Mechanism of Inactivation of α-Amino-ε-caprolactam Racemase by α-Amino-δ-valerolactam

Both d- and l-α-amino-δ-valerolactam inactivated α-amino-ε-caprolactam racemase during incubation with the enzyme. The degree of inactivation increased with increases in pH and the concentration of l-α-amino-δ-valerolactam in the incubation mixture. Pyridoxal 5′-phosphate reactivated the inactivated enzyme, and glyoxylate and other α-keto acids such as pyruvate, phenylpyruvate, and α-ketobutyrate protected the enzyme from inactivation by l-α-amino-δ-valerolactam. Both the enantiomers of methionine were produced when α-keto-γ-methylthiobutyrate was incubated with the enzyme in the presence of l-α-amino-δ-valerolactam. Thus, the inactivation of the enzyme in terms of α-amino-ε-caprolactam racemization activity is due to conversion of the enzyme-bound pyridoxal 5′-phosphate into pyridoxamine 5′-phosphate by a transamination with l-α-amino-δ-valerolactam. Formation of pyridoxamine 5′-phosphate from the enzyme-bound pyridoxal 5′-phosphate was proved by spectrophotometry and thin layer chromatography. The rate of racemization of l-α-amino-δ-valerolactam was calculated to be 48 times faster than that of the transamination with glyoxylate.     

Separation of l-Leucine-pyruvate and l-Leucine-α-ketoglutarate Transaminases in Acetobacter suboxydans and Identification of Their Reaction Products

l-Leucine-pyruvate and l-leucine-α-ketoglutarate(α-KGA) transaminases were separated by DEAE-cellulose column chromatography and partially purified to 200- and 50-fold, respectively, from the cell-free extract of Acetobacter suboxydans (Gluconobacter suboxydans IFO 3172). The optimum pH range of the former was 5.0~5.5 and that of the latter was 8.5~9.0. l-Leucine, l-citrulline, and l-methionine were the most effective amino donors for the l-leucine-pyruvate transaminase. Basic amino acids as well as aromatic amino acids were able to be amino donors for the transamination with pyruvate. α-KGA was effective as an amino acceptor for this enzyme. The l-leucine-α-KGA transaminase had the typical properties of the branched-chain amino acid transaminase in its substrate specificity.

The reaction products of the transaminations were identified. l-Alanine was formed from pyruvate and l-glutamate from α-KGA. α-Keto acids formed from various amino acids by the l-leucine-pyruvate transaminase were also identified.     

Purification and Characterization of l-Leucine-α-ketoglutarate Transaminase from Acetobader suboxydans

l-Leucine-α-ketoglutarate (α-KGA) transaminase from Acetobacter suboxydans was purified to the state of homogeneity by the criteria of ultracentrifugation and electrophoresis on a cellulose acetate membrane. The molecular weight was about 80,000 and one mole of pyridoxal 5′-phosphate was bound per mole of enzyme as a coenzyme. The enzyme exhibited absorption maxima at 280, 337 and 414 nm.

The branched-chain amino acids and α-KGA were specific as amino donors and an acceptor. l-Leucine-α-KGA transaminase is suggested to correspond to the enzyme so-called Transaminase B.     

Studies on Abscisic Acid

Methyl α-cyclocitrylideneacetate was successively oxidized with selenium dioxide and chromium trioxide-pyridine complex to give methyl 1′-hydroxy-α-cyclocitrylideneacetate and a mixture of methyl 3′-keto-β-cyclocitrylideneacetate and methyl 4′-keto-α-cyclocitrylideneacetate. Further, oxidation of methyl α-cyclocitrylideneacetate with tert-butyl chromate afforded methyl 4′-keto-α-cyclocitrylideneacetate and methyl 1′-hydroxy-4′-keto-α-cyclocitry-lineacetate. Similarly, methyl α-cyclogeranate was oxidized to methyl 3-keto-β-cyclogeranate and methyl 4-keto-α-cyclogeranate. Methyl l′-hydroxy-4′-keto-α-cyclocitrylideneacetate, methyl l-hydroxy-4-keto-α-cyclogeranate and their related compounds did not show growth inhibitory activities on rice seedlings.     

Photo-oxygenation of α-Ionone

Photo-oxygenation of α-ionone was studied to clarify the relationship between the maturity of aroma and photo-oxygenative change of α-ionone. α-Ionone was converted to oxygenated derivatives which were identified as 2,3-epoxy-β-ionone, 3,4-epoxy-α-ionone, 4-keto-β-ionone (trans- and cis-form), 5-keto-α-ionone and 3,4-dihydroxy-α-ionone.     

Ionones and βIonylideneacetic Acids: Their Influence on Abscisic Acid Biosynthesis by Cercospora rosicola

Several ionones and β-ionylideneacetic acids inhibited absicisic acid (ABA) biosynthesis in Cercospora rosicola at 100 μm. At lower concentrations, α-ionone, 1′,2′-dihydroxy-l′,2′-dihydro-β-ionone and 4′-keto-α-ionone enhanced ABA biosynthesis. Conversions of ionones by C. rosicola were identified by GC-MS as: α-ionone to 4′-keto-α-ionone, 4′-keto-α-ionol and dehydrovomifoliol; and 1′-hydroxy-α-ionone to dehydrovomifoliol. The oxidations of α-ionone and its analogs parallel those of the α-ionylideneacetic acids. The β-ionylideneacetic acids were generally oxidized to more polar forms. Metabolites identified by GC-MS were 3′-hydroxy-, 3′-keto- and 1′,2′-epoxy-1′,2′-dihydro-β-ionylideneacetic acids. The fungus rapidly metabolized most exogenous materials to more polar forms.     

Occurrence of Transaminations with Pyruvate andα-Ketoglutarate inAcetobacter suboxydans

Transaminations betweenL-amino acids and pyruvate orα-ketoglutarate(α-KGA) were observed in a cell-free extract ofAcetobacter suboxydans(Gluconobacter suboxydans IFO 3172). The level of the activities of transaminations with pyruvate was greatly influenced by the kinds and amounts of nitrogen sources for growth media. The enzymic activities of transaminations with pyruvate in glutamate-grown cells were extremely higher than in yeast ex- tract-grown cells. Nutritional components decreasing the activities were presumed to be some of amino acids and ammonium ion present in yeast extract. The activities of transaminations withα-KGA were not so variable in the presence or absence of the components.

The optimum pH of transaminations with pyruvate was in the range of 5.0 ~ 5.5 and that of the reaction withα-KGA in 8.0 ~ 8.5. The optimum temperature of the former was 65°C and that of the latter about 45°C. Some other different properties were also recognized between the two kinds of reactions.     

Isolation and identification of some guanidino compounds in the urine of patients with hyperargininaemia by liquid chromatography,thin-layer chromatography and gas chromatography-mass spectrometry

Liquid column chromatographic studies of monosubstituted guanidino compounds, which are excreted in the urine of patients with hyperargininaemia are reported. The guanidino-positive peaks, with the highest excretion values, were isolated from urine and the isolated compounds were identified by thin-layer chromatography and gas chromatography—mass spectrometry. Guanidinoacetic acid, N-α-acetylarginine, argininic acid, γ-guanidinobutyric acid, arginine and α-keto-δ-guanidinovaleric acid were found to be excreted at high levels in the urine of patients with hyperargininaemia compared with controls.     

A Gluconobacter oxydans mutant converting glucose almost quantitatively to 5-keto-d-gluconic acid

Gluconobacter oxydans converts glucose to gluconic acid and subsequently to 2-keto-d-gluconic acid (2-KGA) and 5-keto-d-gluconic acid (5-KGA) by membrane-bound periplasmic pyrroloquinoline quinone-dependent and flavin-dependent dehydrogenases. The product pattern obtained with several strains differed significantly. To increase the production of 5-KGA, which can be converted to industrially important l-(+)-tartaric acid, growth parameters were optimized. Whereas resting cells of G. oxydans ATCC 621H converted about 11% of the available glucose to 2-KGA and 6% to 5-KGA, with growing cells and improved growth under defined conditions (pH 5, 10% pO₂, 0.05% pCO₂) a conversion yield of about 45% 5-KGA from the available glucose was achieved. As the accumulation of the by-product 2-KGA is highly disadvantageous for an industrial application of G. oxydans, a mutant was generated in which the membrane-bound gluconate-2-dehydrogenase complex was inactivated. This mutant, MF1, grew in a similar way to the wild type, but formation of the undesired 2-KGA was not observed. Under improved growth conditions, mutant MF1 converted the available glucose almost completely (84%) into 5-KGA. Therefore, this newly developed recombinant strain is suitable for the industrial production of 5-KGA.     

Detection and quantification of α-keto-δ-(N,N-dimethylguanidino)valeric acid: A metabolite of asymmetric dimethylarginine

Nitric oxide is an ubiquitary cell signaling substance. Its enzymatic production rate by nitric oxide synthase is regulated by the concentrations of the substrate l-arginine and the competitive inhibitor asymmetric dimethylarginine (ADMA). A newly recognized elimination pathway for ADMA is the transamination to α-keto-δ-(N^G,N^G-dimethylguanidino)valeric acid (DMGV) by the enzyme alanine-glyoxylate aminotransferase 2 (AGXT2). This pathway has been proven to be relevant for nitric oxide regulation, but up to now no method exists for the determination of DMGV in biological fluids. We have developed a liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for the quantification of DMGV. D₆-DMGV was used as internal standard. Samples were purified online by column switching, and separation was achieved on a porous graphitic carbon column. The calibration was linear over ranges of 10 to 200 nmol/L for plasma and 0.1 to 20 μmol/L for urine. The intra- and interday accuracies and precisions in plasma and urine were better than 10%. In plasma samples, DMGV was present in concentrations between 19.1 and 77.5 nmol/L. In urine samples, concentrations between 0.0114 and 1.03 μmol/mmol creatinine were found. This method can be used as a tool for the scientific investigation of the ADMA conversion to DMGV via the enzyme AGXT2.     

Synthesis of Analogs of Optically Active α-Ionylideneacetic Acid

The Wittig reaction of (?)-α-ionone (VIa) with carbethoxymethylenetriphenylphosphorane afforded (?)-ethyl α-ionylideneacetate (VIIa). tert-Butyl chromate oxidation of the above ester (VIIa) gave (?)-ethyl 4′-keto-α-ionylideneacetate (VIlla). Selenium dioxide oxidation of (?)-α-ionone (IVa) in ethanol afforded (?)-1′-hydroxy-α-ionone (X), which reacted with car-bethoxymethylenetriphenylphosphorane to give (?)-ethyl 1′-hydroxy-α-ionylideneacetate (XI). tert-Butyl chromate oxidation of the hydroxy-ester (XI) gave (?)-ethyl abscisate (XII) and ethyl 3′-keto-β-ionylideneacetate (XIII). The sensitized photooxidation of ethyl dehydro-β-ionylideneacetate (XVI) using chlorophyll was attempted.     

Studies on Abscisic Acid

Photosensitized oxygenation of dehydro-β-ionylidene-ethanol afforded 1′-hydroxy-4′keto-α-ionylidene-ethanol, which was oxidized with active MnO₂ to give 1′-hydroxy-4′-keto-α-ionylidene-acetaldehyde. The Wittig reaction of α-ionylideneacetaldehyde with carbethoxymethylenetriphenylphosphorane or the phosphorane prepared from ethyl γ-bromosenecioate gave ethyl α-ionylidene-crotonate or ethyl α-ionylidenesenecioate. Vitamin A₂ acid ethyl ester was converted to the hydroxy-keto-ester by photosensitized oxygenation. About the above synthesized compounds were examined growth inhibitory activities on rice seedlings.     

The role of the delocalized α,α′-carbanion in vitamin B6 and Al(III) catalyzed transamination of α-amino and α-keto acids

The rates of the transamination reactions of α-amino acids and α-keto acids were followed by measurement of the 200 MHz proton NMR spectra of solution species as a function of time. Reaction systems measured in D₂O at 10 °C consisted of 1:1:1 molar ratios of pyridoxal:α-amino acid:Al(III) or pyridoxamine:α-keto acid:Al(III). Amino and keto acids employed are alanine, α-aminoisobutyric acid, valine, phenylglycine, pyruvic acid, and α-ketobutyric acid. A negatively charged deprotonated Schiff base coordinated to Al(III) was detected in all systems that undergo transamination (i.e., except α-aminoisobutyric acid). The intermediate resembles the aldimine Al(III) chelate with NMR resonances shifted upfield in accordance with its greater negative charge. Its equilibrium concentration is reached in the time required to reach transamination equilibrium and is maintained in solution at a ca. 10–20% of the aldimine Schiff base concentration.     

Farnesyl and α-Ionylideneethyl Tertiary and Quaternary Amines: Their Influence on Abscisic Acid Biosynthesis by Cercospora rosicola

Farnesyl and α-ionylideneethyl compounds with tertiary and quaternary amine functional groups were synthesized and their effects on abscisic acid (ABA) biosynthesis of Cercospora rosicola observed. The trimethylammonium compounds were lethal at 10 μm and inhibitory at 10 μm, but lesser amounts of α-ionylideneethyltrimethylammonium iodide enhanced ABA biosynthesis. N,N-Dimethylfarnesylamine had little effect on ABA biosynthesis. N,N-Dimethyl (2Z,4E)- and (2E,4E)-α-ionylideneethylamines inhibited ABA biosynthesis at 100 μm but had no or little effect at lower concentrations. Farnesol and farnesylpyrophosphate (FPP) enhanced ABA biosynthesis. FPP appears to be both a precursor and an inducer and farnesol is an inducer of ABA biosynthesis. N,N-Dimethyl (2Z,4E)- and (2E,4E)-α-ionylideneethylamines were converted to N,N-dimethyl (2Z,4E)- and (2E,4E)-4′-keto-α-ionylideneethylamines, respectively. These conversions are analogous to those reported for α-ionone and α-ionylideneacetic acids and show that basic as well as acidic and neutral compounds with α-ionone type rings can undergo oxidation at the 4′-position. α-Ionylideneacetic acids inhibited growth of C. rosicola and the dimethylamines enhanced growth. Complete feedback inhibition was obtained with 400 μm of ABA.     

Enhanced lipid peroxidation and inflammation during heat exposure in rats of different ages: Role of α-tocopherol

To investigate early events possibly related to the development of heat shock, we examined whether inflammatory-(interleukin-6, tumor necrosis factor α and 15-keto-13,14-dihydro-PGF_2α) and peroxidative-(8-iso-PGF_2α and malondialdehyde) markers are altered during acute heat exposure and aging. We also studied the relationships between inflammatory and peroxidative markers in these settings. In order to prevent these reactions developed as a consequence of the conditions mentioned above, we tested the effects of α-tocopherol. Our results demonstrated that 15-keto-13,14-dihydro-PGF_2α and malondialdehyde in the liver were altered during acute heat exposure in the young and middle-aged rats and could be predicted by changes in the levels of circulatory cytokines. Regardless of age, the supplementation with α-tocopherol prevented changes in the plasma cytokine levels and 15-keto-13,14-dihydro-PGF_2α and malondialdehyde levels in the liver, during acute heat exposure. This study notably emphasized the ability of α-tocopherol to prevent different heat induced mechanisms, involved in induction of inflammatory or peroxidative reactions.     

Biosynthesis of amino acids from sucrose and Krebs cycle metabolites by Rhizobium lupini bacteroids

Summary The possibility of amino acids biosynthesis from sucrose, metabolites of Krebs cycle or glyoxylate and ammonium by intact bacteroids has been studied. The suspension of intact Rhizobium lupini bacteroids in phosphate buffer solution pH 7.8 was shown to catalyse the biosynthesis from sucrose and ammonium of some amino acids, such as alanine, aspartic and glutamic acids, glycine and serine. The yield of alanine and aspartic acid was 2.5–3 times higher than that of other amino acids, which were formed in almost equal quantities. Intact bacteroids were also found to catalyse the biosynthesis of aspartic and glutamic acids, alanine and glycine from ammonium and Krebs cycle metabolites such as fumaric acid (FA), oxaloacetic acid (OAA), pyruvic acid (PA), a-ketoglutaric acid (a-KGA), malic acid (MA), as well as from glyoxylic acid (GOA). The biosynthesis of aspartic acid from fumaric acid was dominant. Besides that, the suspension of intact bacteroids catalysed transamination of aspartic and glutamic acids, the transamination of aspartic acid being especially intense with -KGA and GOA. Aspartic acid was synthesized most efficiently through the amination of fumaric acid, while glutamic acid was better synthesized through the transamination of aspartic acid with -KGA than through reductive amination of -KGA.The experimental data proved that intact bacteroids posess Krebs cycle enzymes and primary ammonia assimilation enzymes. This enzyme complex permits bacteroids to detoxify ammonia, which they produce using sucrose and metabolites of Krebs cycle as the sources of carbon.The data obtained are of great interest as they prove the importance of bacteroids in the synthesis of amino acids from ammonium which is formed in the course of N₂-fixation, and sucrose available from leaves.     

Metabolism of amino acids in germinating yellow lupin seeds III. Breakdown of arginine in sugar-starved organs cultivated in vitro

The pathways of arginine transformations in organs of yellow lupin (Lupinus luteus L.) cultivated in vitro in the presence and absence of sucrose were investigated. Isolated embryo axes, isolated cotyledons and seeds deprived of their coat were cultured for 96 h on Heller medium with 60 mM saccharose (the fed variant, +S), without sugar (the starved variant, −S) and for 72 h without sugar, followed by 24 h in its presence (the transferred variant, −S→+S). Activities of arginine decarboxylase [EC 4.1.1.19], arginase [EC 3.5.3.1], and urease [EC 3.5.1.5] were assessed in extracts from isolated embryo axes. They were the highest in the sugar-starved variant. Supplementation of the medium with saccharose resulted in decrease in enzyme activities. The level of urea was higher (of ca. 20 %) in starved embryos than in embryos grown in the saccharose-containing medium. Moreover, participation of transamination in arginine catabolism was evidenced.     

The route of lysine breakdown in Candida tropicalis

Candida tropicalis was found to contain high levels of the following enzymes after growth in defined medium on L-lysine as sole nitrogen source: L-lysine N6-acetyltransferase, N6-acetyl-lysine aminotransferase, and aminotransferase activity for 5-aminovalerate and 4-aminobutyrate. Extracts were also capable of converting 5-acetamidovalerate (and 4-acetamidobutyrate) to acetate. N6-Acetyllysine however, only gave rise to acetate in the presence of 2-oxoglutarate, NAD+ and thiamine pyrophosphate. These activities were undetectable or present in much lower concentrations in cells that had been grown on ammonium sulphate as sole nitrogen source. It is concluded that L-lysine is degraded in this organism via N6-acetyllysine, 5-acetamidovalerate and 5-aminovalerate, both nitrogen atoms being removed by transamination.     

Studies on Abscisic Acid

Oxidation of methvl 2-trans-β-ionylideneacetate with X-bromosuccinimide afforded methyl 2-cis and trans-3′-hydroxy-β-ionylideneacetates. NaBH₄ reduction of methyl 2-cis-3′-keto-β-ionylideneacetate and ethyl 4′-keto-α-ionylideneacetate gave methyl 2-cis-3′-hydroxy-β-ionylideneacetate and ethyl 4′-hydroxy-α-ionyiideneacetate respectively. Further, methyl 4′-methoxy-epoxy-α-ionylideneacetate was prepared by epoxidation of methyl 4′-methoxy-α-ionylideneacetate. And then methyl 4′-hydroxy-l′, 2′-dihydro-β-ionylideneacetate was synthesized from ethyl 4-keto-α-cyclogeranate. Growth inhibitory activities of the above compounds on rich seedlings were examined.     

A Facile Synthesis of Conjugated α-Methylene-δ-valerolactones

A facile synthetic method of variously substituted α-methylene-δ-valerolactones was devised and the growth inhibiting effects of synthesized samples were tested on rice seedlings. Alkylation of enamines (II) with methyl α-bromomethylacrylate (III) provided key intermediates, unsaturated keto esters (IV) which were readily converted to substituted α-methylene-δ-valerolactones (V) by a sequence involving saponification, reduction and lactonization. The growth inhibitory activities of the 6-membered α-methylene lactones synthesized closely paralleled that of α-methylene-γ-phenyl-γ-butyrolactone (XIII) which had already been shown in our previous paper to possess the inhibitory activity comparable to that of the natural inhibitors, heliangine and pyrethrosin.