The effect of cosubstrates on tricarboxylic acid cycle dynamics during pyruvate oxidation: the formation of alpha-ketoglutarate and utilization of glutamate by mitochondria from rabbit brain.

—Data comparing tricarboxylic acid cycle dynamics in mitochondria from rabbit brain using [2- or 3-¹⁴C]pyruvate with and without cosubstrates (malate, α-ketoglutarate, glutamate) are reported. With a physiological concentration of an unlabelled cosubstrate, from 90-99% of the isotope remained in cycle intermediates. However, the liberation of ¹⁴CO₂ and the presence of ¹⁴C in the C-1 position of α-ketoglutarate indicated that multiple turns of the cycle occurred. Entry of pyruvate into the cycle was greater with malate than with either α-ketoglutarate or glutamate as cosubstrate. With malate as cosubstrate for [¹⁴C]pyruvate the amount of [¹⁴C]citrate which accumulated averaged 30nmol/ml or 23% of the pyruvate utilized while α-ketoglutarate averaged 45 nmol/ml or 35% of the pyruvate utilized. With α-ketoglutarate as cosubstrate for [¹⁴C]pyruvate, the average amount of [¹⁴C]citrate which accumulated decreased to 8 nmol/ml or 10% of the pyruvate utilized while [¹⁴C]α-ketoglutarate increased slightly to 52 nmol/ml or an increase to 62%, largely due to a decrease in pyruvate utilization. The percentage of ¹⁴C found in α-ketoglutarate was always greater than that found in malate, irrespective of whether α-ketoglutarate or malate was the cosubstrate for either [2- or 3-¹⁴C]pyruvate. The fraction of ¹⁴CO₂ produced was slightly greater with α-ketoglutarate as cosubstrate than with malate. This observation and the fact that malate had a higher specific activity than did α-ketoglutarate when α-ketoglutarate was the cosubstrate, indicated a preferential utilization of α-ketoglutarate formed within the mitochondria. When l -glutamate was a cosubstrate for [¹⁴C]pyruvate the principal radioactive product was glutamate, formed by isotopic exchange of glutamate with [¹⁴C] α-ketoglutarate. If malate was also added, [¹⁴C]citrate accumulated although pyruvate entry did not increase. Due to retention of isotope in glutamate, little [¹⁴C]succinate, malate or aspartate accumulated. When [U-¹⁴C]l -glutamate was used in conjunction with unlabelled pyruvate more ¹⁴C entered the cycle than when unlabelled glutamate was used with [¹⁴C]pyruvate and led to α-ketoglutarate, succinate and aspartate as the major isotopic products. When in addition, unlabelled malate was added, total and isotopic α-ketoglutarate increased while [¹⁴C]aspartate decreased. The increase in [¹⁴C]succinate when [¹⁴C] glutamate was used indicated an increase in the flux through α-ketoglutarate dehydrogenase and was accompanied by a decrease of pyruvate utilization as compared to experiments when either α-ketoglutarate or glutamate were present at low concentration. It is concluded that the tricarboxylic acid cycle in brain mitochondria operates in at least three open segments, (1) pyruvate plus malate (oxaloacetate) to citrate; (2) citrate to α-ketoglutarate and; (3) α-ketoglutarate to malate, and that at any given time, the relative rates of these segments depend upon the substrate composition of the environment of the mitochondria. These data suggest an approach to a steady state consistent with the kinetic properties of the tricarboxylic acid cycle within the mitochondria.     

Microbiological Studies of Coli-aerogenes Bacteria

α-Ketoglutarate was obtained in a very small amount by the oxidative fermentation of acetate with either a growing culture or the washed cells of Escherichia coli. This microorganism was also observed to accumulate a considerable amount of α-ketoglutarate as the oxidation-product of C₄-dicarboxylic acids such as succinate, fumarate, malate and oxalacetate. The addition of acetate to the reaction mixtures containing either C₃- or C₄-acids brought about an increase in the yield of α-ketoglutarate. The bacteria of coli-aerogenes revealed an ability of oxidizing tricarboxylic acids under suitable conditions, but there was no noticeable production of α-ketoglutarate. The formation of glyoxylate was observed to occur during the degradation of citrate by the bacteria of coli-aerogenes. Finally, a cyclic mechanism of aerobic carbon-metabolism in the bacteria was propounded and discussed.     

Studies on the mechanism of acetate oxidation by bacteria. V. evidence for the participation of fumarate,malate, and oxalacetate in the oxidation of acetic acid by Escherichia coli

1. Simultaneous oxidation of C¹⁴-methyl-labeled acetate, and unlabeled malate or fumarate and α-ketoglutarate results in entrapment of labeled carbon in the C₄-dicarboxylic acids, but not in α-ketoglutarate, although all substrates are utilized at comparable rates. 2. A large endogenous reduction of all C₄-dicarboxylic acids (fumarate, oxalacetate, and malate) to succinate is observed under aerobic conditions, and when vigorous oxidation is proceeding. This effect occurs with both freshly harvested young (18 hour) cells and stored (2 week) cells. 3. This reduction can be considerably minimized under high oxygen tensions. 4. The quantitative concordance of these results with a Thunberg-Knoop cyclic mechanism for acetate oxidation is shown. Possible alternative C₄ products formed prior to succinate are not completely excluded, but it appears that the cells can utilize the succinate condensation as a major pathway in acetate oxidation.     

Ionic control of renal gluconeogenesis IV. Effect of extracellular phosphate concentration

Isolated rat renal tubules from glucose from pyruvate, malate, glycerol and α-ketoglutarate. The rate of glucose formation from all but glycerol is enhanced by an increase in Ca²⁺ concentration. Because changes in inorganic phosphate concentrations influence the uptake and retention of calcium by isolated cells, the effect of changes in phosphate concentration upon renal gluconeogenesis was examined. It was found that changing phosphate concentration altered the metabolism of isolated rat renal tubules in three ways which dependend upon the Ca²⁺ concentration. In the absence of Ca²⁺, increasing phosphate concentration from 0.07 to 1.2 mM led to a stimulation of the decarboxylation of [U-¹⁴C]malate, [1-14C]pyruvate, [2-¹⁴C]-pyruvate, α-keto[5-¹⁴C]glutarate and [1,3-¹⁴C₂]glycerol, and to an increase in ATP concentration but had no effect upon the rate of glucose formation from malate, pyruvate, α-ketoglutarate but a slight stimulation of glucose production from glycerol. A further increase in phosphate above 1.2 mM had no effect on any of these parameters. In the presence of either low (0.2 mM) or high (2.0 mM) Ca²⁺, changing phosphate concentration had no effect upon the decarboxylation of any of these substrates except glycerol whose decarboxylation was stimulated by increasing medium phosphate concentration. In the presence of calcium, increasing phosphate concentration led to an inhibition of glucose formation from malate, pyruvate and α-ketoglutarate but not from glycerol. Also in the presence of calcium both parathyroid hormone and cyclic AMP stimulated glucose formation, and under these conditions increasing phosphate concentration led to an inhibition of glucose formation. In tubules treated with parathyroid hormone an increase in phosphate concentration from 0.07 to 6.0 mM led to a significant increase in cyclic AMP concentration even though the rate of glucose formation decreased.Analysis of metabolite concentrations and rates of substrates decarboxylations, under a variety of conditions, revealed that P_i altered renal gluconeogenesis at a site different from those controlled by changes in Ca²⁺ concentration. The P_i-control site was tentatively identified as the glyceraldehyde phosphate dehydrogenase-glycerate kinase reaction sequence. However, the effect of changing P_i concentration upon parathyroid hormone-induced alterations in cyclic AMP concentration could not be explained by this action of P_i, and was probably due to an effect of P_i upon cellular calcium distribution. Thus, changes in P_i concentration appear to have two cellular effects, only one of which is related to a change in cellular calcium metabolism.     

Properties of Wheat Mitochondria. Study of Substrates, Cofactors and Inhibitors

Isolated mitochondria of wheat shoots oxidize α- ketoglutarate, DL-malate succinate and NADH with good relative respiration control and ADP: O ratio. They have high affinity for α-ketoglutarate and NADH as substrates and utilize malate and succinate with a respiration ratio of about one-half of α-ketoglutarate. The average ADP : O ratios approach the expected theoretical values, i.e., 3.6 ± 0.2 for α-ketoglutarate, 1.8 ± 0.2 for succinate, and 2.8 ± 0.2 for malate. The ADP: O ratio with NADH is 1.8 ± 0.2. The maximum coupling of oxidation and phosphorylation is obtained at concentrations of 10 mM, 2 mM, 10 mM and 8 mM for α-ketoglutarate, NADH, malate and succinate, respectively. — Wheat mitochondria have little or no dependence on added cofactors. Mitochondria prepared by our procedure apparently retain sufficient amounts of endogenous cofactors required for NAD-linked systems. FAD⁺ is found to improve succinate oxidation. Cytochrome c does not have any significant effect on respiratory parameters of wheat mitochondria. — Wheat mitochondria are some -what resistant to DNP at 1.7 × 10^-5M. Malonate seems to improve coupling of α-ketoglutarate oxidation. Other Krebs cycle intermediates have been tested on three major substrates of TCA cycle, i.e., α-ketoglutarate, malate and succinate.     

delta-Aminolevulinic acid synthesis in a Cyanidium caldarium mutant unable to make chlorophyll a and phycobiliproteins.

Wild-type cells of the unicellular rhodophyte, Cyanidium caldarium, synthesize chlorophyll a, phycobiliproteins, and heme from δ-aminolevulinic acid during light-dependent chloroplast development but are unable to make photosynthetic pigments in the dark. C. caldarium, mutant GGB-Y, is an obligate heterotroph which, in the light, produces a chloroplast devoid of photosynthetic pigments. The present investigation has shown that δ-aminolevulinic acid is synthesized in cells of mutant GGB-Y incubated with levulinic acid, a competitive inhibitor of δ-aminolevulinic acid dehydrase (the second enzyme in the porphyrin biosynthetic pathway). In vivo, cells of mutant GGB-Y preferentially incorporated C₁ of glutamate and α-ketoglutarate into the C₅ fragment (formaldehyde) of δ-aminolevulinic acid after alkaline periodate degradation. This suggested that δ-aminolevulinic acid arises directly from the carbon skeleton of glutamate and α-ketoglutaric acid. The pattern of incorporation of C₃, C₄, and C₅ of α-ketoglutarate into the C₁–C₄ (succinic acid) fragment of δ-aminolevulinic acid after alkaline periodate degradation was consistent with the origin of δ-aminolevulinic acid from a five-carbon precursor. C₁ and C₂ of glycine and C₂ and C₃ of succinate were incorporated into both the formaldehyde and succinate fragments of δ-aminolevulinic acid in a manner inconsistent with condensation of glycine and succinyl CoA by δ-aminolevulinic acid synthetase, the rate-limiting enzyme in the porphyrin pathway in animals and bacteria. Extracts of the soluble protein from cells of mutant GGB-Y displayed a Soret band at 410 nm indicating the presence of hemoproteins. This shows that mutant GGB-Y cells synthesize heme. The respiration of radiolabeled glutamate, α-ketoglutarate, and glycine to ¹⁴CO₂ is consistent with the existence of mitochondrial cytochromes in cells of mutant GGB-Y and with the ability of the mutant to synthesize δ-aminolevulinic acid. The present results suggest that δ-aminolevulinic acid is synthesized directly from glutamate or α-ketoglutarate and that this is the only process by which the rate-limiting intermediate in the porphyrin pathway is synthesized in C. caldarium. If correct, the rate-limiting, regulative enzyme in the biosynthetic pathway for synthesis of chlorophyll a, bile pigment (phycocyanobilin), and heme must have been completely different in the evolutionary antecedents of modern-day plants and animals.     

Correlation of the effects of citric acid cycle metabolites on succinate oxidation by rat liver mitochondria and submitochondrial particles

1. Succinate dehydrogenase is inhibited by citrate and β-hydroxybutyrate in a complex manner, both in mitochondria and submitochondrial particles. Kinetics of inhibition in the particles points to a competitive component in the mechanism involved.
2. Pyruvate, α-ketoglutarate, malate, and glutamate stimulate oxidation of succinate by mitochondria.
3. Stimulation by α-ketoglutarate and glutamate is not influenced by the presence of rotenone.
4. Stimulation by pyruvate is higher in the absence of rotenone and increases significantly in the presence of K⁺ and valinomycin. Pyruvate supplies in mitochondria reducing equivalents for malate dehydrogenase operating in the reverse direction-reduction of oxaloacetate to malate.
5. Stimulation by malate is higher in the presence of rotenone.

     

Acute effects of ammonia on the enzymes of citric acid cycle in rat brain

Activities of the enzymes of citric acid cycle were determined along with aspartate and alanine aminotransferases and NADP⁺-isocitrate dehydrogenase in the brains of rats treated with an acute dose of ammonium acetate and compared with those of normal animals. Elevation in the activities of pyruvate, α-ketoglutarate and succinate dehydrogenases and citrate synthase was observed in hyperammonemic animals. The activities of malate, NADP⁺-isocitrate dehydrogenases and aminotransferases decreased under these conditions. The results suggest that ammonia toxicity might not be due to the depletion of α-ketoglutarate from citric acid cycle.     

Archenteron formation induced by ascorbate and alpha-ketoglutarate in sea urchin embryos kept in SO2- 4 -free artificial seawater

In permanent blastulae of the sea urchin, which were obtained by culture in SO^2?₄-free artificial seawater from the time of fertilization, ascorbate and α-ketoglutarate, activators of protocollagen proline hydroxylase, induced the formation of archenteron. By adding either ascorbate or α-ketoglutarate to the SO^2?₄-free culture at 12 hr of fertilization, spherical embryos with archenteron were obtained by successive 12 hr cultures at 20°C. The embryos thus obtained did not develop to plutei. Archenteron formation induced by these compounds in SO^2?₄-free-cultured embryos, as well as in the normal embryos, was inhibited by α,α′-dipyridyl, an inhibitor of protocollagen proline hydroxylase. Glutamate, malate, citrate, and fumarate did not stimulate archenteron formation in SO^2?₄-free cultured embryos. In the SO^2?₄-free-cultured embryos exposed to [¹⁴C]proline, considerable radioactivity was found in hot trichloroacetic acid-extractable proteins but the radioactivity of [¹⁴C]hydroxyproline residue, produced by hydroxylation of proline residue of protocollagen, was markedly lower than that in normal embryos. In the presence of ascorbate and α-ketoglutarate, the radioactivity of [¹⁴C]hydroxyproline residue became high and was lowered by α,α′-dipyridyl. Archenteron formation induced by ascorbate and α-ketoglutarate in the embryos kept in SO^2?₄-free artificial seawater probably results from the stimulated protocollagen hydroxylation.     

NADP-malic enzyme from maize leaf: regulatory properties.

The regulatory properties of purified maize leaf NADP-malic enzyme (EC 1.1.1.40) were studied at three different pHs and the following results were obtained. (a) At pH 7.5 enzyme activity reaches a maximum at 0.4–0.8 mm malate depending on the Mg²⁺ concentration, and higher levels of malate result in marked substrate inhibition; with increasing pH the degree of substrate inhibition is reduced to where at pH 8.4 little or no inhibition is observed. (b) The inhibitory effect of malate is more pronounced at 1 mm Mg²⁺ than at 5–10 mm Mg²⁺ in the pH range of 7.5 to 8.4; a plot of enzyme activity vs Mg²⁺ concentration at 3 mm malate follows Michaelis-Menten kinetics at both pH 7.5 and 8.4; the apparent affinity of the enzyme for Mg²⁺ at pH 8.4 was threefold greater than that at pH 7.5. (c) The activity of NADP-malic enzyme decreases as the ratio of $\text{NADPH}\text{NADP}$ increases, and this effect is enhanced at lower pH. (d) Various α-keto acids including glyoxylate, oxaloacetate, and α-ketoglutarate inhibit NADP-malic enzyme activity, whereas HCO₃^?, pyruvate, and other organic acids, sugar phosphates, and amino acids have little or no effect on the activity of the enzyme. Based on these experimental findings, the regulatory properties of maize leaf NADP-malic enzyme are discussed with respect to its key role in net CO₂ fixation in maize bundle sheath chloroplasts during C₄ photosynthesis.     

Activity of liver mitochondrial Krebs cycle NAD<Superscript>+</Superscript>-dependent dehydrogenases in rats with hepatitis induced by acetaminophen under conditions of alimentary protein deficiency

Activity of isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, malate dehydrogenase, and the NAD⁺/NADН ratio were studied in the liver mitochondrial fraction of rats with toxic hepatitis induced by acetaminophen under conditions of alimentary protein deficiency. Acetaminophen-induced hepatitis was characterized by a decrease of isocitrate dehydrogenase, α-ketoglutarate dehydrogenase and malate dehydrogenase activities, while the mitochondrial NAD⁺/NADН ratio remained at the control level. Modeling of acetaminophen-induced hepatitis in rats with alimentary protein deficiency caused a more pronounced decrease in the activity of studied Krebs cycle NAD⁺-dependent dehydrogenases and a 2.2-fold increase of the mitochondrial NAD⁺/NADН ratio.     

Effects of α-ketoglutarate on neutrophil intracellular amino and α-keto acid profiles and ROS production

The aim of this study was to determine the effects of α-ketoglutarate on neutrophil (PMN), free α-keto and amino-acid profiles as well as important reactive oxygen species (ROS) produced [superoxide anion (O₂ ^?), hydrogen peroxide (H₂O₂)] and released myeloperoxidase (MPO) acitivity. Exogenous α-ketoglutarate significantly increased PMN α-ketoglutarate, pyruvate, asparagine, glutamine, asparatate, glutamate, arginine, citrulline, alanine, glycine and serine in a dose as well as duration of exposure dependent manner. Additionally, in parallel with intracellular α-ketoglutarate changes, increases in O₂ ^– formation, H₂O₂-generation and MPO acitivity have also been observed. We therefore believe that α-ketoglutarate is important for affecting PMN “susceptible free amino- and α-keto acid pools” although important mechanisms and backgrounds are not yet completely explored. Moreover, our results also show very clearly that changes in intragranulocytic α-ketoglutarate levels are relevant metabolic determinants in PMN nutrition considerably influencing and modulating the magnitude and quality of the granulocytic host defense capability as well as production of ROS.     

Enzymes of Carbohydrate Metabolism in Four Human Species of Leishmania: A Comparative Survey*

SYNOPSIS. The occurrence and levels of activity of various enzymes of carbohydrate catabolism in culture forms (promastigotes) of 4 human species of Leishmania (L. brasiliensis, L. donovani, L. mexicana, and L. tropica) were compared. These organisms possess enzymes of the Embden-Meyerhof pathway but lack lactate dehydrogenase. No evidence could be found for the production of lactic acid by growing cultures and lactic acid could not be detected either in cell-free preparations or after incubation of cell-free extracts with pyruvate and NADH under appropriate conditions. All 4 species possess α-glycerophosphate dehydrogenase and α-glycerophosphate phosphatase which together could regenerate NAD, thus compensating for the absence of lactate dehydrogenase. The oxidative and nonoxidative reactions of the hexose monophosphate pathway are present in all 4 species. Cell-free extracts have pyruvate dehydrogenase activity which allows the entry of pyruvate into and its subsequent oxidation through the tricarboxylic acid cycle. All enzymes of this cycle, including a thiamine pyrophosphate dependent α-ketoglutarate dehydrogenase are present. Both NAD and NADP-linked malate dehydrogenase activities are present. The isocitrate dehydrogenase is NADP specific. There is an active glutamate dehydrogenase which could compete with α-ketoglutarate dehydrogenase for the common substrate (α-ketoglutarate). Replenishment of C₄ acids is accomplished by heterotrophic CO₂ fixation catalyzed by pyruvate carboxylase. All 4 species have high levels of NADH oxidase activity. Several enzymes thus far not found in any species of Leishmania have been demonstrated. These are: phosphoglucose isomerase, triose phosphate isomerase, fructose-1, 6-diphosphatase, 3-phosphoglycerate kinase, enolase, α-glycerophosphate dehydrogenase, α-glycerophosphate phosphatase, pyruvate dehydrogenase complex, citrate synthase, aconitase, α-ketoglutarate dehydrogenase, glutamate dehydrogenase, and NADH oxidase.     

Glutamate metabolism triggered by oxaloacetate in intact plant mitochondria

In Percoll-purified potato tuber mitochondria, glutamate metabolism can be triggered by oxaloacetate, in the presence of ADP and thiamine pyrophosphate. There is a lag phase before O₂ uptake is initiated. During this lag period, oxaloacetate is rapidly converted into α-ketoglutarate and succinate, or into malate at the expense of the NADH generated by α-ketoglutarate dehydrogenase. The ratio of the flux rates of both pathways is strongly dependent on the glutamate concentration in the medium. When all the oxaloacetate is consumed, a rapid O₂ uptake is initiated. The effects of malonate on glutamate metabolism triggered by oxaloacetate and on α-ketoglutarate oxidation are reported. It is concluded that the inhibition of the succinate dehydrogenase by either malonate or oxaloacetate does not affect the rate of α-ketoglutarate dehydrogenase functioning. All the metabolites accumulated are excreted by the mitochondria in the supernatant. Some of them are then reabsorbed. These results emphasize the importance of the anion carriers in the overall process.     

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.     

Utilization of Hydrocarbons and Vitamin B12 Production by Bacillus badius

The accumulation of vitamin B₁₂ by Bacillus badins grown on hydrocarbon was investigated. The bacterium could assimilate n-alkanes of C₁₁–C₁₈, ethanol, fumarate, α-ketoglutarate and malate. n-Alkanes of C₁₆–C₁₈, were the best for vitamin B₁₂ production. The bacterium utilized well all of the nitrogen sources tested. Above all, ammonium dihydrogen phosphate was the best for the bacteria] growth and vitamin B₁₂ production. Addition of organic nutrients such as malt extract and meat extract, and addition of metal ions such as ferrous and cobalt promoted the growth and vitamin B₁₂ production. Interestingly, vitamin B₁₂ was produced mostly in the supernatant. The cyanoform of the corrinoid predominantly formed in the supernatant would confirm the identity with cobalamin.     

Decarboxylation of malate by isolated bundle-sheath cells of certain plants having the C4-dicarboxylic acid cycle of photosynthesis

Summary Bundle-sheath cells isolated by the grinding and filtration procedure of Edwards and Black (1971b) from species of plants having the C₄-dicarboxylic acid pathway of photosynthesis were tested for the decarboxylation of malate from the C₄-carboxyl position. The bundle-sheath cells, which showed high malic enzyme activity in extracts, decarboxylated 4[¹⁴C]malate at rates sufficient to be involved in photosynthesis. The malate decarboxylation is dependent on the addition of magnesium or manganese and NADP⁺. The activity was increased by raising the temperature from 30 to 50°. The evidence supports the idea that malate may be a carboxyl donor to the reductive pentose-phosphate cycle in bundle-sheath cells in certain C₄-dicarboxylic acid pathway plants such as Zea mays L., Sorghum bicolor L., and Digitaria sanguinalis (L.) Scop.Abbreviations C₄ pathway C₄-dicarboxylic acid pathway - RPP pathway reductive pentose phosphate pathway - C₄ plants plants having the C₄ and the RPP pathways - C₃ plants plants having only the RPP pathway - R5P ribose-5-phosphate - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - Tricine N-tris-(hydroxymethyl)methylglycine     

C4-Dicarboxylic acid metabolism in bundle-sheath chloroplasts,mitochondria and strands of Eriochloa borumensis Hack., a phosphoenolpyruvate-carboxykinase type C4 species

C₄-acid metabolism by isolated bundlesheath chloroplasts, mitochondria and strands of Eriochloa borumensis Hack., a phosphoennolpyruvate-carboxykinase (PEP-CK) species, was investigated. Aspartate, oxaloacetate (OAA) and malate were decarboxylated by strands with several-fold stimulation upon illumination. There was strictly light-dependent decarboxylation of OAA and malate by the chloroplasts, but the chloroplasts did not decarboxylate aspartate in light or dark. PEP was a primary product of OAA or malate decarboxylation by the chloroplasts and its formation was inhibited by 3-(3,4-dichlorophenyl)-1, 1-dimethylurea or NH₄Cl. There was very little conversion of PEP to pyruvate by bundle-sheath chloroplasts, mitochondria or strands. Decarboxylation of the three C₄-acids by mitochondria was light-independent. Pyruvate was the only product of mitochondrial metabolism of C₄-acids, and was apparently transaminated in the cytoplasm since PEP and alanine were primarily exported out of the bundle-sheath strands. Light-dependent C₄-acid decarboxylation by the chloroplasts is suggested to be through the PEP-CK, while the mitochondrial C₄-acid decarboxylation may proceed through the NAD-malic enzyme (NAD-ME) system. In vivo both aspartate and malate are considered as transport metobolites from mesophyll to bundle-sheath cells in PEP-CK species. Aspartate would be metabolized by the mitochondria to OAA. Part of the OAA may be converted to malate and decarboxylated through NAD-ME, and part may be transported to the chloroplasts for decarboxylation through PEP-CK localized in the chloroplasts. Malate transported from mesophyll cells may serve as carboxyl donor to chloroplasts through the chloroplastic NAD-malate dehydrogenase and PEP-CK. Bundle-sheath strands and chloroplasts fixed ¹⁴CO₂ at high rates and exhibited C₄-acid-dependent O₂ evolution in the light. Studies with 3-mercaptopicolinic acid, a specific inhibitor of PEP-CK, have indicated that most (about 70%) of the OAA formed from aspartate is decarboxylated through the chloroplastic PEP-CK and the remaining (about 30%) OAA through the mitochondrial NAD-ME. Pyruvate stimulation of aspartate decarboxylation is discussed; a pyruvate-alanine shuttle and an aspartate-alanine shuttle are proposed between the mesophyll and bundle-sheath cells during aspartate decarboxylation through the PEP-CK and NAD-ME system respectively.Abbreviations CK carboxykinase - -Kg -ketoglutarate - ME malic enzyme - 3-MPA 3-mercaptopicolinic acid - OAA oxaloacetate - PEP phosphoenolpyruvate - R5P ribose-5-phosphate     

Effects of calcium ions and quinolinic acid on rat kidney mitochondrial kynurenine aminotransferase

At pH 6.4, rat kidney mitochondrial kynurenine aminotransferase activity is enhanced several-fold by the addition of CaCl₂, apparently because Ca⁺⁺ facilitates the translocation of α-ketoglutarate, one of the substrates, across the mitochondrial inner membrane. Chloride salts or Mg⁺⁺, Mn⁺⁺, Na⁺, K⁺, and NH₄⁺ did not have this effect. At pH 6.8, the enzyme activity was near maximal even without added Ca⁺⁺ but was strongly depressed by either of two calcium chelating agents, quinolinic acid (Q.A.) and ethyleneglycol-bis(β-aminoethyl ether)N,N′-tetraacetic acid (EGTA). These observations support the view that Ca⁺⁺ is involved in regulating kidney mitochondrial translocation of α-ketoglutarate and that the reported interference of polycarboxylate anion translocation by Q.A. $\text{in vivo}$ depends on the ability of that agent to chelate Ca⁺⁺.     

Utilization of malate or aspartate by a yeast lacking malate enzyme

Summary Hansenula anomala, a yeast lacking malate enzyme, was able to grow in media containing malate or aspartate as sole carbon and energy sources. Both aspartate--ketoglutarate transaminase and pyruvate kinase activities changed their levels when the yeast was grown on different carbon sources. Pyruvate kinase activity was increased by fructose 1,6-diphosphate.These results indicate that in this yeast malate enzyme is not indispensable for the formation of pyruvate from malate or aspartate and that C₄ dicarboxylic acids may provide pyruvate through the combined action of phosphoenolpyruvate carboxykinase and pyruvate kinase. It is also concluded that aspartate--ketoglutarate transaminase and pyruvate kinase are under regulatory control in Hansenula anomala.