Effects of branched chain amino acids,pyruvate, or ketone bodies on the free amino acid pool and release from brain cortex slices of normal and streptozotocindiabetic rats

Brain cortex slices from diabetic rats incubated in Krebs-Ringer-bicarbonate (KRB)-glucose medium show, compared to the normals, a 75% higher glutamine content. Branched chain amino acids (BCAA) added, at 0.5mM each, to this medium increase (53%) the glutamine content in the normal slices but have no effect on the glutamine content in the slices from diabetic rats. When the incubation medium is KRB-pyruvate, glutamine and glutamate contents are lower than in the KRB-glucose medium. The addition of BCAA in the KRB-pyruvate medium partially restores the contents of glutamine in the normal and of glutamine plus glutamate in the diabetic. Keto acids or BCAA added to the incubation medium of normal slices decrease the pool of most of the neutral and acidic amino acids but they do not affect this pool in slices from the diabetic rats. In addition keto acids increase the ratio glutamate in the tissue: glutamate in the medium.Abbreviations used BCAA branched chain amino acids - 3-OHB d,l-3-hydroxybutyrate - AcAc acetoacetate - KRB Krebs-Ringer-bicarbonate     

Branched chain amino acid aminotransferase isoenzymes of Pseudomonas cepacia

Pseudomonas cepacia grew rapidly using a mixture of all three branched chain amino acids as carbon source, but failed to use individual branched chain amino acids as sole carbon source. Extracts of bacteria grown on branched chain amino acids had between 2- and 3-fold higher levels of -ketoglutarate-dependent branched chain amino acid aminotransferase activity than extracts of glucose-grown bacteria. The increase in enzyme activity was due to the presence of a second aminotransferase not detected in extracts of glucose-grown bacteria. The enzyme, which presumably plays a role in branched chain amino acid degradation, had an apparent molecular weight (mol. wt.) of 75,000. The other aminotransferase was formed constitutively and apparently functions in synthesis of branched chain amino acids. It was more stable than the 75,000 mol.wt. enzyme, and was purified to homogeneity and found to be a 180,000 mol.wt. oligomer containing 6 subunits of approximately 30,000 mol.wt. Antiserum prepared against the purified enzyme inhibited its activity but failed to influence the activity of the 75,000 mol.wt. aminotransferase, suggesting that the two isoenzymes are encoded by different genes.     

Factors influencing cell fatty acid composition and A40926 antibiotic complex production in <Emphasis Type="Italic">Nonomuraea</Emphasis> sp. ATCC 39727

A40926 is a glycopeptide antibiotic complex consisting of several structurally related factors. It is produced by fermentation of Nonomuraea sp. ATCC 39727 and the complex components differ in the structure of the fatty acid moiety linked to the aminoglucuronic acid unit. In previous work, we observed that the production of single factors in glycopeptide antibiotic complexes could be selectively enhanced by the addition of suitable precursors to the culture medium. In this contribution, we examine the effects of branched amino acid addition to fermentation of Nonomuraea sp. in a chemically defined minimal medium. The changes in the composition of cell fatty acids correlate to the fatty acid distribution within the A40926 complex in diverse cultivation conditions. Nonomuraea sp. prefers isobutyric, butyric and propionic acids as initiators of fatty acid biosynthesis. The relative amount of the produced fatty acids is significantly influenced by the availability of intermediates or final products from the amino acid catabolic pathways. Antibiotic complex composition closely reflects the cell fatty acid pattern, in agreement with the assumption that the antibiotic fatty acid moieties are synthesized by shortening the chain of cell fatty acids.     

Relative importance of physiological precursors for ketogenesis in the nutritional homeostasis of the development of the embryonic chick

This paper investigates the relative importances of specific amino acids, and, in particular, branched chain amino acids and their keto derivatives as possible ketogenic precursors in suspensions of liver cells isolated from chick embryos. Addition of the branched chain keto acids stimulated ketogenesis. The order of potency was α-ketoisocaproic acid >α-ketoisovaleric acid >DL- α-keto-β-methyl-n-valeric acid. The relative order of effectiveness for branched chain keto acids was maintained at all comparable concentrations, and in each case maximum rates were observed with concentrations of 1–2 mM. In contrast to the stimulation of ketogenesis by their keto derivatives, branched chain amino acids were ineffective as precursors for ketogenesis. Of the other amino acids (utilised at concentrations present in chick embryo plasma) only Tyr, Lys, Phe and Arg produced significant increases in ketone body formation above the endogenous rate. At these physiological concentrations, the effectiveness of the amino acids were in the order of Tyr > Lys = Phe > Arg. The interactions between three groups of ketogenic precursor (fatty acids, amino acids and keto amino acids, all at physiological concentrations), produced rates of ketogenesis that were purely additive. These results indicate that high concentrations of hydroxybutyrate and acetoacetate found in plasma of developing chick embryos may arise from hepatic metabolism of several distinct precursors. The relative importance of each category of precursor may vary with the precise developmental status of animals.     

Branched chain amino acids as source of specific branched chain volatile fatty acids during the fermentation process of fish sauce

Summary.  The source of the formation of branched chain volatile fatty acids (VFA) in fish sauce was investigated. Certain branched VFA were derived from the degradation of specific amino acids as iso-butyric acid from valine and iso-valeric acid from leucine. Short and long straight chain VFA were significantly higher in the linoleic acid added sample than in the control but did not significantly bring changes to the branched chain VFA. It is suggested that straight chain VFA developed from fish fats. Alanine and isoleucine did not have a clear influence on the production of volatile fatty acids. Received November 23, 2001 Accepted June 20, 2002 Published online December 18, 2002 RID="*" ID="*" Part of this paper was presented in the 7th International Congress on Amino Acids and Proteins in Vienna, Austria from August 6–10, 2001. Authors' address: Norlita G. Sanceda, Ph.D., Institute of Environmental Science for Human Life, 2-1-1 Otsuka, Bunkyo-ku, Tokyo 112, Japan, Fax: + 81-3-5978-5805, E-mail: lita@cc.ocha.ac.jp     

Role of mitochondrial transamination in branched chain amino acid metabolism

Oxidative decarboxylation and transamination of 1-14C-branched chain amino and alpha-keto acids were examined in mitochondria isolated from rat heart. Transamination was inhibited by aminooxyacetate, but not by L-cycloserine. At equimolar concentrations of alpha-ketoiso[1-14C]valerate (KIV) and isoleucine, transamination was increased by disrupting the mitochondria with detergent which suggests transport may be one factor affecting the rate of transamination. Next, the subcellular distribution of the aminotransferase(s) was determined. Branched chain aminotransferase activity was measured using two concentrations of isoleucine as amino donor and [1-14C]KIV as amino acceptor. The data show that branched chain aminotransferase activity is located exclusively in the mitochondria in rat heart. Metabolism of extramitochondrial branched chain alpha-keto acids was examined using 20 microM [1-14C]KIV and alpha-ketoiso[1-14C]caproate (KIC). There was rapid uptake and oxidation of labeled branched chain alpha-keto acid, and, regardless of the experimental condition, greater than 90% of the labeled keto acid substrate was metabolized during the 20-min incubation. When a branched chain amino acid (200 microM) or glutamate (5 mM) was present, 30-40% of the labeled keto acid was transaminated while the remainder was oxidized. Provision of an alternate amino acceptor in the form of alpha-keto-glutarate (0.5 mM) decreased transamination of the labeled KIV or KIC and increased oxidation. Metabolism of intramitochondrially generated branched chain alpha-keto acids was studied using [1-14C]leucine and [1-14C]valine. Essentially all of the labeled branched chain alpha-keto acid produced by transamination of [1-14C]leucine or [1-14C]valine with a low concentration of unlabeled branched chain alpha-keto acid (20 microM) was oxidized. Further addition of alpha-ketoglutarate resulted in a significant increase in the rate of labeled leucine or valine transamination, but again most of the labeled keto acid product was oxidized. Thus, catabolism of branched chain amino acids will be favored by a high concentration of mitochondrial alpha-ketoglutarate and low intramitochondrial glutamate.     

Effects of abnormal metabolites of maple syrup urine disease on neurotransmitter receptor binding

The deficiency of keto acid decarboxylase in maple syrup urine disease results in the accumulation of branched chain amino acids and their corresponding keto acids in tissues and body fluids. The effects of abnormal metabolites were investigated on neurotransmitter receptor binding in rat brain. alpha-Keto acids caused selective in vitro decrease in alpha-adrenergic, beta-adrenergic receptor binding in synaptosomal preparations from rat brain. No significant changes were observed in binding of cholinergic, GABA, and dopamine receptors binding in appropriate rat brain preparations. These results indicate that selective inhibition of adrenergic receptor binding by branched chain keto acids may presumably account for neural abnormality in maple syrup urine disease.     

Identification of a potential bottleneck in branched chain fatty acid incorporation into triacylglycerol for lipid biosynthesis in agronomic plants

In plant, unusual fatty acids are produced by a limited number of species. The industrial benefits of these unusual structures have led several groups to study their production in transgenic plants. Their research results led to very modest accumulation in seeds which was largely due to a limited knowledge of the lipid metabolism and fatty acid transfer in plants. More specifically we need to better understand the substrate specificity and selectivity of acyltransferases which are required for the incorporation of these unusual fatty acids into storage triacylglycerols. In our studies we have compared the incorporation of [¹⁴C] Oleoyl-CoA and Branched Chain Acyls-CoA into [³H] LPA-C18:1 by the Lysophosphatidic acid Acyltransferase (LPAAT) from developing seeds of agronomic plants (flax (Linum usitatissimum) and rape (Brassica napus)) and from a plant capable of producing high amounts of hydroxy fatty acids (castor bean (Ricinus communis)). Our assays demonstrate that LPAATs of the three studied species (1) incorporated preferentially oleyl-CoA, (2) could incorporate cyclopropane acyl-CoA when added alone as a substrate, however very weakly for rapeseed and castor bean seeds, (3) presented a low capacity to incorporate methyl branched acyl-CoA when added alone as a substrate (4) weakly incorporated cyclopropane acyl-CoA and was unable to incorporate methyl branched acyl-CoA when presented with an equimolar mix of oleyl-CoA and branched chain acyl-CoA. In all cases, the LPAAT had a low affinity for branched chain acyl-CoAs. The results show that LPAAT activity from agronomic plants constitutes a bottleneck for the incorporation of branched Chain acyl-CoA into PA.     

Effects of insulin secretagogues on the secretion of insulin during thiamine deficiency

The mechanism by which glucose and other nutrient secretagogues induce the insulin secretion, is still controversial. Thiamine deficient rats, having a block in the glucose and branched chain amino acid metabolism at pyruvate and branched chain keto acids dehydrogenases respectively, were used to study the effects of insulin secretagogues. The levels of fasting blood glucose and serum insulin were estimated. Also, the serum insulin was assayed after intravenous administration of leucine, arginine and tolbutamide. The fasting blood glucose was increased and the serum insulin was decreased in thiamine deficiency. Leucine and arginine did not enhance insulin secretion in thiamine deficient animals. Tolbutamide induces the insulin secretion minimally in thiamine deficient rats. These results suggest that the nutrient secretagogues require an unimpaired glucose metabolism to induce insulin secretion.     

Nutritional control of branched chain alpha-ketoacid dehydrogenase in rat hepatocytes

Branched chain alpha-ketoacid dehydrogenase (EC 1.2.4.4) complex, the rate-limiting enzyme of branched chain amino acid catabolism in most tissues, is subject to regulation by covalent modification, with phosphorylation inactivating and dephosphorylation activating the complex. The enzyme complex from liver of chow-fed rats is mainly in the active form but that from liver of rats fed a low-protein diet is mainly in the inactive form. Isolated hepatocytes were used to identify factors that affect interconversion of branched chain alpha-ketoacid dehydrogenase. The enzyme present in hepatocytes of rats fed a low-protein diet appears much more responsive to regulation by covalent modification than the branched chain alpha-ketoacid dehydrogenase present in hepatocytes of normal chow-fed rats. alpha-Chloroisocaproate, a specific inhibitor of the kinase responsible for phosphorylation and inactivation of the complex, greatly stimulates oxidation of alpha-keto[1-14C]isovalerate by hepatocytes prepared from rats fed a low-protein diet but not from normal chow-fed rats. Oxidizable substrates are also much more effective inhibitors of branched chain alpha-ketoacid oxidation with hepatocytes from rats fed a low-protein diet than from normal chow-fed rats. Activity measurements with cell-free extracts suggest that changes in flux through the dehydrogenase with intact hepatocytes prepared from rats fed a low-protein diet are explained in large part by changes in the proportion of the enzyme in the active, dephosphorylated form. Regulation of liver branched chain alpha-ketoacid dehydrogenase by covalent modification functions to conserve branched chain amino acids for protein synthesis during periods of restricted dietary protein intake.     

Measurement of alpha-keto acids in plasma using an amino acid analyzer

A method for measuring keto acid concentrations in physiological fluids using an amino acid analyzer was developed. After preliminary deproteinization and removal of amino acids, reduction with sodium cyanoborohydride at 105 degrees C resulted in efficient conversion of the keto acids to their corresponding amino acids. In applying the technique to plasma samples, the use of MeOH for deproteinization was necessary to avoid the large losses of keto acids that occurred during precipitation of proteins with perchloric acid. The method was used to follow plasma ketoisocaproate concentrations in rat plasma after administration of leucine, and was sufficiently sensitive to detect concomitant changes in other branched-chain keto acid concentrations.     

The role of growth hormone and amino acids on brain protein synthesis in aged rats given proteins of different quantity and quality

Summary. The purpose of the present study was to determine whether the regulation of brain protein synthesis was mediated through changes in the plasma concentrations of insulin and growth hormone (GH), and whether the concentrations of amino acids in the brain and plasma regulate the brain protein synthesis when the quantity and quality of dietary protein is manipulated. Two experiments were done on three groups of aged rats given diets containing 20% casein, 5% casein or 0% casein (Experiment 1), and 20% casein, 20% gluten, or 20% gelatin (Experiment 2) for 1 d (only one 5-h period) after all rats were fed the 20% casein diet for 10 d (only 5-h feeding per day). The aggregation of brain ribosomes, the concentration in plasma GH, and the branched chain amino acids in the plasma and cerebral cortex declined with a decrease of quantity and quality of dietary protein. The concentration of plasma insulin did not differ among groups. The results suggest that the ingestion of a higher quantity and quality of dietary protein increases the concentrations of GH and several amino acids in aged rats, and that the concentrations of GH and amino acids are at least partly related to the mechanism by which the dietary protein affects brain protein synthesis in aged rats.     

Plasma amino acids of lean and obese Zucker rats subjected to a cafeteria diet after weaning.

Plasma amino acids of Zucker obese (fa/fa) and lean (Fa/?) rats fed either a reference nonpurified pellet or a cafeteria diet have been studied from 30 to 60 days after birth. Obese rats showed higher plasma branched chain amino acid levels but similar total amino acids, urea and glucose concentrations. The ingestion of a cafeteria diet induced higher levels in many amino acids, as well as in the composite figure in lean rats, but failed to alter total 2-amino nitrogen concentrations in obese rats, despite high levels in several non-essential amino acids and lower values in essential amino acids; urea levels were much lower in rats fed the cafeteria diet. The results are consistent with an impairment of amino acid nitrogen elimination via urea cycle in cafeteria diet-fed rats. This is independent of the hyperinsulinemia-driven plasma accumulation of several essential amino acids induced by genetic obesity. The effects were, then additive.     

A profile of plasma branched chain amino acids in a totally pancreatectomized patient: effects of glucagon replacement under a steady feeding state

Physiological amounts of glucagon replacement brought about the reduction of plasma branched chain amino acid levels as well as glucogenic amino acid levels in a totally pancreatectomized patient who was under a steady feeding state, i.e., constant administration of an elemental diet combined with continuous subcutaneous insulin infusion. This finding suggests that glucagon may play a physiological role not only on glucogenic amino acids but also branched chain amino acids in plasma in feeding state.     

TRANSAMINATIONS BETWEEN AMINO ACIDS AND KETO ACIDS ELEVATED IN PHENYLKETONURIA AND MAPLE SYRUP URINE DISEASE

Abstract— The transamination between amino acids and aliphatic and aromatic keto acids has been investigated in homogenates of human and rat brain. Tryptophan, phenylalanine and 3,4-dihydroxyphenylalanine (DOPA) at concentrations of 3.6 min and below trans-aminated aromatic keto acids more rapidly than α-ketoglutarate; lower K_m values were found for tryptophan and phenylalanine in the presence of the aromatic keto acid. Rat brain and liver arninotransferases exhibited similar affinities for tryptophan in the presence of different keto acids. Branched chain keto acids were also acceptors of the amino groups of tryptophan and DOPA. In brain homogenates α-ketoglutarate and p -hydroxyphenyl-pyruvate were transaminated by tyrosine and 5-hydroxytryptophan at about equal rates, whereas a-ketoglutarate was transaminated more rapidly with aliphatic amino acids. At concentrations of 1.6 m DOPA and 0.8 mM p -hydroxyphenylpyruvate, transamination was 6-fold greater than the rate of formation of dopamine. The dihydroxyphenylpyruvate formed during arninotransfer from DOPA by brain tissue was not readily decarboxylated, whereas 65–70 per cent of the indolepyruvate formed from tryptophan was decarboxylated. We suggest that an increased rate or degree of transamination between tryptophan and aromatic and branched chain keto acids may explain the increased excretion of non-hydroxylated indolic acids in phenylketonuria and'maple syrup urine'disease, respectively. Increased aminotransfers from tryptophan and DOPA may reduce the amounts of precursors available for the synthesis of serotonin and catecholamines, both of which are at low levels in the sera of untreated phenylketonurics.     

Nonenzymatic assembly of branched polyubiquitin chains for structural and biochemical studies

Polymeric chains of a small protein ubiquitin are involved in regulation of nearly all vital processes in eukaryotic cells. Elucidating the signaling properties of polyubiquitin requires the ability to make these chains in vitro. In recent years, chemical and chemical–biology tools have been developed that produce fully natural isopeptide-linked polyUb chains with no need for linkage-specific ubiquitin-conjugating enzymes. These methods produced unbranched chains (in which no more than one lysine per ubiquitin is conjugated to another ubiquitin). Here we report a nonenzymatic method for the assembly of fully natural isopeptide-linked branched polyubiquitin chains. This method is based on the use of mutually orthogonal removable protecting groups (e.g., Boc- and Alloc-) on lysines combined with an Ag-catalyzed condensation reaction between a C-terminal thioester on one ubiquitin and a specific ε-amine on another ubiquitin, and involves genetic incorporation of more than one Lys(Boc) at the desired linkage positions in the ubiquitin sequence. We demonstrate our method by making a fully natural branched tri-ubiquitin containing isopeptide linkages via Lys11 and Lys33, and a ¹⁵N-enriched proximal ubiquitin, which enabled monomer-specific structural and dynamical studies by NMR. Furthermore, we assayed disassembly of branched and unbranched tri-ubiquitins as well as control di-ubiquitins by the yeast proteasome-associated deubiquitinase Ubp6. Our results show that Ubp6 can recognize and disassemble a branched polyubiquitin, wherein cleavage preferences for individual linkages are retained. Our spectroscopic and functional data suggest that, at least for the chains studied here, the isopeptide linkages are effectively independent of each other. Together with our method for nonenzymatic assembly of unbranched polyubiquitin, these developments now provide tools for making fully natural polyubiquitin chains of essentially any type of linkage and length.     

复方高支链氨基酸对肝硬化大鼠肝功能及营养状况的影响

将SD雄性大鼠用四氯化碳处理建立肝硬化大鼠模型,并随机分为A、B、C三组,A组大鼠给予静脉输注生理盐水,B组、C组大鼠分别给予输注等量的普通氨基酸注射液和复方高支链氨基酸注射液,分别于实验第0d、第14d测定大鼠体质量、肝功能指标及营养学指标水平。实验结束后,B、C两组大鼠体质量明显增加,与A组相比,B、C两组大鼠肝功能各指标水平显著降低,血清蛋白水平显著升高,且C组相比,B组大鼠肝功能水平与血清蛋白水平改善作用更为明显(p<0.05)。说明复方高支链氨基酸能改善肝硬化大鼠的肝功能指标,抑制血浆蛋白分解,有效控制肝硬化病症的进一步恶化。     

Genetic analysis of pathway regulation for enhancing branched‐chain amino acid biosynthesis in plants

The branched‐chain amino acids (BCAAs) valine, leucine and isoleucine are essential amino acids that play critical roles in animal growth and development. Animals cannot synthesize these amino acids and must obtain them from their diet. Plants are the ultimate source of these essential nutrients, and they synthesize BCAAs through a conserved pathway that is inhibited by its end products. This feedback inhibition has prevented scientists from engineering plants that accumulate high levels of BCAAs by simply over‐expressing the respective biosynthetic genes. To identify components critical for this feedback regulation, we performed a genetic screen for Arabidopsis mutants that exhibit enhanced resistance to BCAAs. Multiple dominant allelic mutations in the VALINE‐TOLERANT 1 (VAT1) gene were identified that conferred plant resistance to valine inhibition. Map‐based cloning revealed that VAT1 encodes a regulatory subunit of acetohydroxy acid synthase (AHAS), the first committed enzyme in the BCAA biosynthesis pathway. The VAT1 gene is highly expressed in young, rapidly growing tissues. When reconstituted with the catalytic subunit in vitro, the vat1 mutant‐containing AHAS holoenzyme exhibits increased resistance to valine. Importantly, transgenic plants expressing the mutated vat1 gene exhibit valine tolerance and accumulate higher levels of BCAAs. Our studies not only uncovered regulatory characteristics of plant AHAS, but also identified a method to enhance BCAA accumulation in crop plants that will significantly enhance the nutritional value of food and feed.     

Some physiological functions of the L-leucine dehydrogenase in Bacillus subtilis

Mutants of Bacillus subtilis constitutive for L-leucine dehydrogenase synthesis were selected. Using these mutants we could determine two functional roles for the L-leucine dehydrogenase. This enzyme liberates ammonium ions from branched chain amino acids when supplied as the sole nitrogen source. Another function is to synthesize from L-isoleucine, L-leucine, and L-valine the branched chain -keto acids which are precursors of branched chain fatty acid biosynthesis. These results together with the inducibility of the enzyme suggest that the L-leucine dehydrogenase has primarily a catabolic rather than an anabolic function in the metabolism of Bacillus subtilis.