支链氨基酸对运动大鼠氨基酸代谢和运动能力的影响

观察了支链氨基酸(BCAA)对大鼠运动能力和血清游离氨基酸代谢的影响。实验用21只雄性wistar大鼠,随机分为3组:正常组、游泳对照组和游泳补充BCAA组。2个运动组每天游泳训练1h,10天后游泳6h,观察补充BCAA对大鼠游泳运动能力和血清游离氨基酸水平的影响。实验结果表明,补充BCAA可明显提高大鼠游泳存活率,抑制血清中必需氨基酸、非必需氨基酸和总氨基酸水平升高,游泳运动后大鼠的血清中乳酸和LDH的升高幅度有所降低,抑制骨骼肌LDH活力的下降。说明补充BCAA可明显提高大鼠的运动能力,减少运动造成的蛋白质分解     

Neuronal Metabolism of Branched-Chain Amino Acids: Flux Through the Aminotransferase Pathway in Synaptosomes

Abstract: The metabolism of branched-chain amino acids (BCAAs) was studied in cortical synaptosomes. With [¹⁵N]leucine (1 mM) as precursor, the cumulative appearance of ¹⁵N in [¹⁵N]glutamate and [¹⁵N]aspartate was 0.2 nmol/min/mg of protein without supplemental α-ketoglutarate and 0.3 nmol/min/mg of protein in the presence of α-ketoglutarate (0.5 mM). The BCAA aminotransferase reaction also proceeded in the “reverse” direction [α-ketoisocaproate (KIC) + glutamate → leucine + α-ketoglutarate]. This was documented by incubating synaptosomes with [¹⁵N]glutamate and measuring the formation of [¹⁵N]leucine. Without KIC in the medium, the rate of [¹⁵N]leucine production was 0.13 nmol/min/mg of protein. In the presence of 25 µM KIC the rate was 0.79 nmol/min/mg of protein and even greater (1.0 nmol/min/mg of protein) in the presence of 500 µM KIC. The reamination of KIC was two- to threefold faster with [2-¹⁵N]glutamine as precursor compared with [¹⁵N]glutamate. The ketoacid of valine, α-ketoisovalerate (KIV), was reaminated to [¹⁵N]valine at a rate comparable to that observed with respect to KIC. The BCAA transaminase mediated not only the bidirectional transfer of amino groups between leucine or valine and glutamate, but also the direct transfer of nitrogen between leucine and valine. This was ascertained in studies in which the incubation medium was supplemented with either [¹⁵N]leucine and KIV or [¹⁵N]valine and KIC (amino acids at 1 mM and ketoacids at 25 or 500 µM). The rate was faster in the direction of leucine formation at both the lower (6.1-fold) and higher (1.7-fold) KIC concentration. It is suggested that in synaptosomes the BCAA transaminase (a) functions predominantly in the direction of leucine formation and (b) maintains a constant ratio of BCAAs and ketoacids to one other.     

支链氨基酸的抗疲劳作用

支链氨基酸作为必需氨基酸,不仅是合成机体蛋白质的原料,而且具有特殊的生理、生物学功能。其代谢与抗疲劳作用的机理在本文中进行了详细阐述。     

Uptake of Branched-Chain Amino Acids by Streptococcus thermophilus

The transport of branched-chain amino acids in Streptococcus thermophilus was energy dependent. The metabolic inhibitors of glycolysis and ATPase enzymes were active, but the proton-conducting uncouplers were not. Transport was optimal at temperatures of between 30 and 45°C and at pH 7.0 for the three amino acids leucine, valine, and isoleucine; a second peak existed at pH 5.0 with valine and isoleucine. By competition and kinetics studies, the branched-chain amino acids were found to share at least a common transport system.     

肝硬化疾病与支链氨基酸应用研究进展

蛋白质-营养不良是肝硬化病人最常见的并发症之一。肝脏作为蛋白质、脂类和糖代谢的主要器官,病变后的代谢紊乱随之而来。不适宜的蛋白质-能量摄入只会加重病情最后发生肝性脑病等危及生命的严重后果。因此,肝硬化病人的营养管理显得尤为重要,氨基酸的适宜供给无疑是营养治疗的重中之重。已知支链氨基酸能通过刺激肝细胞合成、减少肝损伤后的分解代谢等诸多方式改善营养状况,但是各种试验结果仍存在争议。最佳适宜量究竟多大,安全性范围的设定以及确切的保护机理等问题仍待进一步深入研究。     

Quantitative Analysis of the Whole-Body Metabolic Fate of Branched-Chain Amino Acids

1. Download high-res image (203KB)
2. Download full-size image

     

Intrauterine Malnutrition and the Brain: Effects on Enzymes and Free Amino Acids Related to Glutamate Metabolism

Abstract: The effect of feeding pregnant rats with wheat and Bengal gram (black chick pea) diets during the later part of pregnancy on brain growth, enzymes, and free amino acids of glutamate metabolism in 1-day-old rats was investigated. These diets did not induce growth dissociation, and the body and brain weights were equally affected. The concentrations of DNA, RNA, protein, and free α-amino nitrogen in brain decreased significantly and the activities of glutamine synthetase, glutamine transferase, glutaminase 1, glutaminase 11, and glutamate decarboxylase and the concentrations of free amino acids, glutamic acid, glutamine, alanine, and GABA were also decreased. The concentration of aspartic acid, however, was increased. Wheat and Bengal gram diets fortified with lysine and with methionine, cystine, and tryptophan respectively showed various beneficial effects on the changes observed in the brain. A 20% casein diet induced higher body and brain weights and better brain protein and free α-amino nitrogen concentrations than those observed on a 10% casein diet.     

Astrocyte Leucine Metabolism: Significance of Branched-Chain Amino Acid Transamination

Abstract: We studied astrocytic metabolism of leucine, which in brain is a major donor of nitrogen for the synthesis of glutamate and glutamine. The uptake of leucine into glia was rapid, with a V _max of 53.6 ± 3.2 nmol/mg of protein/min and a K _m of 449.2 ± 94.9 µ M . Virtually all leucine transport was found to be Na⁺ independent. Astrocytic accumulation of leucine was much greater (3×) in the presence of α-aminooxyacetic acid (5 m M ), an inhibitor of transamination reactions, suggesting that the glia rapidly transaminate leucine to α-ketoisocaproic acid (KIC), which they then release into the extracellular fluid. This inference was confirmed by the direct measurement of KIC release to the medium when astrocytes were incubated with leucine. Approximately 70% of the leucine that the glia cleared from the medium was released as the keto acid. The apparent K _m for leucine conversion to extracellular KIC was a medium [leucine] of 58 µ M with a V _max of ∼2.0 nmol/mg of protein/min. The transamination of leucine is bidirectional (leucine + α-ketoglutarate ↮ KIC + glutamate) in astrocytes, but flux from leucine → glutamate is more active than that from glutamate → leucine. These data underscore the significance of leucine handling to overall brain nitrogen metabolism. The release of KIC from glia to the extracellular fluid may afford a mechanism for the "buffering" of glutamate in neurons, which would consume this neurotransmitter in the course of reaminating KIC to leucine.     

Escherichia coli K-12 Mutants Altered in the Transport of Branched-Chain Amino Acids

Two mutants of Escherichia coli K-12 are described which are resistant to the inhibition that valine exerts on the growth of E. coli. These mutants have lesions at two different loci on the chromosome. One of them, brnP, is linked to leu (87% cotransduction) and is located between leu and azi represented on the map at 1 min; the other, brnQ, is linked to phoA (96% cotransduction), probably between proC and phoA and represented at 10 min. These mutants are resistant to valine inhibition but are sensitive to dipeptides containing valine. Since it is known that dipeptides are taken up by E. coli through a transport system(s) different from those used by amino acids, this sensitivity to the peptides suggests an alteration in the active transport of valine. The mutants are resistant to valine only if leucine is present in the growth medium; the uptake of valine is less in both mutants than it is in wild-type E. coli, and it is reduced even further if leucine is present. Under these conditions the total uptake of valine is almost completely abolished in the brnQ mutant. The brnP mutant takes up about 60% as much valine as does the wild type, but no exogenous valine is incorporated into proteins. The apparent K(m) and V(max) of isoleucine, leucine, and valine for the transport system are reported; the brnP mutant, when compared to the wild type, has a sevenfold higher K(m) for isoleucine and a 17-fold lower K(m) for leucine; the V(max) for the three amino acids is reduced in the brnQ mutant, up to 20-fold for valine. The transport of arginine, aspartic acid, glycine, histidine, and threonine is not altered in the brnQ mutant under conditions in which that of the branched amino acids is. Evidence is reported that O-methyl-threonine enters E. coli through the transport system for branched amino acids, and that thiaisoleucine does not.     

Metabolism of Aromatic Amino Acids in Microorganisms

It was found that when Rhodotorula rubra IFO 0911 was grown in a phenylalanine medium, benzoic acid and p-hydroxybenzoic acid besides cinnamic acid were formed in the cultured both. The conversions of cinnamic acid into benzoic acid and of benzoic acid into p-hydroxybenzoic acid, and the degradation of p-hydroxybenzoic acid were demonstrated in intact cells of Rhodotorula rubra. These activities were observed in the cells grown on various media, including the medium containing no phenylalanine, and were found to be distributed widely in Rhodotorula. The cells of Rhodotorula rubra were also able to degrade p-coumaric acid, 3,4-dihydroxybenzoic acid (protocatechuic acid), p-hydroxyphenyl-acetic acid, 3-methoxy-4-hydroxycinnamic acid (ferulic acid) and 3-methoxy-4-hydroxybenzoic acid (vanillic acid). From these results, the metabolic pathways for phenylalanine and tyrosine in Rhodotorula were discussed.     

YjeH Is a Novel Exporter of l-Methionine and Branched-Chain Amino Acids in Escherichia coli

Amino acid efflux transport systems have important physiological functions and play vital roles in the fermentative production of amino acids. However, no methionine exporter has yet been identified in Escherichia coli. In this study, we identified a novel amino acid exporter, YjeH, in E. coli. The yjeH overexpression strain exhibited high tolerance to the structural analogues of l-methionine and branched-chain amino acids, decreased intracellular amino acid levels, and enhanced export rates in the presence of a Met-Met, Leu-Leu, Ile-Ile, or Val-Val dipeptide, suggesting that YjeH functions as an exporter of l-methionine and the three branched-chain amino acids. The export of the four amino acids in the yjeH overexpression strain was competitively inhibited in relation to each other. The expression of yjeH was strongly induced by increasing cytoplasmic concentrations of substrate amino acids. Green fluorescent protein (GFP)-tagged YjeH was visualized by total internal reflection fluorescence microscopy to confirm the plasma membrane localization of YjeH. Phylogenetic analysis of transporters indicated that YjeH belongs to the amino acid efflux family of the amino acid/polyamine/organocation (APC) superfamily. Structural modeling revealed that YjeH has the typical “5 + 5” transmembrane α-helical segment (TMS) inverted-repeat fold of APC superfamily transporters, and its binding sites are strictly conserved. The enhanced capacity of l-methionine export by the overexpression of yjeH in an l-methionine-producing strain resulted in a 70% improvement in titer. This study supplements the transporter classification and provides a substantial basis for the application of the methionine exporter in metabolic engineering.     

Acute and Chronic Administration of the Branched-Chain Amino Acids Decreases Nerve Growth Factor in Rat Hippocampus

Maple syrup urine disease (MSUD) is a neurometabolic disorder caused by deficiency of the activity of the mitochondrial enzyme complex branched-chain α-keto acid dehydrogenase leading to accumulation of the branched-chain amino acids (BCAA) and their corresponding branched-chain α-keto acids. In this study, we examined the effects of acute and chronic administration of BCAA on protein levels and mRNA expression of nerve growth factor (NGF) considering that patients with MSUD present neurological dysfunction and cognitive impairment. Considering previous observations, it is suggested that oxidative stress may be involved in the pathophysiology of the neurological dysfunction of MSUD. We also investigated the influence of antioxidant treatment (N-acetylcysteine and deferoxamine) in order to verify the influence of oxidative stress in the modulation of NGF levels. Our results demonstrated decreased protein levels of NGF in the hippocampus after acute and chronic administration of BCAA. In addition, we showed a significant decrease in the expression of ngf in the hippocampus only following acute administration in 10-day-old rats. Interestingly, antioxidant treatment was able to prevent the decrease in NGF levels by increasing ngf expression. In conclusion, the results suggest that BCAA is involved in the regulation of NGF in the developing rat. Thus, it is possible that alteration of neurotrophin levels during brain maturation could be of pivotal importance in the impairment of cognition provoked by BCAA. Moreover, the decrease in NGF levels was prevented by antioxidant treatment, reinforcing that the hypothesis of oxidative stress can be an important pathophysiological mechanism underlying the brain damage observed in MSUD.     

Glycine and L-Alanine as Antagonists of Growth Inhibition by the Branched-Chain Amino Acids in Spirodela polyrhiza

Growth inhibition of the duckweed Spirodela polyrhiza (L.) Schleidenby exogenously supplied L-leucine, L-valine, or L-isoleucinewas antagonized by simultaneously supplied glycine or L-alanine.Calculations, using the kinetic parameters for the uptake ofthese amino acids, indicated that the antagonisms to a considerabledegree resulted from competitive inhibition of the uptake ofthe growth-inhibiting amino acids. However, the antagonismsresulted partly from an interaction between growth inhibitorand antagonist inside the cells.     

支链氨基酸合成调控机制及菌种选育策略研究进展

提高国内支链氨基酸产生菌的高产菌株选育水平有助于缩短与国外生产之间的差距,满足国内市场需求。根据支链氨基酸生物合成途径及代谢调节,重点阐述了合成过程中关键酶的代谢调控,介绍了诱变育种、代谢工程、基因组改组及全局转录机器工程四种育种策略的研究进展。在支链氨基酸选育方面,全局转录机器工程育种目前虽无成功实例,但具有很大的潜力,而其他育种策略在氨基酸的选育中均发挥重要作用,可供国内相关育种工作者参考使用。     

富含谷氨酰胺和支链氨基酸的肠外制剂对创伤大鼠的效用

研究普通氨基酸注射液 (17AA)与富含谷氨酰胺及支链氨基酸注射液 (2 0AA)对创伤大鼠的营养效用。以Wistar大鼠为创伤模型 ,分别输注两种配方的氨基酸注射液 ,以日立L - 85 0 0氨基酸自动分析仪测定动物血浆游离氨基酸 ,并测定创伤处海绵内羟脯氨酸含量。结果显示创伤后大鼠血浆牛磺酸、谷氨酸、谷氨酰胺和支链氨基酸含量较术前下降 ,但 2 0AA组血浆氨基酸恢复优于 17AA组 ,创伤处海绵内羟脯氨酸含量 2 0AA组显著高于 17AA组 (1.2 9± 0 .2 1vs 0 .83± 0 .16mg/块海绵 ,P <0 .0 5 )。提示 ,创伤后给予富含谷氨酰胺和支链氨基酸的营养制剂能提高血浆氨基酸浓度并有利于创伤的恢复     

Biosynthesis of the Branched-Chain Amino Acids in Yeast: a Leucine-Binding Component and Regulation of Leucine Uptake

Use of an ion-exchange resin assay has shown that leucine is bound to a component of a dialyzed extract of yeast. Leucine binding may be related to in vivo uptake of the amino acid. A yeast strain with a 30-fold lower affinity for leucine uptake in vivo has a parallel reduction in affinity for in vitro leucine binding; the rate of leucine uptake in wild-type yeast can be increased four- to fivefold by growth on leucine as a sole nitrogen source. Under these conditions, the specific activity of the leucine-binding component also increases over threefold. Regulation of leucine uptake was studied by using wild-type strain 60615 and a mutant 60615/fl(2) with a constitutively elevated leucine uptake system. Leucine pool formation in the mutant was accompanied by an overshoot, leading to a loss of leucine from the pool. The phenomenon could be observed in the wild type under certain conditions. The mechanism of this process was examined. The leucine uptake system was found to be stable in the absence of protein synthesis. The rate of leucine uptake increased on reduction of the pool of amino acids, and in strain 60615/fl(2) the ability to overshoot was rapidly recovered on depletion of the leucine pool. The results suggest a control of leucine uptake by feedback inhibition, in which leucine or other amino acids, e.g., isoleucine, inhibit leucine uptake. The results do not exclude control by a rapidly activated-inactivated system.     

Creatine Kinase Activity from Rat Brain Is Inhibited by Branched-Chain Amino Acids in Vitro

Maple syrup urine disease (MSUD) is an inherited metabolic disorder biochemically characterized by the accumulation of branched-chain amino acids (BCAAs) and their branched-chain keto acids (BCKAs) in blood and other tissues. Neurological dysfunction is usually present in the affected patients, but the mechanisms of brain damage in this disease are not fully understood. Considering that brain energy metabolism seems to be altered in MSUD, the main objective of this study was to investigate the in vitro effect of BCAAs and BCKAs on creatine kinase activity, a key enzyme of energy homeostasis, in brain cortex of young rats. BCAAs, but not their BCKAs, significantly inhibited creatine kinase activity at concentrations similar to those found in the plasma of MSUD patients (0.5–5 mM). Considering the crucial role creatine kinase plays in energy homeostasis in brain, if this effect also occurs in the brain of MSUD patients, it is possible that inhibition of this enzyme activity may contribute to the brain damage found in this disease.     

Biosynthesis of Branched-Chain Amino Acids in Schizosaccharomyces pombe: Properties of Acetohydroxy Acid Synthetase1

The regulatory properties of acetohydroxy acid synthetase (AHAS), the first enzyme in the biosynthetic pathway to valine and the second in the isoleucine pathway, were investigated in the fission yeast Schizosaccharomyces pombe. The enzyme was partially purified from crude extracts by protamine sulfate treatment, ammonium sulfate fractionation, and gel filtration through Sephadex G-25. AHAS from S. pombe is unique in that its activity shows a single peak around pH 6.5; high sensitivity to feedback inhibition by valine at this pH (K(i) = 0.1 mM) indicates that the enzyme is involved in valine biosynthesis. Pyruvate saturation kinetics of AHAS extracted from cells grown on glycerol as sole carbon and energy source were normal and hyperbolic. In contrast, the enzyme from glucose-grown cells exhibited sigmoidal saturation kinetics, an effect which disappeared when the synthetase from such cells was partially purified. This phenomenon was shown to be due to competition for pyruvate between AHAS and pyruvate decarboxylase; the latter enzyme is present in large amounts in cells fermenting glucose. Valine inhibition is noncompetitive in nature, and this effector exhibits homotropic cooperative effects; isoleucine is a less-potent inhibitor of AHAS activity. Mercurial treatment reversibly desensitized the enzyme to valine inhibition. On the basis of these data, the S. pombe AHAS appears to be an allosteric regulatory enzyme with the properties of a negative V system.