丝孢酵母脂肪酶的酶学性质和化学修饰

实验室筛选到的一株丝孢酵母Trichosporon sp.脂肪酶经过硫酸铵沉淀和一系列的层析步骤分离纯化到电泳纯。对纯酶的酶学性质研究表明,此酶的分子量为28kD,pI为pH8.7,最适作用温度40℃,最适作用pH为8.0。随后利用不同的化学修饰剂对酶蛋白进行修饰,通过酶催化活力的改变对位于酶活性位的氨基酸残基进行分析,结果表明酶活性位可能含有组氨酸、丝氨酸和谷氨酸(或天冬氨酸)。最后,对此酶的氨基酸成分进行了分析,结果表明,此酶分子中天冬氨酸、丝氨酸、谷氨酸、甘氨酸、丙氨酸含量高,两种碱性氨基酸精氨酸、赖氨酸及组氨酸的含量也比较高,而脯氨酸、色氨酸的含量相对较低。     

不同晶型、旋光型氨基酸原料药的红外图谱分析

分析比较了 33种不同来源的氨基酸产品红外图谱的差异 ,其中丝氨酸、门冬氨酸、醋酸赖氨酸、谷氨酸 (白色结晶性粉末 )、苏氨酸、缬氨酸、丙氨酸、亮氨酸、脯氨酸、盐酸组氨酸、盐酸精氨酸、酪氨酸、胱氨酸等 13种与标准图谱完全一致 ;甲硫氨酸、盐酸赖氨酸、甘氨酸、谷氨酸 (白色结晶 )等 4种与标准图谱不一致 ,其原因是 :甘氨酸和谷氨酸由晶型不同造成 ,甲硫氨酸因旋光性不同而造成 ,盐酸赖氨酸与相应的生化试剂图谱一致。     

癌肿与氨基酸代谢的研究

研究了癌肿与氨基酸代谢的关系。这些癌肿包括喉癌HepⅡ细胞 ,急性非淋巴细胞白血病和急性淋巴细胞白血病 ,结果表明 :( 1 )喉癌细胞株培养过程中亮氨酸、赖氨酸、丝氨酸、天冬酰胺、异亮氨酸、甘氨酸以及苏氨酸等水平明显降低 ,而色氨酸水平明显增加 ,说明喉癌细胞的生长繁殖必须依赖以上 7种氨基酸同时释放了色氨酸 ;( 2 )急性非淋巴细胞白血病 (ANLL)患者血浆中的谷氨酸、甘氨酸、亮氨酸、苯丙氨酸、酪氨酸和色氨酸等水平明显升高 ,而苏氨酸、组氨酸、丙氨酸等水平明显降低 ,这些结果与国际报道相一致 ;( 3)经治疗后 ,ANLL患者血浆中甘氨酸、色氨酸和苯丙氨酸等水平明显降低 ,而丙氨酸、组氨酸等水平明显升高 ,表明肿瘤细胞处在无氧代谢。患者经治疗后色氨酸和苯丙氨酸水平降低和组氨酸水平的升高对患者预后是有益的 ;( 4)急性淋巴细胞白血病患者血浆中苯丙氨酸、赖氨酸、色氨酸和酪氨酸水平提高 ,这些氨基酸能促进肿瘤生长 ,而门冬酰胺、谷氨酰胺以及天冬氨酸水平降低 ,说明这 3种氨基酸为肿瘤生长所必须。此外还发现ALL患者外周淋巴细胞中精氨酸水平增加 ,精氨酸对癌肿细胞有直接杀伤作用。     

枯萎病不同抗性黄瓜(Cucumis sativus L.)根系分泌物氨基酸组分与抗病的相关性

以5种对枯萎病不同抗性黄瓜品种为试材,对其根系分泌物氨基酸组分进行测定,并对氨基酸组分与黄瓜品种枯萎病抗病性之间的相关性进行了分析。结果表明：在中抗品种根系分泌中检测到的16种氨基酸：半胱氨酸Cys、苏氨酸Thr、丙氨酸Ala、缬氨酸Val、异亮氨酸Lle、天冬氨酸Asp、亮氨酸Leu、苯丙氨酸Phe、甘氨酸Gly、甲硫氨酸Met、组氨酸His、谷氨酸Glu、酪氨酸Tyr、赖氨酸Lys、丝氨酸Ser和精氨酸Arg。其中的精氨酸在感病品种中没有被检出,组氨酸和精氨酸组分在抗病品种中没有被检出。根系分泌物中总氨基酸含量随品种抗性的增加而降低;精氨酸、丝氨酸和赖氨酸的含量与品种对枯萎病的病情指数呈负相关,其他13种氨基酸组分含量与品种对枯萎病的病情指数呈正相关,其中苯丙氨酸含量与病情指数呈显著正相关。丝氨酸与苯丙氨酸、天冬氨酸、丙氨酸、甘氨酸的比值均与品种对枯萎病的病情指数呈显著负相关,其中Ser／Phe与品种对枯萎病的病情指数呈极显著负相关。     

光滑球拟酵母中α-酮戊二酸脱氢酶系生理作用解析

[目的]研究α-酮戊二酸脱氢酶系在光滑球拟酵母碳代谢流、能量代谢和氨基酸代谢中的生理作用.[方法]通过敲除光滑球拟酵母中编码α-酮戊二酸脱氢酶系中E1酶的基因kgd1,构建α-酮戊二酸脱氢酶活性缺失菌株T.glabrata kgd1::kan,并考察KGDH缺失引起TCA循环关键酶活性,碳代谢流量以及胞内氨基酸和能荷水平等方面的变化.[结果]光滑球拟酵母中α-酮戊二酸脱氢酶活性的缺失导致:(1)细胞启动乙醛酸途径,通过形成TCA-乙醛酸循环实现TCA循环的正常代谢;(2)胞内NADH/NAD+水平下降33.7%,ATP/ADP水平下降31.8%,而与NADH代谢相关的丙酮酸脱氢酶、异柠檬酸脱氢酶和苹果酸脱氢酶的活性分别提高58.1%、33.3%和32.5%;(3)胞内丙酮酸含量下降50.1%,而胞内琥珀酸、苹果酸和α-酮戊二酸含量则分别增加了172.7%、66.1%和41.1%;(4)丙酮酸族氨基酸含量下降29.3%,而胞内谷氨酸族氨基酸和天冬氨酸族氨基酸含量则提高了34.7%和26.8%.[结论]上述研究结果表明,α-酮戊二酸脱氢酶系在微生物细胞中心碳代谢、能量代谢和氨基酸代谢中发挥着重要作用.     

蚯蚓活动对土壤氨基酸组分及含量的影响

通过室内培养试验,研究了赤子爱胜蚓(Eisenia foetida)和威廉环毛蚓(Metaphire guillelmi)对土壤氨基酸组分及含量的影响,并探讨了两种不同生活型蚯蚓作用效果的异同。结果表明:蚯蚓活动可显著改变土壤氨基酸含量,爱胜蚓作用下土壤酸解氨基酸和游离氨基酸分别增加5.08 g/kg和7.72 mg/kg,环毛蚓作用下土壤酸解氨基酸和游离氨基酸分别增加3.86 g/kg和4.44mg/kg。各处理酸解氨基酸均以中性氨基酸所占比例为最大(平均51.9%),酸性氨基酸次之(平均23.3%),而含硫氨基酸(平均14.4%)及碱性氨基酸最少(平均10.4%)。各处理游离氨基酸同样以中性氨基酸为主,平均54.4%,而以碱性氨基酸含量最少,平均仅为7.2%。蚯蚓活动并未改变土壤氨基酸可检出种类,各处理分别检测出16种酸解氨基酸和14种游离氨基酸。土壤酸解氨基酸和游离氨基酸组分含量在蚯蚓作用下均有明显改变:加入爱胜蚓后土壤酸解氨基酸组分中天冬氨酸、精氨酸、甲硫氨酸、丙氨酸、赖氨酸和甘氨酸增幅较高,均在85.7%以上,缬氨酸、苏氨酸、丝氨酸、谷氨酸、亮氨酸、酪氨酸和组氨酸增幅较小在40.7%—62.7%间波动;加入环毛蚓后土壤酸解氨基酸组分中甲硫氨酸、赖氨酸、天冬氨酸、酪氨酸和丙氨酸增幅较大,均在71.9%以上,甘氨酸、精氨酸、异亮氨酸增幅适中,分别为56.8%、55.6%和54.9%;丝氨酸、亮氨酸、苏氨酸、谷氨酸、组氨酸和苯丙氨酸增幅最小,均在40%以下;游离氨基酸组分中组氨酸、精氨酸、甘氨酸、亮氨酸、异亮氨酸和丙氨酸在加入爱胜蚓后增加的幅度较大,增幅在150.0%以上,增幅较为缓和的氨基酸组分有天冬氨酸、苏氨酸、丝氨酸、缬氨酸、谷氨酸和苯丙氨酸,介于58.8%—92.1%之间;环毛蚓作用下,天冬氨酸、精氨酸、丝氨酸和异亮氨酸增幅最大,分别为184.2%、173.3%、163.0%和116.6%;苏氨酸、亮氨酸、缬氨酸和甘氨酸增幅较缓,介于52.3%—92.7%之间;谷氨酸、组氨酸、苯丙氨酸、丙氨酸和甲硫氨酸增幅较低,均在33.1%之下;而半胱氨酸在蚯蚓作用下显著降低,降幅为11.8%。对比两种生活型蚯蚓作用效果可知,土壤氨基酸总含量及各组分含量在爱胜蚓和环毛蚓作用下的增加或减少趋势相同(土壤酸解氨基酸组分缬氨酸除外),但改变幅度却存在明显差异,总体而言,爱胜蚓作用效果优于环毛蚓。     

11种非必需氨基酸对离体植物生长的胁迫作用

研究了11种非必需氨基酸对小麦、水稻等植物离体胚植株以及拟南芥、白菜等植物种子植株生长的影响,结果显示,L-半胱氨酸、L-酪氨酸和L-丝氨酸在浓度达到3mmol·L“时开始抑制植株生长,而L-谷氨酸、L-谷氨酰胺、L-甘氨酸和L-天冬氨酸4种氨基酸在5—7mmol·L^-1时才有轻微抑制作用;L-胱氨酸、L-天冬酰胺和L-丙氨酸3种氨基酸,在5．7mmol·L^-1内没有抑制作用。脯氨酸的3种异构体在9mmol·L^-1浓度下没有抑制作用发生,且还表现出有约1个生长级别（3．4cm左右）的生长促进作用。丝氨酸和丙氨酸的D-异构体在3mmol·L^-1浓度即有强烈的生长抑制作用。这些结果表明非必需氨基酸胁迫植物的途径有多态性,这些多态性对研究植物氨基酸代谢有重要意义。     

抗坏血酸—Fe^3＋对牛红细胞铜锌超氧化物歧化酶氧化修…

用抗坏血酸－Ｆｅ＾３＋作用于牛红细胞铜锌超氧化物歧化酶,发现酶分子中有羰基和二酪氨酸生成,天冬氨酸,谷氨酸和甘氨酸含量相对增加,而丝氨酸,赖氨酸,组氨酸,精氨酸和脯氨酸的含量相对减少,酶的部分亚基被降解成肽段,且肽链断裂与肽键水解无关,等电点下降。进一步研究发现金属辅基铜和锌丢失,酶的紫外吸收表现为增色效应,内源性荧光减弱,酶分子中β－折叠含量有相对减少无规则卷曲有相对增加趋势。以上结果提示抗坏血     

白地霉的氧化代谢

1．自地霍完整静息幼胞氧化乙醇、乙酸、丙酮酸及草酰乙酸的速度很高,对其他三羧酸循环各酸如：琥珀酸、延胡索酸、a-酮戊二酸、苹果酸、柠檬酸、顺岛头酸及异柠檬酸也能氧化,但速度甚低。 2．在白地霉无细胞提取液中测出了三羧酸循环中以下各种酶活力：柠檬酸精合酶、异柠檬酸脱氢酶、顺岛头酸酶、a-酮戊二酸脱氢酶、琥珀酸脱氢酶、延胡索酸酶、苹果酸脱氢酶、苹果酸酶、草酰乙酸羧化酶等,并间接得知有乙酸激活酶及L-谷氨酸脱氢酶存在。 3．在白地霉无捆胞提取液中测出了乙醛酸循环的两个关键的酶皂口异柠檬酸酶及苹果酸合成酶。 4．由以上结果可知白地霉可利用三羧酸循环及乙醛酸循环作为末端呼吸途径。     

Bromine derivatives of amino acids as intermediates in the peroxidase-catalyzed formation of singlet oxygen

Recently, J. R. Kanofsky et al. (1988, J. Biol. Chem. 263, 9692-9696) reported that human eosinophils generated modest amounts of singlet oxygen. In the mechanism proposed, hypobromous acid (made from the peroxidase-catalyzed oxidation of bromide ion) reacted with hydrogen peroxide to form singlet oxygen. In contrast, human neutrophils, which generate both hypochlorous acid and hydrogen peroxide, do not make singlet oxygen. The failure of human neutrophils to generate singlet oxygen is due in part to the trapping of hypochlorous acid by endogenous amines. In this paper, I show that amino acids are much more effective traps for hypochlorous acid than for hypobromous acid. Glycine totally inhibits singlet oxygen generation from a model enzyme system composed of chloroperoxidase, hydrogen peroxide, and chloride ion, but causes only a 35% reduction in singlet oxygen generation from an analogous enzyme system containing bromide ion instead of chloride ion. The products of the reaction of hypobromous and glycine (presumably an equilibrium mixture of N-bromoglycine, N,N-dibromoglycine, and hypobromous acid) retain the ability to react with hydrogen peroxide to form singlet oxygen. In contrast, the products of the reaction of hypochlorous acid and glycine do not react with hydrogen peroxide to produce singlet oxygen. Similar results were obtained for L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cystine, L-glutamic acid, L-glutamine, L-histidine, L-lysine, L-phenylalanine, L-proline, L-serine, and L-tyrosine. Thus, bromine derivatives of amino acids may act as intermediates in the peroxidase-catalyzed generation of singlet oxygen.     

Influence of amino acids on the growth of Bacteroides melaninogenicus.

Addition of individual amino acids to a Trypticase-yeast extract-hemin medium affected growth rates and final yields of an asaccharolytic strain and a saccharolytic strain of Bacteroides melaninogenicus. L-Aspartate or L-asparagine produced maximal growth enhancement for both strains. L-[14C]aspartate was fermented by resting cells of the asaccharolytic strain. L-Cysteine or L-serine also enhanced growth for the saccharolytic strain. However, growth of the saccharolytic strain was inhibited by L-lysine, L-glutamate, L-glutamine, L-isoleucine, L-leucine, and L-proline; growth of the asaccharolytic strain was inhibited by DL-valine and L-serine. Both strains were inhibited by L-histidine, DL-methionine, L-tryptophan, L-arginine, and glycine.     

Effects of amino acids on the isolated chicken retina, and on its response to glutamate stimulation

Abstract— The effect of a number of amino acids on the transparency and on the release of of [¹⁴C]glutamate from isolated chicken retinae charged with this compound was investigated. Also the effect of various amino acids on the response of the retina to stimulation with unlabelled glutamate, which causes an increase in transparency and a release of the label, was examined. In parallel experiments the effect of these same amino acids on the transparency and spreading depression (SD) was investigated in preparations consisting of the posterior part of the eye. A number of amino acids such as L-leucine, L-phenylalanine, L-tryptophan, L-lysine, L-histidine, L-arginine and others had little or no effect on these preparations. DL-valine and DL-homoserine caused an increase in transparency but no release of the label and did not affect the response to glutamate. Another group of amino acids comprising DL-a-alanine, L-serine, L-threonine, L-proline and glycine also caused an increase in the transparency of the retina without a release of labelled glutamate, but prevented the increase in transparency resulting from glutamate stimulation without affecting the release of the label. A final group of amino acids which included L-glutamic acid diethyl ester, DL-a-methyl glutamate, L-glutamine, L-asparagine, DL-homocysteate and L-cysteine caused a change in transparency of the retina accompanied by a release of the label; they prevented the change in transparency as well as the release of the label during stimulation by glutamate. Some amino acids, L-serine, L-threonine, DL-a-methyl glutamate, L-asparagine, DL-homocysteate and L-cysteine, caused wrinkling and folding of the retinae which furthermore became opaque. Of the amino acids investigated, proline gave promise of being a practical antagonist to the action of glutamate on the retina.     

Determination of bacterial decarboxylases of some amino acids by means of high voltage paper electrophoresis

The author describes a method, for the determination of bacterial L-histidine, L-tyrosine, L-tryptophan, L-arginine, L-lysine, L-ornithine, L-serine, L-phenyl-alanine, L-glutamic acid and L-aspartic acid decarboxylase by means of high voltage paper electrophoresis, using electrolytes of low ionic strength. For the group of test microorganisms (Enterobacteriaceae, Achromobacteriaceae andPseudomonas), L-histidine, L-arginine, L-lysine, L-ornithine and L-glutamic acid decarboxylases were found to be of the greatest practical significance. The optimal conditions for maximum formation of these decarboxylases by some of the test strains ofEscherichia coli, Hafnia andProteus are discussed.     

Synthesis, spectroscopic, mutagenic, and cytotoxicity studies of some mixed-ligand platinum(II) complexes of 2,2'-bipyridine and amino acids

Seven platinum(II) complexes of the type [Pt(bipy)(AA)]n+ (where n = 1 or 0 and AA is anion of L-valine, L-isoleucine, L-aspartic acid (dianion), L-glutamic acid (dianion), L-glutamine, L-proline, or S-methyl-L-cysteine) have been prepared and characterized. The modes of binding of amino acids in these complexes have been ascertained particularly by infrared and 1H NMR spectral studies. The L-glutamine complex shows a ID50 value (50% inhibitory dose) in the range of greater than 20 micrograms/ml to 100 micrograms/ml of the complex. However, the complexes of L-valine, L-isoleucine, L-aspartic acid, L-glutamic acid, L-proline, and S-methyl-L-cysteine show ID50 values greater than 100 micrograms/ml of the complex. The above complexes also show inferior growth inhibition of P-388 cells than platinum(II) complexes of 2,2'-bipyridine with L-alanine, L-leucine, L-methionine, and L-aspargine as reported earlier. The platinum(II) complexes of 2,2'-bipyridine with glycine (Gly), L-alanine (Ala), L-leucine (leu), L-valine (Val), L-methionine (Met), L-phenylalanine (Phe), L-serine (Ser), L-tyrosine (Tyr) and L-tryptophan (Trp) have been tested for mutagenesis using TA 100 and TA 98 strains. They show nonmutagenicity. This is in contrast to the cis-[Pt(NH3)2Cl2] showing a base pair substitution mutagenesis.     

Consequences of aspartase deficiency in Yersinia pestis.

Growing cells of Yersinia pseudotuberculosis, but not those of closely related Yersinia pestis, rapidly destroyed exogenous L-aspartic and L-glutamic acids, thus prompting a comparative study of dicarboxylic amino acid catabolism. Rates of amino acid metabolism by resting cells of both species were determined at pH 5.5, 7.0, and 8.5. Regardless of pH, Y. pseudotuberculosis destroyed L-glutamic acid, L-glutamine, L-aspartic acid, and L-asparagine at rates greater than those observed for Y. pestis. Although rates of proline degardation were similar, its metabolism by Y. pestis at pH 8.5 resulted in excretion of glutamic and aspartic acids. Similarly, Y. pestis excreted aspartic acid when incubated with L-glutamic acid (pH 8.5) or L-asparagine (pH 5.5, 7.0, and 8.5). Aspartase activity was not detected in extracts of 10 strains of Y. pestis but was present in all 11 isolates of Y. pseudotuberculosis. The latter contained significantly more glutaminase, asparaginase, and L-glutamate-oxalacetate transminase activity than did extracts of Y. pestis; specific activities of L-glutamate dehydrogenase and alpha-ketoglutarate dehydrogenase were similar. The observed differences in dicarboxylic amino acid metabolism are traceable to asparatase deficiency in Y. pestis and may account for the slow doubling time of this organism relative to Y. pseudotuberculosis.     

Selectivity properties of a Na-dependent amino acid cotransport system in adult alveolar epithelial cells

We investigated the amino acid specificity of a Na-dependent amino acid cotransport system that contributes to transepithelial Na absorption in the apical membrane of cultured adult rat alveolar epithelial cell monolayers. Short-circuit current was increased by basic, uncharged polar, and nonpolar amino acids but not by L-aspartic acid or L-proline. EC(50) values for L-lysine and L-histidine were 0.16 and 0.058 mM, respectively. The L-lysine-stimulated short-circuit current was Na dependent, with a concentration causing a half-maximal stimulation by Na of 44.24 mM. L-Serine, L-glutamine, and L-cysteine had EC(50) values of 0.095, 0.25, and 0.12 mM, respectively. L-Alanine had the highest affinity, with an EC(50) of 0.027 mM. We conclude that monolayer cultures of adult rat alveolar epithelial cells possess a broad-specificity Na-dependent amino acid cotransport system with properties consistent with system B(0,+). We suggest that this cotransport system plays a critical role in recycling of constituent amino acids that make up glutathione, thus ensuring efficient replenishment of this important antioxidant within the alveolar fluid.     

Amperometric biosensor based on diamond paste for the enantioanalysis of L-lysine

An amperometric biosensor was proposed for the enantioanalysis of L-lysine. The biosensor is based on the impregnation of L-lysine oxidase in diamond paste. The potential used for the determination of l-lysine was 650 mV. The biosensor exhibited a linear concentration range between 1 and 100 nmol/L with a limit of detection of 4 pmol/L. The selectivity of the biosensor is high over other amino acids, such as L-serine, L-leucine, L-aspartic acid, L-glutamic acid, histamine, glycine. The proposed biosensor can be applied for the determination of L-lysine in serum samples and pharmaceutical compounds.     

Effects of various kinds of dietary amino acids on the hepatotoxic action of D-galactosamine in rats

The protective effects of various kinds of dietary amino acids against the hepatotoxic action of D-galactosamine (GalN) were examined. Male Wistar rats fed with 20% casein diets containing 10% or 5% amino acid for one week were injected with GalN (800 mg/kg body weight), and the serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactate dehydrogenase (LDH) activities, the hepatic glycogen concentration, and the serum glucose-level were examined 20 hours after the injection. In the groups with the 10% amino acid diets, activities of AST, ALT, and LDH in serum of 10% L-glutamine (Gln), 10% L-asparagine (Asn), and 10% L-serine (Ser) groups were significantly lower than those of the control group, and in the groups with the 5% amino acid diets, those activities of 5% L-histidine (His), 5% L-tyrosine (Tyr), 5% L-lysine (Lys), and 5% L-glycine (Gly) groups were also lower than those of the control group. The concentration of liver glycogen of 10% Gln-, 10% Asn-, and 10% Ser- groups and those levels of 5% His-, 5% Tyr-, 5% Lys-, and 5% Gly-groups were also significantly higher than that of the control group. As a result, it was found that some kinds of dietary amino acid such as L-Ser, L-Asn, L-His, L-Lys, L-Tyr, and L-Gly, in addition to L-Gln were effective to protect the rats from GalN-induced injury.     

Evidence for functional asymmetry in the olfactory system of the Senegalese sole (Solea senegalensis)

The two olfactory epithelia of flatfish of the family Soleidae are essentially in contact with two distinct environments; the upper (right) side samples open water while the lower (left) side samples interstitial water. This study assessed whether there are differences in the responsiveness of the two epithelia by use of the electro-olfactogram in the Senegalese sole (Solea senegalensis). The upper epithelium was significantly more responsive to the basic amino acids (L-lysine and L-arginine), glycine, and L-threonine than the lower epithelium. The lower epithelium was significantly more responsive to aromatic amino acids (L-tryptophan, L-tyrosine, L-DOPA, and L-phenylalanine), L-leucine, and L-asparagine than the upper. Both epithelia had similar responsiveness to the sulphur-containing amino acids (L-cysteine and L-methionine), L-alanine, L-serine, and L-glutamine. Neither side was responsive to the acidic amino acids (L-aspartate and L-glutamate) or the D-isomers of any amino acid tested. The upper olfactory organ was much more responsive to conspecific-derived stimuli (bile and intestinal fluid) than the lower organ. We suggest that these differences in responsiveness may be related to different functional roles of the upper and lower epithelia in feeding and chemical communication.