was supported by the Deutsche Forschungsgemeinschaft. B. Baur was a recipient of a doctoral grant, and L. Leport recipient of a post-doctoral grant of the DFG. The skilled technical assistance of Eva Wirth and Maria Lesch is gratefully acknowledged.     

Carbon Dioxide Fixation by Lupin Root Nodules: I. Characterization, Association with Phosphoenolpyruvate Carboxylase, and Correlation with Nitrogen Fixation during Nodule Development

In vivo CO₂ fixation and in vitro phosphoenolpyruvate (PEP) carboxylase levels have been measured in lupin (Lupinus angustifolius L.) root nodules of various ages. Both activities were greater in nodule tissue than in either primary or secondary root tissue, and increased about 3-fold with the onset of N₂ fixation. PEP carboxylase activity was predominantly located in the bacteroid-containing zone of mature nodules, but purified bacteroids contained no activity. Partially purified PEP carboxylases from nodules, roots, and leaves were identical in a number of kinetic parameters. Both in vivo CO₂ fixation activity and in vitro PEP carboxylase activity were significantly correlated with nodule acetylene reduction activity during nodule development. The maximum rate of in vivo CO₂ fixation in mature nodules was 7.9 nmol hour⁻¹ mg fresh weight⁻¹, similar to rates of N₂ fixation and reported values for amino acid translocation.     

Carbon dioxide fixation in isolated kalanchoe chloroplasts

Chloroplasts isolated from Kalanchoe diagremontiana leaves were capable of photosynthesizing at a rate of 5.4 μmoles of CO₂ per milligram of chlorophyll per hour. The dark rate of fixation was about 1% of the light rate. A high photosynthetic rate was associated with low starch content of the leaves. Ribose 5-phosphate, fructose 1,6-diphosphate, and dithiothreitol stimulated fixation, whereas phosphoenolpyruvate and azide were inhibitors. The products of CO₂ fixation were primarily those of the photosynthetic carbon reduction cycle.     

Diel Interactions of Oxygenic Photosynthesis and N2 Fixation (Acetylene Reduction) in a Marine Microbial Mat Community

Diel variations in N₂ fixation (acetylene reduction), CO₂ fixation, and oxygen concentrations were measured, on three separate occasions, in a marine microbial mat located on Shackleford Banks, North Carolina. Nitrogenase activity (NA) was found to be inversely correlated with CO₂ fixation and, in two of the three diel periods studied, was higher at night than during the day. Oxygen concentrations within the top 3 mm of the mat ranged from 0 to 400 μM on a diel cycle; anaerobic conditions generally persisted below 4 mm. NA in the mat was profoundly affected by naturally occurring oxygen concentrations. Experimentally elevated oxygen concentrations resulted in a significant depression of NA, whereas the addition of the Photosystem II inhibitor 3(3,4-dichlorophenyl)-1,1-dimethylurea decreased oxygen concentrations within the mat and resulted in a significant short-term enhancement of NA. Mat N₂-fixing microorganisms include cyanobacteria and heterotrophic, photoautotrophic, and chemolithotrophic eubacteria. Measured (whole-mat) NA is probably due to a combination of the NA of each of these groups of organisms. The relative contributions of each group to whole-mat NA probably varied during diel and seasonal (successional) cycles. Reduced compounds derived from photosynthetic CO₂ fixation appeared to be an important source of energy for NA during the day, whereas heterotrophic or chemolithotrophic utilization of reduced compounds appeared to be an important source of energy for NA at night, under reduced ambient oxygen concentrations. Previous estimates of N₂ fixation calculated on the basis of daytime measurements may have seriously underestimated diel and seasonal nitrogen inputs in mat systems.     

Contemporaneous N2 Fixation and Oxygenic Photosynthesis in the Nonheterocystous Mat-Forming Cyanobacterium Lyngbya aestuarii

The nonheterocystous filamentous cyanobacterial genus Lyngbya is a widespread and frequently dominant component of marine microbial mats. It is suspected of contributing to relatively high rates of N₂ fixation associated with mats. The ability to contemporaneously conduct O₂-sensitive N₂ fixation and oxygenic photosynthesis was investigated in Lyngbya aestuarii isolates from a North Carolina intertidal mat. Short-term (<4-h) additions of the photosystem II (O₂ evolution) inhibitor 3(3,4-dichlorophenyl)-1,1-dimethylurea stimulated light-mediated N₂ fixation (nitrogenase activity), indicating potential inhibition of N₂ fixation by O₂ production. However, some degree of light-mediated N₂ fixation in the absence of 3(3,4-dichlorophenyl)-1,1-dimethylurea was observed. Electron microscopic immunocytochemical localization of nitrogenase, coupled to microautoradiographic studies of ¹⁴CO₂ fixation and cellular deposition of the tetrazolium salt 2,4,5-triphenyltetrazolium chloride, revealed that (i) nitrogenase was widely distributed throughout individual filaments during illuminated and dark periods, (ii) ¹⁴CO₂ fixation was most active in intercalary regions, and (iii) daylight 2,4,5-triphenyltetrazolium chloride reduction (formazan deposition) was most intense in terminal regions. Results suggest lateral partitioning of photosynthesis and N₂ fixation during illumination, with N₂ fixation being confined to terminal regions. During darkness, a larger share of the filament appears capable of N₂ fixation.     

Interrelationships between Water and Cellular Metabolism in Artemia cysts. V. 14CO2 incorporation

The ability of cysts of the brine shrimp, Artemia salina, to incorporate ¹⁴CO₂ into organic compounds soluble in cold-trichloroacetic acid was examined over a broad range of cellular water concentrations. Carbon dioxide was not incorporated by cysts containing less than about 0.3 g H₂O/g dried cysts, the “critical hydration” for CO₂-fixation. This relationship held whether the cysts were hydrated from the liquid or the vapor phase. The incorporation of radioactivity was shown to be due exclusively to metabolic activity in the cellular component of the cyst. Above the critical hydration, the amount of ¹⁴CO₂ incorporated was a function of cyst water content, but the kinds of metabolites labelled with this precursor, and their relative proportions, were found to be similar in cysts of greatly different hydration. Almost all of the radioactivity was associated with amino acids, Krebs cycle intermediates and related acids, and pyrimidine nucleotides. The fact that the pathway involved with CO₂-fixation, and subsequent metabolism of the fixation products are all initiated in cysts containing as little as 0.3 g H₂O/g is particularly noteworthy since this hydration level is well within the range of the amounts of “bound water” described in the literature for a wide array of cells and tissues.     

A pathway of the autotrophic CO2 fixation in Chloroflexus aurantiacus

Autotrophically grown cells of Chloroflexus aurantiacus B-3 were shown to possess activity of ATP-dependent malate lyase (acetylating CoA). ATP: malate lyase is supposed to be the specific enzyme of the cycle of the autotrophic CO₂ fixation, in which pyruvate synthase, pyruvate phosphate dikinase, phosphoenolpyruvate (PEP) carboxylase and malate dehydrogenase are involved as well. The main product of the CO₂ fixation cycle is glyoxylate, which could further be converted into 3-phosphoglyceric acid (3-PGA) in the reactions of either glycerate or serine pathway. The enzymes of both pathways were detected in C. auratiacus B-3. The results of the in vivo studies of glyxoylate and glycine metabolism, as well as the inhibitor analysis using fluoroacetate (FAc), isonicotinic acid hydrazide (INH), and 4-aminopterin (4-AP) confirm the operation of the proposed pathway in Chloroflexus.Abbreviations 3-PGA 3-phosphoglyceric acid - 4-AP 4-aminopterin - FAc fluoroacetate - INH isonicotinic acid hydrazide - MV methyl viologen - PEP phosphoenolpyruvate - THF tetrahydrofolate - TPP thiamine pyrophosphate     

Autotrophic growth and CO2 fixation of Chloroflexus aurantiacus

Chlorofluexus aurantiacus OK-70 fl was grown photoautotrophically with hydrogen as the electron source. The lowest doubling time observed was 26 h.The mechanism of CO₂ fixation in autotrophically grown cells was studied. The presence of ribulose-1,5-bis-phosphate carboxylase and phosphoribulokinase could not be demonstrated. Carbon isotope fractionation (¹³C) was small, and alanine and aspartate but not 3-phosphoglycerate were the major labelled compounds in short term ¹⁴CO₂ labelling. Thus CO₂ is not fixed by the Calvin cycle.Fluoroacetate (FAc) completely inhibited protein synthesis in cultures and caused a slight citrate accumulation. However, CO₂ fixation continued and increased polyglucose formation occurred. Under these conditions added acetate was metabolized to polyglucose, as were glycine, serine, glyoxylate and succinate, but to a lesser extent; little or no formate or CO was utilised.Glyoxylate inhibited CO₂ fixation in vivo, indicating that pyruvate is formed from acetyl-CoA and CO₂ by pyruvate synthase. Two key enzymes of the reductive TCA cycle, citrate lyase and -ketoglutarate synthase were not detected in cell free extracts, but pyruvate synthase and phosphoenolpyruvate carboxylase were demonstrated. It is concluded that acetyl-CoA is a central intermediate in the CO₂ fixation process, but the mechanism of its synthesis is not clear.Abbreviations Rubisco ribulose-1,5-bisphosphate carboxylase - TCA cycle tricarboxylic acid cycle - FAc monofluoroacetate - PEP phosphoenolpyruvate - MV methyl viologen - TTC triphenyltetrazolium chloride - PMS phenazine methosulfate     

Carbon Metabolism of Filamentous Anoxygenic Phototrophic Bacteria of the Family Oscillochloridaceae

The carbon metabolism of representatives of the family Oscillochloridaceae (Oscillochloris trichoides DG6 and the recent isolates Oscillochloris sp. R, KR, and BM) has been studied. Based on data from an inhibitory analysis of autotrophic CO₂ assimilation and measurements of the activities of the enzymes involved in this process, it is concluded that, in all Oscillochloris strains, CO₂ fixation occurs via the operation of the Calvin cycle. Phosphoenolpyruvate (PEP), which is formed in this cycle, can be involved in the metabolism via the following reaction sequence: PEP (+CO₂) å oxalacetate å malate å fumarate å succinate å succinyl-CoA (+CO₂) å 2-oxoglutarate. Acetate, utilized as an additional carbon source, can be carboxylated to pyruvate by pyruvate synthase and further involved in the metabolism via the above reaction sequence. Propionyl-CoA synthase and malonyl-CoA reductase, the key enzymes of the 3-hydroxypropionate cycle, have not been detected in Oscillochloris representatives.__________Translated from Mikrobiologiya, Vol. 74, No. 3, 2005, pp. 305–312.Original Russian Text Copyright © 2005 by Berg, Keppen, Krasil’nikova, Ugol’kova, Ivanovsky.     

CARBOHYDRATE AND AMINO ACID METABOLISM IN RAT CEREBRAL CORTEX IN MODERATE AND EXTREME HYPERCAPNIA

—The time course of changes in glycolytic and citric acid cycle intermediates and in amino acids was studied in acute and steady state hypercapnia. Experiments on unanaesthetized animals exposed to 10% CO₂ for 10, 20 and 60s showed that there was a transient decrease in glycogen concentration, progressive increases in glucose-6-phosphate and fructose-6-phosphate and decreases in pyruvate and lactate. During this time the levels of amino acids and Krebs cycle intermediates did not change, except for a small fall in malate at 60s. The results indicate that there was a decrease in glycolytic flux due to an inhibition of the phosphofructokinase reaction. Since the tissue levels of phosphocreatine, ATP, ADP and AMP were unchanged inhibition of phosphofructokinase was probably due to the fall in pH. Anaesthetized animals were exposed to about 5% CO₂ (for 2, 5, 15, 30 and 60 min) or to about 45% CO₂ (for 5 and 15 min). Except for succinate, which increased, all citric acid cycle metabolites analysed (citrate, α-ketoglutarate, fumarate and malate) decreased with the rise in CO₂-tension. The sum of the amino acids analysed (glutamate, glutamine, aspartate, asparagine, alanine and GABA) decreased at extreme hypercapnia. The results suggest that Krebs cycle intermediates and amino acids are partly used as substrates for energy production when there is reduced pyruvate availability due to hypercapnia. It is proposed that amino acid carbon is made available for oxidation via transamination (aspartate aminotransferase reaction) and deamination (glutamate dehydrogenase reaction) and that citric acid cycle intermediates are metabolized following a reversal of reactions usually leading to CO₂ fixation.     

Crassulacean acid metabolism (CAM) in leaves of Aloe arborescens mill

In the succulent leaves of Aloe arborescens Mill diurnal oscillations of the malic acid content, being indicative of Crassulacean Acid Metabolism (CAM), were exhibited only by the green mesophyll. In contrast, the malic acid level of the central chloroplast-free water-storing tissue remained constant throughout the day-night cycle. Apart from malate, the green tissue contained high amounts of isocitrat which was lacking in the water tissue. There was no significant transfer from the green mesophyll to the water tissue of ¹⁴C fixed originally via dark ¹⁴CO₂ fixation in the mesophyll. Both isolated mesophyll and water tissue were capable of dark CO₂ fixation yielding mainly malate as the first stable product. Both tissues have phosphoenolpyruvate carboxylase. However, the enzymes derived from the both sources could be distinguished by their molecular weights and by their kinetic properties, suggesting different phosphoenolpyruvate carboxylase proteins. The conclusion drawn from the experiments is that in a. arborescens the CAM cycle proceeds exclusively in the green mesophyll and that the water tissue, though capable of malate synthesis via -carboxylation of phosphoenolpyruvate, behaves as an independent metabolic system where CAM is lacking. This view is supported by the finding that the cell walls bordering the green mesophyll from the water tissue lack plasmodesmata, hence conveniant pathways of metabolite transport.Abbreviations CAM Crassulacean acid metabolism - PEP phosphoenolpyruvate - PEP-C phosphoenolpyruvate carboxylase     

ATP-dependent carbon transport in perfused Chara cells

Carbon transport across the plasma membrane, and carbon fixation were measured in perfused Chara internodal cells. These parameters were measured in external media of pH 5·5 and pH 8·5, where CO₂ and HCO₃^- are, respectively, the predominant carbon species in both light and dark conditions. Cells perfused with medium containing ATP could utilize both CO₂ and HCO₃^- from the external medium in the light. Photosynthetic carbon fixation activity was always higher at pH 5·5 than at pH 8·5. When cells were perfused either with medium containing hexokinase and 2-deoxyglucose to deplete ATP from the cytosol (HK medium) or with medium containing vanadate, a specific inhibitor of the plasma membrane H⁺_-ATPase (V medium), photosynthetic carbon fixation was strongly inhibited at both pH 5·5 and 8·5. Perfusion of cells with medium containing pyruvate kinase and phosphoenolpyruvate (PEP) to maximally activate the H⁺_-ATPase (PK medium), stimulated the photosynthetic carbon fixation activities. Oxygen evolution of isolated chloroplasts and the carbon fixation of cells supplied ¹⁴C intracellularly were not inhibited by perfusion media containing either hexokinase and 2-deoxyglucose or vanadate. The results indicate that Chara cells possess CO₂ and HCO₃^- transport systems energized by ATP and sensitive to vanadate in the light. In the dark, intact cells also fix carbon. By contrast, in cells perfused with medium containing ATP, no carbon fixation was detected in 1 mol m ^-3 total dissolved inorganic carbon (TDIC) at pH 8·5. By increasing TDIC to 10 mol m^-3, dark fixation became detectable, although it was still lower than that of intact cells at 1mol m^-3 TDIC. Addition of PEP or PEP and PEP carboxylase to the perfusion media significantly increased the dark-carbon fixation. Perfusion with vanadate had no effect on the dark-carbon fixation.     

Metabolic Interactions Between Glucose, Glycerol, Alanine and Acetate in Leishmania braziliensis panamensis Promastigotes

¹³C-nuciear magnetic resonance (NMR) spectroscopy was used to investigate the products of glycerol and acetate metabolism released by Leishmania braziliensis panamensis promastigotes and also to examine the interaction of each of these substrates with glucose or alanine. The NMR data were supplemented by measurements of the rates of oxygen consumption and substrate utilization, and of ¹⁴CO₂ production from ¹⁴C-labeIed substrate. Cells incubated with [2-¹³C]glycerol released acetate, succinate and D-lactate in addition to CO₂. Cells incubated with acetate released only CO₂. More succinate C-2/C-3 than C-l/C-4 was released from both [2-¹³C]glycerol and [2-¹³C]glucose, indicating that succinate was formed predominantly by CO₂ fixation followed by reverse flux through part of the Krebs cycle. Some redistribution of the position of labeling was also seen in alanine and pyruvate, suggesting cycling through pyruvate/oxaloacetate/phosphoenolpyruvate. Cells incubated with combinations of 2 substrates consumed oxygen at the same rate as cells incubated with 1 or no substrate, even though the total substrate utilization had increased. When promastigotes were incubated with both glycerol and glucose, the rate of glucose consumption was unchanged but glycerol consumption decreased about 50%, and the rate of ¹⁴CO₂ production from [l,(3)-¹⁴C]glycerol decreased about 60%. Alanine did not affect the rates of consumption of glucose or glycerol, but decreased ¹⁴CO₂ production from these substrates by increasing flow of label into alanine. Although glucose decreased alanine consumption by 70%, it increased the rate of ¹⁴CO₂ production from [U-¹⁴C]- and [l-¹⁴C]alanine by about 20%. This is consistent with rapid equilibration of alanine with pyruvate derived from glucose and yet little decrease in the specific activity of the large alanine pool.     

Regulation of oxaloacetate, aspartate, and malate formation in mesophyll protoplast extracts of three types of c(4) plants

The use of mesophyll protoplast extracts from various C₄ species has provided an effective method for studying light-and substrate-dependent formation of oxaloacetate, malate, and asparate at rates equivalent to whole leaf C₄ photosynthesis. Conditions regulating the formation of the C₄ acids were studied with protoplast extracts from Digitaria sanguinalis, an NADP-malic enzyme C₄ species, Eleusineindica, an NAD-malic enzyme C₄ species, and Urochloa panicoides, a phosphoenolpyruvate (PEP) carboxykinase C₄ species. Light-dependent induction of CO₂ fixation by the mesophyll extracts of all three species was relatively low without addition of exogenous substrates. Pyruvate, alanine and α-ketoglutarate, or 3-phosphoglycerate induced high rates of CO₂ fixation in the mesophyll extracts with oxaloacetate, malate, and aspartate being the primary products. In all three species, it appears that pyruvate, alanine, or 3-phosphoglycerate may serve as effective precursors to the formation of PEP for carboxylation through PEP-carboxylase in C₄ mesophyll cells. Induction by pyruvate or alanine and α-ketoglutarate was light-dependent, whereas 3-phosphoglycerate-induced CO₂ fixation was not.     

The role of the Krebs cycle in the generation of intramitochondrial reducing equivalents for the 11 beta-hydroxylation of deoxycorticosterone in isolated rat adrenal cells

Isolated rat adrenal cells were used to study the possible pathways of intramitochondrial NADPH generation for 11β-hydroxylation of 11-deoxycorticosterone. Pyruvate was efficiently utilized by the mitochondria as shown by evolution of ¹⁴CO₂ from [1-¹⁴C]- and [2-¹⁴C]pyruvate. Citrate, isocitrate, succinate, and malate were not utilized by intact cells due to their inability to permeate the plasma membrane. For every mole of corticosterone formed, 1.9 and 0.8 moles of ¹⁴CO₂ were formed from [1-¹⁴C]- and [2-¹⁴C]pyruvate, respectively, indicating that pyruvate dehydrogenase was quite active and supplied acetyl C?oA to the Krebs cycle. Fluorocitrate and 2,4-dinitrophenol inhibited 11β-hydroxylation of 11-deoxycorticosterone as well as the production of ¹⁴CO₂ from [2-¹⁴C]pyruvate. Comparison of data with the two inhibitors showed that for the same percentage of inhibition of ¹⁴CO₂ production, the inhibition of 11β-hydroxylation was greater with 2,4-dinitrophenol than with fluorocitrate. It is concluded that operation of the Krebs cycle may be essential for 11β-hydroxylation to occur primarily because NADH generated by the cycle provides ATP, via the respiratory chain, as well as the substrate for the energy-linked transhydrogenase that forms NADPH. The NADPH required for 11β-hydroxylation seems to be derived to a large extent via the energy-linked transhydrogenase.     

Conditionally lethal mutations in Chinese hamster cells. Characterization of a cell line with a possible defect in the krebs cycle

A variant Chinese hamster cell line has been isolated from a mutagenized population that has a markedly reduced ability to oxidize a variety of substrates via the Krebs cycle. The production of ¹⁴CO₂ from ¹⁴C-labeled compounds was measured using pyruvate, acetate, β-hydroxybutyrate, palmitate and glutamate, and in all cases it was negligible in the mutant. In contrast to this, significant amounts of ¹⁴CO₂ were produced from ¹⁴C-aspartate and ¹⁴C-succinate which suggests that some reactions of the Krebs cycle can take place and this conclusion is supported by tracer experiments with labeled compounds. The rate of respiration measured with a Clark oxygen electrode in the mutant was compared to several normal Chinese hamster cell lines and was found to be only 8%. Mitochondria appear to be present in normal numbers and with only minor differences in morphology. The measurement of difference spectra between oxidized and reduced states permits us to conclude that the cytochromes are all present and functional. These results lead us to believe that there may be a defect in the Krebs cycle between α-ketoglutarate and succinate. Alternatively a defect in a structural component of the mitochondria or in the electron-transport chain itself may be causing pleiotropic effects in the Krebs cycle and respiration.