Biosynthesis of Branched-Chain Amino Acids in Yeast: Correlation of Biochemical Blocks and Genetic Lesions in Leucine Auxotrophs

The three enzymatic steps in the conversion of alpha-ketoisovalerate to alpha-ketoisocaproate were examined in wild-type and in leucine auxotrophic stocks of yeast. Procedures for the reliable assay of each of the enzymatic steps in crude extracts were devised. Crude extracts of the prototrophic haploid stock catalyzed all three enzymatic steps. Examination of a series of leucine auxotrophs permitted a correlation between the three enzymatic steps and the genetic lesions affecting 10 different loci. This examination revealed that a single locus (le-6) affected primarily alpha-isopropylmalate synthetase, the first step in the pathway. Lesions in six loci (le-1, le-4, le-5, le-7, le-8, and le-10) lead primarily to a deficiency in the activity of the second enzyme in the pathway, alpha-isopropylmalate isomerase. Stocks with lesions in three loci (le-2, le-3, and le-9) were primarily blocked in the third step of the pathway, catalyzed by beta-isopropylmalate dehydrogenase. The results with the mutants provide strong evidence that the pathway for leucine biosynthesis proposed by Strassman and his colleagues is the sole significant pathway in yeast.     

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

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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。     

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     

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。     

氨基酸与生化药物

回顾中国氨基酸类药物的发展历程,并对２０００年后的发展提出意见。     

Role of CodY in regulation of the Bacillus subtilis hut operon.

Bacillus subtilis mutants deficient in amino acid repression of the histidine utilization (hut) operon were isolated by transposon mutagenesis. Genetic characterization of these mutants indicated that they most likely contained transposon insertions within the codVWXY operon. The codY gene is required for nutritional regulation of the dipeptide permease (dpp) operon. An examination of hut expression in a delta codY mutant demonstrated that amino acid repression exerted at the hutOA operator, which lies immediately downstream of the hut promoter, was defective in a delta codY mutant. The codY gene product was not required for amino acid regulation of either hut induction or the expression of proline oxidase, the first enzyme in proline degradation. This indicates that more than one mechanism of amino acid repression is present in B. subtilis. An examination of dpp and hut expression in cells during exponential growth in various media revealed that the level of CodY-dependent regulation appeared to be related to the growth rate of the culture.     

Genetic and Biochemical Characterization of Mutants of Bacillus subtilis Defective in Succinate Dehydrogenase

Eleven succinate-accumulating mutants of Bacillus subtilis have been mapped by transformation and transduction crosses and characterized with respect to activities of citric acid cycle enzymes. These mutants could be divided into three genetic groups. Nine of the mutants were found to map between argA and leu in the citF locus. A second group was located between lys-1 and trpC2 and the third group could not be located on the B. subtilis chromosome in extensive transduction crosses. All of the citF mutants lack detectable succinate dehydrogenase activity, whereas both of the other groups show a reduced level of this enzyme. In addition, most of the mutants in the citF locus lack cytochrome a, whereas the level of this cytochrome is normal in the other two groups. A procedure has been devised for the solubilization of the succinate dehydrogenase from the membrane of B. subtilis with the non-ionic detergent Brij 58. Some properties of the soluble and bound forms of succinate dehydrogenase are described.     

Genetic and Biochemical Characterization of the MinC-FtsZ Interaction in Bacillus subtilis

Cell division in bacteria is regulated by proteins that interact with FtsZ and modulate its ability to polymerize into the Z ring structure. The best studied of these regulators is MinC, an inhibitor of FtsZ polymerization that plays a crucial role in the spatial control of Z ring formation. Recent work established that E. coli MinC interacts with two regions of FtsZ, the bottom face of the H10 helix and the extreme C-terminal peptide (CTP). Here we determined the binding site for MinC on Bacillus subtilis FtsZ. Selection of a library of FtsZ mutants for survival in the presence of Min overexpression resulted in the isolation of 13 Min-resistant mutants. Most of the substitutions that gave rise to Min resistance clustered around the H9 and H10 helices in the C-terminal domain of FtsZ. In addition, a mutation in the CTP of B. subtilis FtsZ also produced MinC resistance. Biochemical characterization of some of the mutant proteins showed that they exhibited normal polymerization properties but reduced interaction with MinC, as expected for binding site mutations. Thus, our study shows that the overall architecture of the MinC-FtsZ interaction is conserved in E. coli and B. subtilis. Nevertheless, there was a clear difference in the mutations that conferred Min resistance, with those in B. subtilis FtsZ pointing to the side of the molecule rather than to its polymerization interface. This observation suggests that the mechanism of Z ring inhibition by MinC differs in both species.