Evidence for a 2-oxoglutarate dehydrogenase complex activity in mitochondria of Neurospora crassa and compared localization with the pyruvate dehydrogenase complex

A 2-oxoglutarate dehydrogenase complex activity is demonstrated in Neurospora crassa mitochondria. A submitochondrial fractionation by digitonin treatment followed by freeze-thawing enables measurement of a well preserved activity in the mitochondrial matrix. In contrast to other reports, the pyruvate dehydrogenase activity is also found to be localized in the matrix.     

Implication of glutamate, isocitrate and malate deshydrogenases in nitrogen assimilation in the cadmium-stressed tomato

Tomato seedlings grown on nitric medium and treated with various cadmium concentrations (0 to 50 microM) were used. Results obtained show that cadmium remains predominantly located in the roots, which then seem to play the role of trap-organs. Increasing cadmium concentration in the medium leads particularly to a decrease in NO3- accumulation, together with a decrease in the activity of glutamine synthetase and in the quantity of plastidic isoform ARNm (GS2), and, on the contrary, to an increase of the cytosolic isoform ARNm (GS1). On the other hand, stimulations were observed for NADH-dependent glutamate synthase, NADH-dependent glutamate dehydrogenase, ARNm quantity of this enzyme, ammonium accumulation, and protease activity. In parallel, stimulations were observed for NAD+ and NADP+-dependent malate dehydrogenase and NADP+-dependent isocitrate dehydrogenase. These results were discussed in relation to the hypothesis attributing to the dehydrogenase enzymes (GDH, MDH, ICDH) an important role in the plant defence processes against cadmium-induced stresses.     

Solubilisation of D-amino acid dehydrogenase of Escherichia coli K12 and its re-binding to envelope preparations

D-amino acid dehydrogenase was found to be solubilised from envelope preparations of Escherichia coli by treatment with detergents but not with aqueous buffer solutions. Triton X-100-solubilised dehydrogenase was found to rebind to envelope preparations from the wild strain (AB 259) and its D-amino acid dehydrogenase-less mutant (DAD 13), and activities higher than those of native envelopes could be obtained. Re-binding was stimulated by magnesium. D-alanine stimulated cytochrome reduction and oxygen uptake were reconstituted when the solubilised dehydrogenase rebound to AB 259 envelopes. Re-binding of solubilised dehydrogenase to DAD 13 envelopes was independent of the growth medium used for DAD 13.     

Bromopyruvate, a potential affinity label for octopine dehydrogenase

Bromopyruvate, an analogue of pyruvate, one of the substrates of octopine dehydrogenase, was tested as an inhibitor of the enzyme. Provided both the coenzyme and the second substrate, arginine, were present, bromopyruvate rapidly inactivated the enzyme. This inactivation was irreversible, obeyed pseudo-first order kinetics and exhibited a rate saturation effect. Pyruvate protected the enzyme against inactivation by bromopyruvate and these compounds competed for the same site. Bromopyruvate also behaved as a true substrate for the enzyme. This reagent thus exhibits the kinetic characteristics of a good affinity label for octopine dehydrogenase.     

Generation of hydrogen peroxide during the oxidation of L-phenylalanine by Proteus mirabilis isolated membranes

In the process of L-phenylalanine oxidation by Proteus mirabilis cytoplasmic membrane, hydrogen peroxide was produced at a rate corresponding to 1-3 per cent of the total electron flow (30-110 nmoles min-1mg-1). Peroxide was estimated using a fluorimetric assay with horseradish peroxidase, or by anodic oxidation on a platinum electrode. When using the former method, superoxide dismutase decreased the apparent yield of peroxide, a fact suggesting that H2O2 was in part the dismutation product of superoxide radicals. However the superoxide dismutase effect could be an artefact due to the generation of some superoxide during the peroxidatic reaction in the assay. Adrenaline was the reagent used for the detection of superoxide. There was no significant emergence of superoxide as the result of phenylalanine oxidation by the membrane (specific activity lower than 1-2 nmoles min-1mg-1). Thus it seemed that superoxide was not an intermediate for the bulk of H2O2 formed in this system. According to the results, peroxide was probably formed at a stage of electron transport earlier than the cytochrome level. The membrane phenylalanine dehydrogenase could be a site where peroxide was evolved in these experiments.     

Quaternary structure and proteolysis of the polynucleotide phosphorylase from C. perfringens]

This report describes structural studies on purified polynucleotide phosphorylase from C. perfringens. A method is described for the purification of the enzyme which yields a product equivalent in activity to the native polynucleotide phosphorylase from E. coli. These studies revealed a molecular heterogeneity arising from successive stages of proteolysis, to which this enzyme is especially sensitive; unusally, the enzyme is obtained as a mixture of variable proportions of the native and proteolysed forms. We found in all cases a trimeric basic structure composed of the native (alpha) or proteolysed (lapha) or proteolysed (alpha', alpha") catalytic sub-units, However, the enzyme is rather easily dissociated into its sub-units, a phenomenon which seems to accompany proteolysis (Table). Under the action of either endogenous proteases or trypsin, two enzymatic forms are obtained: their quaternary structures seem analogous, but they differ in their catalytic properties from each other and from the initial enzyme. With some care at each step of purification, the polynucleotide phosphorylase of E. coli can be obtained exclusively in its native form. The greater susceptibility to proteolysis of the enzyme from C. perfrigens and the relationship between such degradation and quaternary structure seem to be at the origin of the peculiar behavior of this polynucleotide phosphorylase.     

Ammonium ions enhance the flow through the pyruvate dehydrogenase in Ehrlich ascites tumor cells

The flow through pyruvate dehydrogenase was assayed in glycolysing cells by the evolution of 14CO2 from [1-14C] pyruvate. Parallel incubations were carried out in high bicarbonate buffer (25 mM) and in bicarbonate-free buffer. The activation of the complex by NH+4 was only observed in high bicarbonate buffer, because the dilution of labelled CO2 in the presence of an excess of bicarbonate enables the quantitative determination of labelled CO2 evolved from pyruvate in the decarboxylase step. In the bicarbonate-free buffer the activation of the complex was not observed, because the 14CO2 evolved from pyruvate was consumed by biosynthetic processes inside the cell. On the contrary in isolated hepatocytes the NH+4 activation of the pyruvate dehydrogenase was observed in both buffers. In Ehrlich ascites cells, in common with other mammalian tissues, pyruvate dehydrogenase activity was found to be inversely correlated to the intramitochondrial ATP/ADP ratio.     

Inactivation of yeast alcohol dehydrogenase by a reactive coenzyme analogue: 3-chloroacetyl pyridine adenine dinucleotide

Yeast alcohol dehydrogenase is very rapidly and irreversibly inactivated by 3-chloroacetyl pyridine adenine dinucleotide, a reactive NAD+-analogue (Biellmann et al., 1974, FEBS Lett. 40, 29-32). Kinetic investigations with this compound, and structurally related compounds, show that this inactivation, against which NAD+ provides a complete protection, corresponds to an affinity label. The incorporation of the coenzyme analogue correlates linearly with the enzyme inactivation, the total inactivation corresponding to one mole of inactivator per coenzyme binding site. The pH-dependence of the inactivation rates of the enzyme by this coenzyme analogue and by its reduced form reflects exactly the pH variation of their respective dissociation constants. In spite of a good stability of the label in the non denatured inactivated enzyme, no modified amino-acid residue could be identified. Considering the affinity of this analogue for yeast alcohol dehydrogenase and the strict steric requirements of this enzyme towards its ligands, the nature of the inactivation reaction as well as different possibilities of the loss of the label in the inactivated enzyme are discussed.     

Modification of redox equilibria between heme and flavin within yeast flavocytochrome b2 (l-lactate cytochrome c reductase) upon binding of pyruvate,the reaction product

Direct determinations of the concentration of semiquinone spin in redox equilibrium with the cytochrome b₂ moiety were carried out at room temperature in the presence of added pyruvate or in its absence. Results show that redox potentials of the one-electron couples of the prosthetic flavin are markedly affected by binding of pyruvate the reaction product in the oxidation of l-lactate. The proportion of flavin semiquinone nearly reaches then 100 per cent.     

A new bacterial dehydrogenase oxidizing the lignin model compound guaiacylglycerol beta-O-4-guaiacyl ether

A lignin model compound, named in short guaiagylglycerol beta-guaiacyl ether (GGE), contains the beta-0-4 ether linkage that is common in the chemical structure of lignin. A Pseudomonas sp. (GU5) had been isolated as an organism able to grow with GGE as the sole source of carbon and energy. When grown on vanillate, the bacteria contained a NAD+ -dependent dehydrogenase converting GGE to a 355 nm absorbing product. The enzyme, named GGE-dehydrogenase, was purified about 160-fold using gel permeation, ion exchange on DEAE-Sephadex, and dye-ligand affinity chromatography. The new protein was about 52 kDa in apparent size with but one polypeptide chain after denaturation and reduction. According to several criteria, the product of GGE oxidation (Km = 12 microM) was identified as the corresponding conjugated ketone at the alpha-carbon of the C3 side-chain. The secondary alcohol function in GGE was apparently the sole target of the enzyme action. However the conversion of GGE into ketone catalyzed by the enzyme was only partial, and did not exceed 50%, probably because only one of the alpha-enantiomers was susceptible to enzyme attack. In contrast the ketone, either made by organic synthesis or by enzymic oxidation of GGE, could be totally reduced back to GGE (Km = 13 microM at pH 8.4, 8 microM at neutral pH), with NADH as the reductant, as confirmed by UV absorption and NMR spectra. Other model compounds with no primary alcoholic function, ether linkage or phenolic group were also substrates for the enzyme, confirming the specificity of GGE-dehydrogenase for the alpha-carbon position. Conjugation of the alpha-ketone with an adjacent phenolic nucleus interfered strongly with equilibrium constants and redox potentials of the system according to pH, and the enzyme displayed widely different optima with pH over 9 when oxidizing GGE, below 7 when reducing the ketone. Equilibrium studies showed that the ketone/GGE potential was -0.37 volt at pH 8.7, -0.23 volt at pH 7 (30 degrees C). The significance of this new dehydrogenase and its properties are discussed, especially in the general concern of lignin biodegradation.     

Genetic and molecular basis of grass cell-wall degradability. I. Lignin-cell wall matrix interactions

Lignification limits grass cell-wall digestion by herbivores. Lignification is spatially and temporally regulated, and lignin characteristics differ between cell walls, plant tissues, and plant parts. Grass lignins are anchored within walls by ferulate and diferulate cross-links, p-coumarate cyclodimers, and possibly benzyl ester and ether cross-links. Cell-wall degradability is regulated by lignin concentration, cross-linking, and hydrophobicity but not directly by most variations in lignin composition or structure. Genetic manipulation of lignification can improve grass cell-wall degradability, but the degree of success will depend on genetic background, plant modification techniques employed, and analytical methods used to characterize cell walls.     

Premiers cas de brachyptérisme dans le genre Sisyphus Latreille (Coleoptera,Scarabaeidae, Sisyphini)

Les espèces du genre Sisyphus Latreille, 1807, présentant des touffes de soies sur les élytres sont étudiées. Sept nouvelles espèces appartenant à ce groupe sont décrites, illustrées et comparées aux espèces proches: deux espèces aux ailes normalement développées, S. manni n. sp. et S. perissinottoi n. sp., et cinq espèces brachyptères, S. jossoi n. sp., S. janssensi n. sp., S. bouyeri n. sp., S. felschei n. sp. et S. usambaricus n. sp. Une autre espèce appartenant au groupe étudié, S. alveatus Boucomont, 1935, se révèle également être brachyptère. Elles constituent les premiers exemples de brachyptérisme au sein de ce genre. Une clé d’identification des espèces à touffes de soies sur les élytres est donnée.