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.     

Microbiological Studies of Coli-aerogenes Bacteria

During investigations on the metabolisms of glucose by coli-aerogenes bacteria, it was found that the bacteria accumulated a large amount of α-ketoglutaric acid under aerobic conditions such as shaking culture, while lactic acid was ascertained to be produced anaerobically by the bacteria as was already known.     

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.     

Acetate assimilation and the synthesis of alanine,aspartate and glutamate inMethanobacterium thermoautotrophicum

Cultures of the autotrophic bacteriumMethanobacterium thermoautotrophicum were shown to assimilate acetate when grown on CO₂ and H₂ in the presence of acetate. At 1 mM acetate 10% of the cell carbon came from acetate, the rest from CO₂. At higher concentrations the percentage increased to reach a maximum of 65%at acetate concentrations higher than 20 mM. The data suggest that acetate may be an important carbon source under physiological conditions.The incorporation of acetate into alanine, aspartate and glutamate was studied in more detail. The cells were grown on CO₂ and H₂ in the presence of 1 mM U-¹⁴C-acetate. The three amino acids were isolated from the labelled cells by a simplified procedure. Alanine, aspartate and glutamate were found to have the same specific radioactivity. Degradation studies showed that C₁ of alanine C₁ and C₄ of aspartate, and C₁ and C₅ of glutamate were exclusively derived from CO₂, whereas C₂ and C₃ alamine and aspartate, and C₃ and C₄ of glutamate were partially derived from acetate. These findings and the presence of pyruvate synthase, phosphoenolpyruvate carboxylase and -ketoglutarate synthase inM. thermoautotrophicum indicate that CO₂ is assimilated into the three amino acids via acetyl CoA carboxylation to pyruvate, phosphoenolpyruvate carboxylation to oxaloacetate, and succinyl CoA carboxylation to -ketoglutarate.     

Conversion of alpha-ketoglutaric-1,2-C214 acid to malic acid in pigeon breast muscle

Conversion of C¹⁴-labeled α-ketoglutarate to malate was tested with a pigeon breast muscle preparation. Complete randomization between the labeled carboxyl groups of the C₄-acids occurred during one pass from α-ketoglutarate to malate.     

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.     

Fermentation of n-Paraffins by Yeast

Nutritional requirement of Candida lipolytica AJ 5004 and its productivity of α-ketoglutarate were further studied.

It became clear that this yeast required only thiamine as grown factor, and even if the yeast was cultured in chemically defined medium containing adequate amount of thiamine, it was able to produce as high yield of α-ketoglutarate as in the medium containing 0.02% of corn steep liquor.

It was also shown that the rate of convertion of n-paraffin to α-ketoglutarate gradually increased as the concentration of n-paraffins was decreased or as the incubation time was prolonged. A very high rate of conversion, 71%, was obtained after prolonged culture, for 5 days, with a culture medium containing 8% of n-paraffins.

The productivity of α-ketoglutarate from C₉- to C₂₀-alkanes by the yeast was maximum in the range from C₁₅ to C₁₉, especially from C₁₇ to C₁₉.     

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.     

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.     

Similarity Between Rat Brain Nicotinic α-Bungarotoxin Receptors and Stably Expressed α-Bungarotoxin Binding Sites

Abstract: The present results demonstrate stable expression of α-bungarotoxin (α-BGT) binding sites by cells of the GH₄C₁ rat pituitary clonal line. Wild-type GH₄C₁ cells do not express α-BGT binding sites, nor do they contain detectable mRNA for nicotinic receptor α2, α3, α4, α5, α7, β2, or β3 subunits. However, GH₄C₁ cells stably transfected with rat nicotinic receptor α7 cDNA (α7/GH₄C₁ cells) express the transgene abundantly as mRNA, and northern analysis showed that the message is of the predicted size. The α7/GH₄C₁ cells also express saturable, high-affinity binding sites for ¹²⁵I-labeled α-BGT, with a K_D of 0.4 nM and B_max of 3.2 fmol/10⁶ intact cells. ¹²⁵I-α-BGT binding affinities and pharmacological profiles are not significantly different for sites in membranes prepared either from rat brain or α7/GH₄C₁ cells. Furthermore, K_D and K_i values for ¹²⁵I-α-BGT binding sites on intact α7/GH₄C₁ cells are essentially similar to those for hippocampal neurons in culture. Sucrose density gradient analysis showed that the size of the α-BGT binding sites expressed in α7/GH₄C₁ cells was similar to that of the native brain α-BGT receptor. Chronic exposure of α7/GH₄C₁ cells in culture to nicotine or an elevated extracellular potassium concentration induces changes in the number of α-BGT binding sites comparable to those observed in cultured neurons. Collectively, the present results show that the properties of α-BGT binding sites in transfected α7/GH₄C₁ cells resemble those for brain nicotinic α-BGT receptors. If the heterologously expressed α-BGT binding sites in the present study are composed solely of α7 subunits, the results could suggest that the rat brain α-BGT receptor has a similar homooligomeric structure. Alternatively, if α-BGT binding sites exist as heterooligomers of α7 plus some other previously identified or novel subunit(s), the data would indicate that the α7 subunits play a major role in determining properties of the α-BGT receptor.     

Decrease of phosphate concentration in the medium by <Emphasis Type="Italic">Brevibacterium casei</Emphasis> cells

Brevibacteria able to decrease phosphate concentration in the medium are of interest for the study of the role of bacteria in the phosphorus cycle and for development of biotechnology of phosphate removal from waste. Brevibacterium casei, Brevibacterium linens, and Brevibacterium epidermidis grown in media with initial phosphorus concentrations of 1–11 mM were shown to decrease its concentration by 90%. The composition of the incubation medium required for B. casei to carry out this process was established. This process occurs in the absence of glucose but requires the presence of Mg²⁺, NH ₄ ⁺ , and α-ketoglutarate. The latter two components may be replaced by amino acids metabolized to NH ₄ ⁺ and α-ketoglutarate: histidine, arginine, glutamine, proline, or glutamic acid. No formation of insoluble phosphate salts was observed when the media were incubated under the same conditions with heat-inactivated cells or without cells at pH 7–8.5.     

Generation of glyoxylate in methylotrophic bacteria

The mode of glyoxylate production from acetyl-CoA was investigated in three strains of methylotrophic bacteria,Pseudomonas MA,Pseudomonas AM1 and organism PAR. This investigation was prompted by the recently reported discovery of a homoisocitrate lyase in methylotrophic bacteria and the suggested involvement of this novel enzyme in assimilation of C₁ and C₂ compounds as part of a homoisocitrate-glyoxylate cycle. We were unable to detect cleavage of any of the four stereoisomers of homoisocitric acid by cell-free extracts of C₁-or C₂-grown bacteria. Extracts of C₁-grown bacteria did not catalyze condensation of glyoxylate with glutarate or production of glyoxylate from acetyl-CoA and 2-ketoglutarate. Extracts of C₁-grownPseudomonas MA catalyzed cleavage of isocitrate;threo-homoisocitrate was a potent competitive inhibitor of this reaction. These results indicate that homoisocitrate cleavage does not occur in any of the methylotrophs tested. The pathway for oxidation of acetyl-CoA to glyoxylate inPseudomonas AM1 and organism PAR therefore remains obscure.     

Total synthesis of acetyl coenzyme a involved in autotrophic CO2 fixation inAcetobacterium woodii

The Gram positive anaerobeAcetobacterium woodii is able to grow autotrophically with a mixture of H₂ and CO₂ as the energy and carbon source. The question, by which pathway CO₂ is assimilated, was studied using long term isotope labeling.Autotrophically growing cultures produced acetate parallel to cell proliferation, and, when U-[¹⁴C]acetate was present as tracer, incorporated radioactivity into all cell fractions. The specific radioactivity and the label positions were determined for those representative cell compounds which biosynthetically originated directly from acetyl CoA (N-acetyl groups), pyruvate (alanine), oxaloacetate (aspartate), -ketoglutarate (glutamate), and hexosephosphates (glucosamine). Per mol compound the same amount of labeled acetate was incorporated into N-acetyl groups, alanine (C-2, C-3), aspartate (C-2, C-3), and twice the amount into glutamate (C-2, C-3, C-4, C-5) and into glucosamine. Consequently, the unlabeled carbon atoms of the C₃–C₆ compounds must have been derived from CO₂ by carboxylation subsequent to acetyl CoA synthesis. When 0.2 mM 2-[¹⁴C]pyruvate was added to autotrophically growing cultures, also a substantial amount of radioactivity was incorporated. Two important differences in comparison to the acetate experiment were observed: The N-acetyl groups were almost unlabeled and glutamate contained the same specific radioactivity as alanine or aspartate.These data showed that acetyl CoA is the central intermediate for biosynthesis and excluded the operation of the Calvin cycle inA. woodii. The results were consistent with the operation of a different autotrophic CO₂ fixation pathway in which CO₂ is converted into acetyl CoA by total synthesis via methyltetrahydrofolate; acetyl CoA is then further reductively carboxylated to pyruvate.     

FATTY ACIDS AND HYDROXY FATTY ACIDS IN THREE SPECIES OF FRESHWATER EUSTIGMATOPHYTES

The monocarboxylic fatty acids and hydroxy fatty acids of three species of freshwater microalgae—Vischeria punctata Vischer, Vischeria helvetica (Vischer et Pascher) Taylor, and Eustigmatos vischeri (Hulbert) Taylor, all from the class Eustigmatophyceae— were examined. Each species displayed a very similar distribution of fatty acids, the most abundant of which were 20:5n-3, 16:0, and 16:1n-7; C₁₈ polyunsaturated fatty acids were minor components. These fatty acid distributions closely resemble those found in marine eustigmatophytes but are quite distinct from those found in most other algal classes. These microalgae also contain long-chain saturated and unsaturated monohydroxy fatty acids. Two distinct types of hydroxy fatty acids were found: a series of saturated α-hydroxy acids ranging from C₂₄ to C₃₀ with a shorter series of monounsaturated α-hydroxy acids ranging from C₂₆ to C₃₀ together with a series of saturated β-hydroxy acids ranging from C₂₆ to C₃₀. The latter have not previously been reported in either marine or freshwater microalgae, although C₃₀ to C₃₄ midchain (ω-18)-hydroxy fatty acids have been identified in hydrolyzed extracts from marine eustigmatophytes of the genus Nannochloropsis, and C₂₂ to C₂₆ saturated and monounsaturated α-hydroxy fatty acids have been found in three marine chlorophytes. These findings have provided a more complete picture of the lipid distributions within this little studied group of microalgae as well as a range of unusual compounds that might prove useful chemotaxonomic markers. The functions of the hydroxy fatty acids are not known, but a link to the formation of the lipid precursors of highly aliphatic biopolymers is suggested.     

Distribution of C22-, C24- and C26-α-Unit-Containing Mycolic Acid Homologues in Mycobacteria

There are three mycolic acid homologues with C₂₂-, C₂₄- and C₂₆-α-units in Mycobacterium. In order to reveal the composition and distribution of these homologues in each subclass and molecular species of mycolic acids and to compare them with the composition of constitutive non-polar fatty acids (free and bound forms), we have separated non-polar fatty acids and each subclass of mycolic acids from 21 mycobacterial species by thin-layer chromatography, and analyzed non-polar fatty acid methyl esters by gas chromatography (GC) and the cleavage products of methyl mycolate by pyrolysis GC. We further performed mass chromatographic analysis of trimethylsilyl (TMS) ether derivatives of mycolic acid methyl esters by monitoring [B-29]⁺ ions (loss of CHO from the α-branched-chain structure of mycolic acids) of m/z 426, 454 and 482 which are attributed to C₂₂-, C₂₄- and C₂₆-α-units of TMS ether derivatives of methyl mycolates, respectively, (Kaneda, K. et al, J. Clin. Microbiol. 24: 1060-1070, 1986). By pyrolysis GC, C_22:0, C_24:0 and C_26:0 fatty acid methyl esters generated by the C₂-C₃ cleavage of C₂₂-, C₂₄- and C₂₆-α-unit-containing mycolic acid methyl esters, respectively, were detected. Their proportion was almost the same among subclasses of mycolic acids in every Mycobacterium and also similar to the proportion of constitutive non-polar C_22:0, C_24:0 and C_26:0 fatty acids. By mass chromatography, the composition and distribution of C₂₂- and C₂₄-α-unit-containing homologues were revealed to be similar between α- and α'-mycolic acids in every Mycobacterium. We further analyzed in detail M. vaccae and demonstrated that the mass chromatogram of C₂₂-α-unit-containing homologue was analogous in shape to that of the C₂₄-α-unit-containing one, with the latter mass chromatogram being up-shifted from the former by two carbon numbers, in every subclass of α-, α'-, keto and dicarboxy mycolic acids. The present study suggests that the compositions of three homologues of both mycolic acids and constitutive non-polar fatty acids, which are characteristic to each mycobacterial species, may reflect the proportion of the amount of free C_22:0, C_24:0 and C_26:0 fatty acids synthesized in the cell. It is further demonstrated that intermolecular condensation of two fatty acids which become α- and β-units of mycolic acids will occur independently of the carbon chain length or kinds of polar moieties of fatty acids.     

Desacetoxycephalosporin C synthetase: Importance of order of cofactor/ reactant addition

The ring expansion enzyme (desacetoxycephalosporin C synthetase) of cephalosporin C biosynthesis in Cephalosporium acremonium has been purified 40-fold, using gel filtration on LKB Ultrogel AcA54. Purity was about twice that previously attained. The purified enzyme showed an almost absolute requirement for α-ketoglutarate, and most other acids were inactive or only weakly active. The order of addition of the cofactors and reactants was found to exert a major effect on enzyme activity. The major negative effect was caused by α-ketoglutarate or penicillin N being added to the enzyme first. The most effective order of addition to the enzyme was found to be the simultaneous addition of Fe²⁺, ascorbate and ATP, followed by α-ketoglutarate and then penicillin N. The negative effect of the required reactant, α-ketoglutarate, when added too early, coincides with observations made about other α-ketoglutarate-dependent dioxygenases even though, in the case of the synthetase, one atom of oxygen does not end up in the product, desacetoxycephalosporin C.     

Fatty-Acid Composition of Candida utilis as Affected by Growth Temperature and Dissolved-Oxygen Tension

Analyses were made of the fatty-acid composition of Candida utilis NCYC 321 grown in a chemostat at a dilution rate (equal to growth rate) of 0.1 hr⁻¹ and at temperatures in the range of 30 to 15 C and dissolved oxygen tensions between 75 and <1 mm of Hg. Cells grown under glucose limitation or NH₄⁺ limitation contained mainly C_16:0, C_16:1, C_18:0, C_18:1, C_18:2, and C_18:3 acids as detected by gas-liquid chromatography of methyl esters of the acids from lipids extracted with chloroform-methanol. The relative proportions of these acids varied with the growth temperature and the dissolved-oxygen tension in the culture. A decrease in growth temperature from 30 to 20 C led to an increased synthesis of unsaturated acids in cells grown under either limitation at a fixed-oxygen tension in the range of 75 to 5 mm of Hg. In cultures with a dissolved-oxygen tension of 1 and <1 mm of Hg, a further decrease in temperature to 15 C caused an increased synthesis of unsaturated fatty acids. A decrease in dissolved-oxygen tension led to a diminished synthesis of unsaturated fatty acids in cells grown at a fixed temperature under either limitation. Cells grown at a fixed temperature under glucose limitation synthesized a greater proportion of C₁₆ acids at the expense of C₁₈ acids as the dissolved oxygen tension was decreased from 75 to <1 mm of Hg. A preferential synthesis of C₁₆ acids also occurred as the growth temperature was decreased from 30 to 15 C in cells grown under glucose limitation at a fixed-oxygen tension. The same effect was observed in cells grown under NH₄⁺ limitation when the temperature was lowered from 30 to 20 C; but when the temperature was decreased further to 15 C, the cells synthesized a slightly greater proportion of C₁₈ acids. Synthesis of a large proportion of C₁₆ acids was accompanied by an excretion of pyruvate, and occasionally traces of 2-ketoglutarate, and an increased intracellular accumulation of certain amino acids.     

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.     

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.     

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.