Synthesis of optically active amino acids from alpha-keto acids with Escherichia coli cells expressing heterologous genes.

We describe a simple method for enzymatic synthesis of L and D amino acids from alpha-keto acids with Escherichia coli cells which express heterologous genes. L-amino acids were produced with thermostable L-amino acid dehydrogenase and formate dehydrogenase (FDH) from alpha-keto acids and ammonium formate with only an intracellular pool of NAD+ for the regeneration of NADH. We constructed plasmids containing, in addition to the FDH gene, the genes for amino acid dehydrogenases, including i.e., leucine dehydrogenase, alanine dehydrogenase, and phenylalanine dehydrogenase. L-Leucine, L-valine, L-norvaline, L-methionine, L-phenylalanine, and L-tyrosine were synthesized with the recombinant E. coli cells with high chemical yields (> 80%) and high optical yields (up to 100% enantiomeric excess). Stereospecific conversion of various alpha-keto acids to D amino acids was also examined with recombinant E. coli cells containing a plasmid coding for the four heterologous genes of the thermostable enzymes D-amino acid aminotransferase, alanine racemase, L-alanine dehydrogenase, and FDH. Optically pure D enantiomers of glutamate and leucine were obtained.     

NAD(H) enhances the Cu(II)-mediated inactivation of lactate dehydrogenase by increasing the accessibility of sulfhydryl groups

Copper ions are known to inactivate a variety of enzymes, and lactate dehydrogenase (LDH) is exceptionally sensitive to the presence of this metal. We now found that NADH strongly enhances the Cu(II)-mediated loss of LDH activity. Surprisingly, NADH was not oxidized in this process and also NAD⁺ promoted the Cu(II)-dependent inactivation of LDH. Catalase only partly protected the enzyme, whereas hypoxia even enhanced LDH inactivation. NAD(H) accelerated sulfhydryl (SH) group oxidation of LDH by 5,5-dithio-bis(2-nitrobenzoic acid) (DTNB), and, vice versa, LDH-mediated Cu(II) reduction. LDH activity was preserved by thiol donators and pyruvate and partially preserved by lactate and oxamate. Our results suggest that reactive oxygen species (ROS) are of minor importance for the inactivation of LDH induced by Cu(II)/NADH. We propose that conformational changes of the enzymes' active sites induced by NAD(H)-binding increase the accessibility of active sites' cysteine residues to Cu(II) thereby accelerating their oxidation and, consequently, loss of catalytic activity.     

亮氨酸脱氢酶偶联NADH再生体系合成L-2-氨基丁酸

文中以大肠杆菌BL21(DE3)为宿主,构建两株分别共表达亮氨酸脱氢酶(LDH,来源蜡样芽孢杆菌)/甲酸脱氢酶(FDH,来源水生弯杆菌)和亮氨酸脱氢酶(LDH,来源蜡样芽孢杆菌)/醇脱氢酶(ADH,来源红球菌)的重组大肠杆菌。通过偶联两种不同NADH再生体系,以L-苏氨酸为起始原料,利用苏氨酸脱氨酶(L-TD)与LDH-FDH或LDH-ADH一锅法合成L-2-氨基丁酸,并对LDH-FDH工艺和LDH-ADH工艺进行对比优化。LDH-FDH工艺的最适反应pH为7.5,最适反应温度为35℃,通过加入50 g/L甲酸铵、0.3 g/L NAD+、10%LDH-FDH粗酶液(V/V)和7 500 U/L的L-TD酶液,对L-苏氨酸进行分批补加,以便控制2-丁酮酸浓度小于15 g/L,反应28 h,实现了L-2-氨基丁酸的产量为161.8 g/L,产率97%。LDH-ADH工艺的最适pH为8.0,最适反应温度为35℃,通过加入0.3 g/L NAD+、10%LDH-ADH粗酶液(V/V)及7 500 U/L的L-TD酶液,分批补加L-苏氨酸及1.2倍摩尔量异丙醇,以便控制2-丁酮酸浓度小于15g/L,且每生成约40g/L的L-2-氨基丁酸,抽真空去除丙酮,反应24h,实现了L-2-氨基丁酸的产量为119.6 g/L,产率98%。文中所采用的工艺及结果可为L-2-氨基丁酸的工业化提供一定的参考依据。     

Bifunctional isocitrate-homoisocitrate dehydrogenase: a missing link in the evolution of beta-decarboxylating dehydrogenase

Beta-decarboxylating dehydrogenases comprise 3-isopropylmalate dehydrogenase, isocitrate dehydrogenase, and homoisocitrate dehydrogenase. They share a high degree of amino acid sequence identity and occupy equivalent positions in the amino acid biosynthetic pathways for leucine, glutamate, and lysine, respectively. Therefore, not only the enzymes but also the whole pathways should have evolved from a common ancestral pathway. In Pyrococcus horikoshii, only one pathway of the three has been identified in the genomic sequence, and PH1722 is the sole beta-decarboxylating dehydrogenase gene. The organism does not require leucine, glutamate, or lysine for growth; the single pathway might play multiple (i.e., ancestral) roles in amino acid biosynthesis. The PH1722 gene was cloned and expressed in Escherichia coli and the substrate specificity of the recombinant enzyme was investigated. It exhibited activities on isocitrate and homoisocitrate at near equal efficiency, but not on 3-isopropylmalate. PH1722 is thus a novel, bifunctional beta-decarboxylating dehydrogenase, which likely plays a dual role in glutamate and lysine biosynthesis in vivo.     

The N Terminus of Pro-endothelial Monocyte-activating Polypeptide II (EMAP II) Regulates Its Binding with the C Terminus,Arginyl-tRNA Synthetase,and Neurofilament Light Protein

Pro-endothelial monocyte-activating polypeptide II (EMAP II), one component of the multi-aminoacyl tRNA synthetase complex, plays multiple roles in physiological and pathological processes of protein translation, signal transduction, immunity, lung development, and tumor growth. Recent studies have determined that pro-EMAP II has an essential role in maintaining axon integrity in central and peripheral neural systems where deletion of the C terminus of pro-EMAP II has been reported in a consanguineous Israeli Bedouin kindred suffering from Pelizaeus-Merzbacher-like disease. We hypothesized that the N terminus of pro-EMAP II has an important role in the regulation of protein-protein interactions. Using a GFP reporter system, we defined a putative leucine zipper in the N terminus of human pro-EMAP II protein (amino acid residues 1–70) that can form specific strip-like punctate structures. Through GFP punctum analysis, we uncovered that the pro-EMAP II C terminus (amino acids 147–312) can repress GFP punctum formation. Pulldown assays confirmed that the binding between the pro-EMAP II N terminus and its C terminus is mediated by a putative leucine zipper. Furthermore, the pro-EMAP II 1–70 amino acid region was identified as the binding partner of arginyl-tRNA synthetase, a polypeptide of the multi-aminoacyl tRNA synthetase complex. We also determined that the punctate GFP pro-EMAP II 1–70 amino acid aggregate colocalizes and binds to the neurofilament light subunit protein that is associated with pathologic neurofilament network disorganization and degeneration of motor neurons. These findings indicate the structure and binding interaction of pro-EMAP II protein and suggest a role of this protein in pathological neurodegenerative diseases.     

Efficient production of L-Lactic acid by metabolically engineered Saccharomyces cerevisiae with a genome-integrated L-lactate dehydrogenase gene

We developed a metabolically engineered yeast which produces lactic acid efficiently. In this recombinant strain, the coding region for pyruvate decarboxylase 1 (PDC1) on chromosome XII is substituted for that of the l-lactate dehydrogenase gene (LDH) through homologous recombination. The expression of mRNA for the genome-integrated LDH is regulated under the control of the native PDC1 promoter, while PDC1 is completely disrupted. Using this method, we constructed a diploid yeast transformant, with each haploid genome having a single insertion of bovine LDH. Yeast cells expressing LDH were observed to convert glucose to both lactate (55.6 g/liter) and ethanol (16.9 g/liter), with up to 62.2% of the glucose being transformed into lactic acid under neutralizing conditions. This transgenic strain, which expresses bovine LDH under the control of the PDC1 promoter, also showed high lactic acid production (50.2 g/liter) under nonneutralizing conditions. The differences in lactic acid production were compared among four different recombinants expressing a heterologous LDH gene (i.e., either the bovine LDH gene or the Bifidobacterium longum LDH gene): two transgenic strains with 2microm plasmid-based vectors and two genome-integrated strains.     

Studies on the regulation of leucine catabolism: Mechanism responsible for oxidizable substrate inhibition and dichloroacetate stimulation of leucine oxidation by the heart

In the absence of any other oxidizable substrate, the perfused rat heart oxidizes [1-¹⁴C]leucine to ¹⁴CO₂ at a rapid rate and releases only small amounts of α-[1-¹⁴C]ketoisocaproate into the perfusion medium. The branched-chain α-keto acid dehydrogenase complex, assayed in extracts of mitochondria prepared from such perfused hearts, is very active. Under such perfusion conditions, dichloroacetate has almost no effect on [1-¹⁴C]leucine oxidation, α-[1-¹⁴C]ketoisocaproate release, or branched-chain α-keto acid dehydrogenase activity. Perfusion of the heart with some other oxidizable substrate, e.g., glucose, pyruvate, ketone bodies, or palmitate, results in an inhibition of [1-¹⁴C]leucine oxidation to ¹⁴CO₂ and the release of large amounts of α-[1-¹⁴C]ketoisocaproate into the perfusion medium. The branched-chain α-keto acid dehydrogenase complex, assayed in extracts of mitochondria prepared from such hearts, is almost completely inactivated. The enzyme can be reactivated, however, by incubating the mitochondria at 30 °C without an oxidizable substrate. With hearts perfused with glucose or ketone bodies, dichloroacetate greatly increases [1-¹⁴C]leucine oxidation, decreases α-[1-¹⁴C]ketoisocaproate release into the perfusion medium, and activates the branched-chain α-keto acid dehydrogenase complex. Pyruvate may block dichloroacetate uptake because dichloroacetate neither stimulates [1-¹⁴C]leucine oxidation nor activates the branched-chain α-keto acid dehydrogenase complex of pyruvate-perfused hearts. It is suggested that leucine oxidation by heart is regulated by the activity of the branched-chain α-keto acid dehydrogenase complex which is subject to interconversion between active and inactive forms. Oxidizable substrates establish conditions which inactivate the enzyme. Dichloroacetate, known to activate the pyruvate dehydrogenase complex by inhibition of pyruvate dehydrogenase kinase, causes activation of the branched-chain α-keto acid dehydrogenase complex, suggesting the existence of a kinase for this complex.     

Gene cloning and sequence determination of leucine dehydrogenase from Bacillus stearothermophilus and structural comparison with other NAD(P)+-dependent dehydrogenases

The gene for leucine dehydrogenase (EC 1.4.1.9) from Bacillus stearothermophilus was cloned and expressed in Escherichia coli. The selection for the cloned gene was based upon activity staining of the replica printed E. coli cells. A transformant showing high leucine dehydrogenase activity was found to carry an about 9 kilobase pair plasmid, which contained 4.6 kilobase pairs of B. stearothermophilus DNA. The nucleotide sequence including the 1287 base pair coding region of the leucine dehydrogenase gene was determined by the dideoxy chain termination method. The translated amino acid sequence was confirmed by automated Edman degradation of several peptide fragments produced from the purified enzyme by trypsin digestion. The polypeptide contained 429 amino acid residues corresponding to the subunit (Mr 49,000) of the hexameric enzyme. Comparison of the amino acid sequence of leucine dehydrogenase with those of other pyridine nucleotide dependent oxidoreductases registered in a protein data bank revealed significant sequence similarity, particularly between leucine and glutamate dehydrogenases, in the regions containing the coenzyme binding domain and certain specific residues with catalytic importance.     

Cloning and overexpression of Lactobacillus helveticus D-lactate dehydrogenase gene in Escherichia coli.

NAD(+)-dependent D-lactate dehydrogenase from Lactobacillus helveticus was purified to apparent homogeneity, and the sequence of the first 36 amino acid residues determined. Using forward and reverse oligonucleotide primers, based on the N-terminal sequence and amino acid residues 220-215 of the Lactobacillus bulgaricus enzyme [Kochhar, S., Hunziker, P. E., Leong-Morgenthaler, P. & Hottinger, H. (1992) J. Biol. Chem. 267, 8499-8513], a 0.6-kbp DNA fragment was amplified from L. helveticus genomic DNA by the polymerase chain reaction. This amplified DNA fragment was used as a probe to identify two recombinant clones containing the D-lactate dehydrogenase gene. Both plasmids overexpressed D-lactate dehydrogenase (greater than 60% total soluble cell protein) and were stable in Escherichia coli, compared to plasmids carrying the L. bulgaricus and Lactobacillus plantarum genes. The entire nucleotide sequence of the L. helveticus D-lactate dehydrogenase gene was determined. The deduced amino acid sequence indicated a polypeptide consisting of 336 amino acid residues, which showed significant amino acid sequence similarity to the recently identified family of D-2-hydroxy-acid dehydrogenases [Kochhar, S., Hunziker, P. E., Leong-Morgenthaler, P. & Hottinger, H. (1992) Biochem. Biophys. Res. Commun. 184, 60-66]. The physicochemical and catalytic properties of recombinant D-lactate dehydrogenase were identical to those of the wild-type enzyme, e.g. alpha 2 dimeric subunit structure, isoelectric pH, Km and Kcat for pyruvate and other 2-oxo-acid substrates. The kinetic profiles of 2-oxo-acid substrates showed some marked differences from that of L-lactate dehydrogenase, suggesting different mechanisms for substrate binding and specificity.     

Dihydrolipoamide dehydrogenase: functional similarities and divergent evolution of the pyridine nucleotide-disulfide oxidoreductases

Dihydrolipoamide dehydrogenase (E3) is the common component of the three alpha-ketoacid dehydrogenase complexes oxidizing pyruvate, alpha-ketoglutarate, and the branched-chain alpha-ketoacids. E3 also participates in the glycine cleavage system. E3 belongs to the enzyme family called pyridine nucleotide-disulfide oxidoreductases, catalyzing the electron transfer between pyridine nucleotides and disulfide compounds. This review summarizes the information available for E3 from a variety of species, from a halophilic archaebacterium which has E3 but no alpha-ketoacid dehydrogenase complexes, to mammalian species. Evidence is reviewed for the existence of two E3 isozymes (one for pyruvate dehydrogenase complex and alpha-ketoglutarate dehydrogenase complex and the other for branched-chain alpha-ketoacid dehydrogenase complex) in Pseudomonas species and for possible mammalian isozymes of E3, one associated with the three alpha-ketoacid dehydrogenase complexes and one for the glycine cleavage system. The comparison of the complete amino acid sequences of E3 from Escherichia coli, yeast, pig, and human shows considerable homologies of certain amino acid residues or short stretches of sequences, especially in the specific catalytic and structural domains. Similar homology is found with the limited available amino acid sequence information on E3 from several other species. Sequence comparison is also presented for other member flavoproteins [e.g., glutathione reductase and mercury(II) reductase] of the pyridine nucleotide-disulfide oxidoreductase family. Based on the known tertiary structure of human glutathione reductase it may be possible to predict the domain structures of E3. Additionally, the sequence information may help to better understand a divergent evolutionary relationship among these flavoproteins in different species.     

Lipoic acid-dependent oxidative catabolism of alpha-keto acids in mitochondria provides evidence for branched-chain amino acid catabolism in Arabidopsis

Lipoic acid-dependent pathways of alpha-keto acid oxidation by mitochondria were investigated in pea (Pisum sativum), rice (Oryza sativa), and Arabidopsis. Proteins containing covalently bound lipoic acid were identified on isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis separations of mitochondrial proteins by the use of antibodies raised to this cofactor. All these proteins were identified by tandem mass spectrometry. Lipoic acid-containing acyltransferases from pyruvate dehydrogenase complex and alpha-ketoglutarate dehydrogenase complex were identified from all three species. In addition, acyltransferases from the branched-chain dehydrogenase complex were identified in both Arabidopsis and rice mitochondria. The substrate-dependent reduction of NAD(+) was analyzed by spectrophotometry using specific alpha-keto acids. Pyruvate- and alpha-ketoglutarate-dependent reactions were measured in all three species. Activity of the branched-chain dehydrogenase complex was only measurable in Arabidopsis mitochondria using substrates that represented the alpha-keto acids derived by deamination of branched-chain amino acids (Val [valine], leucine, and isoleucine). The rate of branched-chain amino acid- and alpha-keto acid-dependent oxygen consumption by intact Arabidopsis mitochondria was highest with Val and the Val-derived alpha-keto acid, alpha-ketoisovaleric acid. Sequencing of peptides derived from trypsination of Arabidopsis mitochondrial proteins revealed the presence of many of the enzymes required for the oxidation of all three branched-chain amino acids. The potential role of branched-chain amino acid catabolism as an oxidative phosphorylation energy source or as a detoxification pathway during plant stress is discussed.     

Synthesis, physicochemical and spectroscopic characterization of copper(II)-polysaccharide pullulan complexes by UV-vis, ATR-FTIR, and EPR

Bioactive copper(II) complexes with polysaccharides, like pullulan and dextran, are important in both veterinary and human medicine for the treatment of hypochromic microcitary anemia and hypocupremia. In aqueous alkaline solutions, Cu(II) ion forms complexes with the exopolysaccharide pullulan and its reduced low-molecular derivative. The metal content and the solution composition depend on pH, temperature, and time of the reaction. The complexing process begins in a weak alkali solution (pH >7) and involves OH groups of pullulan monomer (glucopyranose) units. Complexes of Cu(II) ion with reduced low-molecular pullulan (RLMP, M_w 6000 g mol⁻¹) were synthesized in water solutions, at the boiling temperature and at different pH values ranging from 7.5 to 12. The Cu(II) complex formation with RLMP was analyzed by UV–vis spectrophotometry and other physicochemical methods. Spectroscopic characterizations (ATR-FTIR, FT-IRIS, and EPR) and spectra–structure correlation of Cu(II)–RLMP complexes were also carried out.     

亮氨酸脱氢酶研究进展及其工业应用

微生物脱氢酶催化羰基不对称还原制备光学纯氨基酸及其衍生物具有非常大的优势。亮氨酸脱氢酶能选择性地催化α-酮酸,氨化还原得到α-氨基酸及其衍生物。本文综述了亮氨酸脱氢酶的来源,理化性质,底物特异性,酶基因工程菌构建等方面的内容及研究进展。从辅酶再生策略,酶膜反应器两方面讨论了其工业化应用,并展望了今后的发展前景。     

Glutamate dehydrogenase contributes to leucine sensing in the regulation of autophagy

Amino acids, leucine in particular, are known to inhibit autophagy, at least in part by their ability to stimulate MTOR-mediated signaling. Evidence is presented showing that glutamate dehydrogenase, the central enzyme in amino acid catabolism, contributes to leucine sensing in the regulation of autophagy. The data suggest a dual mechanism by which glutamate dehydrogenase activity modulates autophagy, i.e., by activating MTORC1 and by limiting the formation of reactive oxygen species.     

Aminoacidopathies: Prevalence,Etiology, Screening,and Treatment Options

Inborn errors of metabolism (IEMs) are a group of inherited metabolic disorders which are caused by mutations in the specific genes that lead to impaired proteins or enzymes production. Different metabolic pathways are perturbed due to the deficiency or lack of enzymes. To date, more than 500 IEMs have been reported with most of them being untreatable. However, fortunately 91 such disorders are potentially treatable, if diagnosed at an earlier stage of life. IEMs have been classified into different categories and one class of IEMs, characterized by the physiological disturbances of amino acids is called as aminoacidopathies. Out of 91 treatable IEM, thirteen disorders are amino acid related. Aminoacidopathies can be detected by chromatography and mass spectrometry based analytical techniques (e.g., HPLC, GC–MS, LC–MS/MS) for amino acid level changes, and through genetic assays (e.g., PCR, TaqMan Genotyping, DNA sequencing) at the mutation level in the corresponding genes. Hence, this review is focused to describe thirteen common aminoacidopathies namely: Phenylketonuria (PKU), Maple Syrup Urine Disease (MSUD), Homocystinuria/Methylene Tetrahydrofolate Reductase (MTHFR) deficiency, Tyrosinemia type II, Citrullinemia type I and type II, Argininosuccinic aciduria, Carbamoyl Phosphate Synthetase I (CPS) deficiency, Argininemia (arginase deficiency), Hyperornithinemia–Hyperammonemia–Homocitrullinuria (HHH) syndrome, N-Acetylglutamate Synthase (NAGS) deficiency, Ornithine Transcarbamylase (OTC) deficiency, and Pyruvate Dehydrogenase (PDH) complex deficiency. Furthermore, the etiology, prevalence and commonly used analytical techniques for screening of aminoacidopathies are briefly described. This information would be helpful to researchers and clinicians especially from developing countries to initiate newborn screening programs for aminoacidopathies.     

Desilicification from the High Silica Containing Kraft Black Liquor by the Improved Carbon Dioxide Method

Addition of NADH to crude but not to pure branched-chain α-keto acid decarboxylase decreased the CO₂ production from α-keto-β-methylvalerate (KMV) suggesting the presence of an NADH dependent inhibitor in the crude enzyme from Bacillus subtilis. This NADH-dependent decarboxylase inhibitor was purified to homogeneity by a fast protein liquid chromatography system.

The purified inhibitor was identical with leucine dehydrogenase as to N-terminal amino acid squence (35 residues) and molecular weight, and catalyzed the oxidative deamination of three branched chain amino acids (BCAAs), valine, leucine, and isoleucine. The decarboxylase inhibitor was therefore identified as leucine dehydrogenase. A decreased substrate availability caused by leucine dehydrogenase thus reasonably accounted for the NADH dependent inhibition of the decarboxylation. In turn, the observation that leucine dehydrogenase competes with the decarboxylase for branched-chain α-keto acid (BCKA) suggested an involvement of this enzyme in the branched chain fatty acid (BCFA) biosynthesis. This view was supported by the observation that addition of NAD to crude fatty acid synthetase increased the incorporation of isoleucine into BCFAs. Pyridoxal-5′-phosphate and α-ketoglutarate, cofactors for BCAA transaminase, modulated BCFA biosynthesis from isoleucine in vitro, suggesting also the involvement of transaminase reaction in BCFA biosynthesis.     

A spectrophotometric coupled enzyme assay to measure the activity of succinate dehydrogenase

Respiratory complex II (succinate:ubiquinone oxidoreductase) connects the tricarboxylic acid cycle to the electron transport chain in mitochondria and many prokaryotes. Complex II mutations have been linked to neurodegenerative diseases and metabolic defects in cancer. However, there is no convenient stoichiometric assay for the catalytic activity of complex II. Here, we present a simple, quantitative, real-time method to detect the production of fumarate from succinate by complex II that is easy to implement and applicable to the isolated enzyme, membrane preparations, and tissue homogenates. Our assay uses fumarate hydratase to convert fumarate to malate and uses oxaloacetate decarboxylating malic dehydrogenase to convert malate to pyruvate and to convert NADP⁺ to NADPH; the NADPH is detected spectrometrically. Simple protocols for the high-yield production of the two enzymes required are described; oxaloacetate decarboxylating malic dehydrogenase is also suitable for accurate determination of the activity of fumarate hydratase. Unlike existing spectrometric assay methods for complex II that rely on artificial electron acceptors (e.g., 2,6-dichlorophenolindophenol), our coupled assay is specific and stoichiometric (1:1 for succinate oxidation to NADPH formation), so it is suitable for comprehensive analyses of the catalysis and inhibition of succinate dehydrogenase activities in samples with both simple and complex compositions.     

Disruptions in valine degradation affect seed development and germination in Arabidopsis

We have functionally characterized the role of two putative mitochondrial enzymes in valine degradation using insertional mutants. Prior to this study, the relationship between branched‐chain amino acid degradation (named for leucine, valine and isoleucine) and seed development was limited to leucine catabolism. Using a reverse genetics approach, we show that disruptions in the mitochondrial valine degradation pathway affect seed development and germination in Arabidopsis thaliana. A null mutant of 3‐hydroxyisobutyryl‐CoA hydrolase (CHY4, At4g31810) resulted in an embryo lethal phenotype, while a null mutant of methylmalonate semialdehyde dehydrogenase (MMSD, At2g14170) resulted in seeds with wrinkled coats, decreased storage reserves, elevated valine and leucine, and reduced germination rates. These data highlight the unique contributions CHY4 and MMSD make to the overall growth and viability of plants. It also increases our knowledge of the role branched‐chain amino acid catabolism plays in seed development and amino acid homeostasis.     

α-Tocopherol and Ascorbic Acid Administration Prevents the Impairment of Brain Energy Metabolism of Hyperargininemic Rats

Summary 1 We have previously demonstrated that arginine administration induces oxidative stress and compromises energy metabolism in rat hippocampus. In the present study we initially investigated the influence of pretreatment with α-tocopherol and ascorbic acid on the effects produced by arginine on hippocampus energy metabolism. We also tested the effect of acute administration of arginine on various parameters of energy metabolism, namely glucose uptake, lactate release and on the activities of succinate dehydrogenase, complex II and cytochrome c oxidase in rat cerebellum, as well as the influence of pretreatment with α-tocopherol and ascorbic acid on the effects elicited by arginine on this structure.2. Sixty-day-old female Wistar rats were treated with a single i.p. injection of saline (control) or arginine (0.8 g/kg) and were killed 1 h later. In another set of experiments, the animals were pretreated for 1 week with daily i.p. administration of saline (control) or α-tocopherol (40 mg/kg) and ascorbic acid (100 mg/kg). Twelve hours after the last injection of the antioxidants the rats received one i.p. injection of arginine (0.8 g/kg) or saline and were killed 1 h later.3. Results showed that arginine administration significantly increased lactate release and diminished glucose uptake and the activities of succinate dehydrogenase and complex II in rat cerebellum. In contrast, complex IV (cytochrome c oxidase) activity was not changed by this amino acid. Furthermore, pretreatment with α-tocopherol and ascorbic acid prevented the impairment of energy metabolism caused by hyperargininemia in cerebellum and hippocampus of rats.     

Ternary copper(II) complexes involving 2-(aminomethyl)-benzimidazole and some bio-relevant ligands. Equilibrium studies and kinetics of hydrolysis for glycine methyl ester under complex formation

Formation equilibria of copper(II) complexes of 2-(aminomethyl)-benzimidazole (AMBI) and the ternary complexes Cu(AMBI)L (L = amino acid, amide, dicarboxylic acid or DNA constituents) have been investigated. Ternary complexes of amino acids or amides are formed by a simultaneous mechanism. Amino acids form the complex Cu(AMBI)L, whereas amides form two complex species Cu(AMBI)L and Cu(AMBI)(LH₋₁). The ternary complexes of copper(II) with AMBI and dicarboxylic acids or DNA units are formed by a stepwise mechanism, whereby binding of copper(II) to AMBI is followed by ligation of the dicarboxylic acids or DNA components. The values of Δ log K indicate that the ternary complexes containing aromatic amino acids are significantly more stable than the complexes containing alkyl- and hydroxyalkyl-substituted amino acids. This may be taken as an evidence for a stacking interaction between the aromatic moiety of AMBI and the aromatic side chains of the bio-active ligands. The solid complexes Cu(AMBI)L where L = 1,1-cyclobutanedicarboxylic acid (CBDCA) and malonic acid were separated and identified by elemental analysis and infrared spectroscopy and magnetic moment. The decomposition course and steps for the isolated complexes were analyzed and the kinetic parameters of the non-isothermal decomposition were calculated. The hydrolysis of glycine methyl ester (MeGly) is catalyzed by the Cu(AMBI)²⁺ complex. The kinetic data is fitted assuming that the hydrolysis reaction proceeds in two steps. The first step, involving coordination of the amino acid ester by the amino and carbonyl groups, is followed by rate-determining attack by OH⁻ ion. The second step involves the equilibrium formation of the hydroxo-complex Cu(AMBI)(MeGly)(OH) followed by intramolecular OH⁻ attack.