肽链及蛋白质N-末端喹喔啉类荧光衍生物的形成

蛋白质及肽链N-末端经Dixon转氨后的羰酰基与邻苯二胺作用可形成喹喔啉类荧光衍生物.形成这一发色团的过程较为缓慢;喹喔啉短肽的激发(Ex 293-305nm)与发射(Em 360-365nm)波长随肽链氨基酸残基组成不同而有一定变化;喹喔啉胰岛素荧光衍生物的激发光波长为Ex318nm,发射波长为Em 353nm.喹喔啉三肽的荧光在乙二醇溶液中的变化因其氨基酸残基组成的不同而有差异.以上结果提示:喹喔啉类荧光衍生物可能作为研究蛋白和肽结构与功能的一种手段.     

不同一级结构寡肽的α-羰酰及喹喔啉衍生物的荧光性质

通过对寡肽N-末端α-羰酰及喹喔啉衍生物和荧光在碘化钾、乙二醇、盐酸胍和氯化钠溶液中的变化测定结果表明:不同一级结构寡肽的羰酰荧光物的碘化钾淬灭过程彼此有差异.在不同浓度的乙二醇和盐酸胍溶液中,第三位氨基酸残基的种类和构型不同的寡肽羰酰衍生物的荧光变化亦有不同.乙二醇引起羰酰甘氨寡肽荧先发射峰位红移.丙氨寡肽的发射峰位蓝移;并且不同链长的甘氨短肽及丙氨短肽羰酰衍生物在不同浓度的乙二醇或盐酸胍溶液中各自的荧光变化有差异,但这种差异随肽链的延长逐渐减小.以上结果提示:尽管(含有2-6个氨基酸残基的)寡肽在溶液中难以形成二级结构,但它们的空间构象可能不是随机的;寡肽链越长,结构相对稳定.     

不同一级结构寡肽的α-羰酰及喹喔啉衍生物的荧光性质

通过对寡肽N-末端α-羰酰及喹喔啉衍生物和荧光在碘化钾、乙二醇、盐酸胍和氯化钠溶液中的变化测定结果表明:不同一级结构寡肽的羰酰荧光物的碘化钾淬灭过程彼此有差异.在不同浓度的乙二醇和盐酸胍溶液中,第三位氨基酸残基的种类和构型不同的寡肽羰酰衍生物的荧光变化亦有不同.乙二醇引起羰酰甘氨寡肽荧先发射峰位红移.丙氨寡肽的发射峰位蓝移;并且不同链长的甘氨短肽及丙氨短肽羰酰衍生物在不同浓度的乙二醇或盐酸胍溶液中各自的荧光变化有差异,但这种差异随肽链的延长逐渐减小.以上结果提示:尽管(含有2-6个氨基酸残基的)寡肽在溶液中难以形成二级结构,但它们的空间构象可能不是随机的;寡肽链越长,结构相对稳定.     

肽链及蛋白质N-末端羰酰荧光衍生物的形成

短肽及胰岛素的N-末端经Dixpn转氨后具有荧光发色性质,其激发与发射波长随肽链氨基酸残基的种类和数量的不同而有差异,其范围大约为Ex 312—333nm;Em 398—408nm;量子产率为0.020—0.03.荧光发色团的基本化学结构可能是N-末端α-羰酰基及其相连的酰胺基,并且N-末端第二及第三位氨基酸残基对其荧光性质有影响.在碱性溶液中(pH>9.0)它的荧光强度降低,但在NaCl溶液中随盐浓度的增加而增强.在不同浓度的CuHCl溶液中,羰酰三肽的荧光强度变化随其氨基酸残基的种类和构型的不同而有差异.以上结果提示;α-羰酰荧光衍生物可能作为蛋白及肽N-末端的荧光探剂,可能成为研究蛋白和肽结构与功能的一种手段.     

非酶糖基化对α-synuclein分子构象的影响

将纯化后的α－synuclein分别与果糖和葡萄糖孵育,通过内源荧光、非酶糖基化衍生物特征荧光、圆二色光谱以及电子显微镜等技术进行检测发现：α—synuclein与还原糖共同孵育后,308nm内源荧光强度明显降低,同时在447nm产生一个非酶糖基化衍生物特征荧光．与果糖孵育的蛋白质样品其非酶糖基化特征荧光的出现速度快于葡萄糖孵育样品．内源荧光与非酶糖基化特征荧光之间存在能量传递现象,提示Tyr残基与非酶糖基化特征荧光发色团在空间距离上彼此接近．圆二色光谱测定结果显示,α－synuclein与果糖孵育后,其α－螺旋含量增加．非酶糖基化的α－synuclein在电子显微镜下表现为短纤维状．非酶糖基化可以诱导α－synuclein蛋白分子聚集,且果糖较葡萄糖更容易使α－synuclein发生非酶糖基化．以上结果提示,非酶糖基化似乎可以导致α－synuclein在细胞内的错误折叠和分子聚集．     

高灵敏显色降解试剂4-N，N-二甲氨基偶氮苯-4′-异硫氰酸酯(DABITC)在蛋白质顺序分析中的应用

一、序言 Edman降解是蛋白质顺序分析最有效的方法。它由三步反应组成,首先,异硫氰酸苯酯(PITC)在碱性pH下与肽或蛋白质的N-端氨基酸反应生成苯基氨荒酰(PTC)衍生物,然后在酸性pH下生成比原先的肽少一个氨基酸的肽与苯基噻唑啉酮(PTZ)衍生物,后者在酸性     

黄原胶寡糖的抗氧化性能和非酶糖基化抑制作用研究

分别在酸性和碱性条件下通过氧化降解制备了两种黄原胶寡糖XG-H和XG-OH.红外光谱法对黄原胶寡糖的结构进行表征,凝胶渗透色谱法测定黄原胶寡糖的分子量,紫外可见分光光度法测定黄原胶寡糖的丙酮酸和还原糖含量,考察了两种黄原胶寡糖的抗氧化性能和非酶糖基化(NEG)的抑制作用.结果表明,XG-H和XG-OH都表现出一定的抗氧化能力且XG-OH强于XG-H; XG-OH促进5-羟甲基糠醛(非酶糖基化中间产物)的生成,但可显著抑制非酶糖基化荧光末端产物的生成.而XG-H表现出非酶糖基化促进作用.这可能与两种黄原胶寡糖的丙酮酸和还原糖含量有关.     

氧化棉子糖分子内交联猪血红蛋白对其结构功能的影响

用高碘酸盐氧化法制得氧化开环棉子糖,并用它分别修饰处在氧合及脱氧构象的猪血红蛋白得到两种不同的修饰衍生物。与天然蛋白质相比,两种修饰后的猪血红蛋白衍生物均具有明显的抗α２β２四聚体解聚的特性。经ＳＤＳ－ＰＡＧＥ和反相ＨＰＬＣ分析发现这两种修饰衍生物均为分子内交联产物,而且交联都发生于２个β亚基之间,两种交联产物的紫外－可见光谱没有明显差别,但在相同浓度下,脱氧构象下被修饰蛋白质的荧光发射光强度明显     

血卟啉及其衍生物在水溶液和细胞内的时间分辨荧光的研究

本文报道了血叶啉衍生物(Hpd)及其由高压液相色谱法分离制备的4种主要成分以及血叶啉,血叶啉二盐酸盐和原叶啉(Ⅸ)二钠盐等8种叶啉衍生物分别在水溶液和CHO细胞内的荧光衰变动力学的研究,实验表明,除Hpd分离组分d的荧光寿命略短外,其余7种在水落液里的荧光寺命均在15ns左右,并发现它们的荧光态在细胞内衰变加快,然而,分离组分d在细胞内的荧光寿命却明显加长.根据实验结果,本文对叶啉衍生物的最低激发单线态在细胞内的驰豫过程提出了一种可能的模式,它反映了叶啉衍生物在细胞内与某种细胞组分的结合以及它们之间的能量传递.分析表明,荧光寿命的缩短意味着光敏效率的降低.     

芝田硫化叶菌麦芽寡糖基海藻糖合酶基因在大肠杆菌中的克隆和表达

用ＰＣＲ 方法从芝田硫化叶菌中扩增了编码一种新酶,即麦芽寡糖基海藻糖合酶（ ＭＴＳａｓｅ） 的基因,扩增的２－２ｋｂ ＤＮＡ 插入到原核表达载体ｐＢＶ２２０ 中,构建成重组质粒ｐＳＢＧＴ１ 。ｐＳＢＧＴ１ 中ＭＴＳａｓｅ 基因在大肠杆菌中得到表达。ＳＤＳＰＡＧＥ 分析表达产物ＭＴＳａｓｅ蛋白的分子量约为７４ｋＤａ ,同核苷酸序列测定所推导的值相符。表达产物占细胞总蛋白约４－４ ％ 。ｐＳＢＧＴ１ 产生的重组酶作用于淀粉部分水解物,使ＤＥ 值降低,得到非还原糖或低还原糖。     

The effect of starvation on branched-chain 2-oxo acid oxidation in rat muscle.

Oxidative-decarboxylation rates of branched-chain amino acids in rat hemidiaphragm and of branched-chain 2-oxo acids in hemidiaphragm, soleus muscle and heart slices of 110-120 g rats were increased considerably by 3-4 days of starvation, when they were calculated from the specific radioactivity in the medium. When the supply from endogenous protein degradation to the oxidation-precursor pool was severely limited by transaminase inhibitors, oxidative-decarboxylation rates of branched-chain 2-oxo acids rose significantly. Since this apparent increase was relatively larger in preparations from fed rats than from 3-days-starved rats, the differences in oxidation rates with nutritional state became less or even not significant. With rat heart the smaller dilution of the oxidation precursor pool after starvation is in accordance with the reported decrease in protein breakdown. Since protein degradation increases with starvation in skeletal muscles, we suggest that the amino acid pool arising from protein degradation is more segregated from the oxidation precursor pool in muscles from starved than from fed rats. We conclude that starvation increases branched-chain amino acid and 2-oxo acid oxidation in skeletal and cardiac muscle considerably less than has been suggested by previous studies.     

Aspartate: 2-oxoglutarate aminotransferase from trichomonas vaginalis. Identity of aspartate aminotransferase and aromatic amino acid aminotransferase.

Aspartate: 2-oxoglutarate aminotransferase from the anaerobic protozoon Trichomonas vaginalis was purified to homogeneity and characterized. It is a dimeric protein of overall Mr approx. 100000. Only a single isoenzyme was found in T. vaginalis. The overall molecular and catalytic properties have features in common with both the vertebrate cytoplasmic and mitochondrial isoenzymes. The purified aspartate aminotransferase from T. vaginalis showed very high rates of activity with aromatic amino acids as donors and 2-oxoglutarate as acceptor. This broad-spectrum activity was restricted to aromatic amino acids and aromatic 2-oxo acids, and no significant activity was seen with other common amino acids, other than with the substrates and products of the aspartate: 2-oxoglutarate aminotransferase reaction. Co-purification and co-inhibition, by the irreversible inhibitor gostatin, of the aromatic amino acid aminotransferase and aspartate aminotransferase activities, in conjunction with competitive substrate experiments, strongly suggest that a single enzyme is responsible for both activities. Such high rates of aromatic amino acid aminotransferase activity have not been reported before in eukaryotic aspartate aminotransferase.     

Inhibition by the branched-chain 2-oxo acids of the 2-oxoglutarate dehydrogenase complex in developing rat and human brain

1. The effect of the branched-chain amino acids, namely leucine, isoleucine and valine and their corresponding 2-oxo acids on the metabolism of 2-oxoglutarate by developing rat and human brain preparations was investigated. 2. The decarboxylation of 2-oxo[1-(14)C]glutarate to (14)CO(2) by mitochondria from adult rat brain was inhibited by the branched-chain 2-oxo acids whereas the branched-chain amino acids had no inhibitory effect on this process. 3. The activity of 2-oxoglutarate dehydrogenase complex was about 0.2unit/g of brain from 2-day-old rats and increased by about fourfold reaching an adult value by the end of the third postnatal week. 4. The K(m) value for 2-oxoglutarate of the 2-oxoglutarate dehydrogenase complex in rat and human brain was 100 and 83mum respectively. 5. The branched-chain 2-oxo acids competitively inhibited this enzyme from suckling and adult rats brains as well as from foetal and adult human brains, whereas the branched-chain amino acids had no effect on this enzyme. 6. Approximate K(i) values for the branched-chain 2-oxo acids found for this enzyme were in the range found for these 2-oxo acids in plasma from patients with maple-syrup-urine disease. 7. The possible significance of the inhibition by the branched-chain 2-oxo acids of the 2-oxoglutarate dehydrogenase complex in brains of untreated patients with maple-syrup-urine disease is discussed in relation to the energy metabolism and the biosynthesis of lipids from ketone bodies.     

Metabolism of valine and 3-methyl-2-oxobutanoate by the isolated perfused rat kidney.

Metabolism of branched-chain amino and 2-oxo acids was studied in the isolated perfused kidney. Significant amounts of 2-oxo acids were released by perfused kidney with all concentrations of amino acids tested (0.1-1.0 mM each), despite the high activity of branched-chain 2-oxo acid dehydrogenase in kidney. As perfusate valine concentration was increased from 0.2 to 1.0 mM, [1-14C]valine transamination (2-oxo acid oxidized + released) increased roughly linearly; [1-14C]valine oxidation, however, increased exponentially. Increasing perfusate concentration of 3-methyl-2-oxo[1-14C]butanoate from 0 to 1.0 mM resulted in a linear increase in the rate of its oxidation and a rise in perfusate valine concentration; at the same time significant decreases occurred in perfusate isoleucine and leucine concentrations, with corresponding increases in rates of release of their respective 2-oxo acids. Comparison of rates of oxidation of [1-14C]valine and 3-methyl-2-oxo[1-14C]butanoate suggests that 2-oxo acid arising from [1-14C]valine transamination has freer access to the 2-oxo acid dehydrogenase than has the 2-oxo acid from the perfusate. The observations indicate that, when branched-chain amino and 2-oxo acids are present in perfusate at near-physiological concentrations, rates of transamination of the amino and 2-oxo acids by isolated perfused kidney are greater than rates of oxidation.     

Structure-function relationships in the 2-oxo acid dehydrogenase family: substrate-specific signatures and functional predictions for the 2-oxoglutarate dehydrogenase-like proteins

Structural relationship within the family of the thiamine diphosphate-dependent 2-oxo acid dehydrogenases was analyzed by combining different methods of sequence alignment with crystallographic and enzymological studies of the family members. For the first time, the sequence similarity of the homodimeric 2-oxoglutarate dehydrogenase to heterotetrameric 2-oxo acid dehydrogenases is established. The presented alignment of the catalytic domains of the dehydrogenases of pyruvate, branched-chain 2-oxo acids and 2-oxoglutarate unravels the sequence markers of the substrate specificity and the essential residues of the family members without the 3D structures resolved. Predicted dual substrate specificity of some of the 2-oxo acid dehydrogenases was confirmed experimentally. The results were used to decipher functions of the two hypothetical proteins of animal genomes, OGDHL and DHTKD1, similar to the 2-oxoglutarate dehydrogenase. Conservation of all the essential residues confirmed their catalytic competence. Sequence analysis indicated that OGDHL represents a previously unknown isoform of the 2-oxoglutarate dehydrogenase, whereas DHTKD1 differs from the homologs at the N-terminus and substrate binding pocket. The differences suggest changes in heterologous protein interactions and accommodation of more polar and/or bulkier structural analogs of 2-oxoglutarate, such as 2-oxoadipate, 2-oxo-4-hydroxyglutarate, or products of the carboligase reaction between a 2-oxodicarboxylate and glyoxylate or acetaldehyde. The signatures of the Ca2+-binding sites were found in the Ca2+-activated 2-oxoglutarate dehydrogenase and OGDHL, but not in DHTKD1. Mitochondrial localization was predicted for OGDHL and DHTKD1, with DHTKD1 probably localized also to nuclei. Medical implications of the obtained results are discussed in view of the possible associations of the 2-oxo acid dehydrogenases and DHTKD1 with neurodegeneration and cancer.     

Microbial metabolism of amino alcohols. Formation of coenzyme B12-dependent ethanolamine ammonia-lyase and its concerted induction in Escherichia coli.

1. A branched-chain 2-oxo acid dehydrogenase was partially purified from ox liver mitochondria. 2. The preparation oxidized 4-methyl-2-oxopentanoate, 3-methyl-2-oxobutyrate and D- and L-3-methyl-2-oxopentanoate. The apparent Km values for the oxo acids and for thiamin pyrophosphate, CoA, NAD+ and Mg2+ were determined. 3. The oxidation of each oxo acid was inhibited by isovaleryl (3-methylbutyryl)-CoA (competitive with CoA) and by NADH (competitive with NAD+); Ki values were determined. 4. The preparation showed substrate inhibition with each 2-oxo acid. The oxidative decarboxylation of 4-methyl-2-oxo[1-14C]pentanoate was inhibited by 3-methyl-2-oxobutyrate and DL-3-methyl-2-oxopentanoate, but not by pyruvate. The Vmax. with 3-methyl-2-oxobutyrate as variable substrate was not increased by the presence of each of the other 2-oxo acids. 5. Ox heart pyruvate dehydrogenase did not oxidize these branched-chain 2-oxo acids and it was not inhibited by isovaleryl-CoA. The branched-chain 2-oxo acid dehydrogenase activity (unlike that of pyruvate dehydrogenase) was not inhibited by acetyl-CoA. 6. It is concluded that the branched-chain 2-oxo acid dehydrogenase activity is distinct from that of pyruvate dehydrogenase, and that a single complex may oxidize all three branched-chain 2-oxo acids.     

Theoretical studies on the mechanism of conversion of androgens to estrogens by aromatase.

Semiempirical molecular orbital calculations (AM1) were used to model several possible reaction mechanisms for the third oxidation of the aromatase-catalyzed conversion of androgens to estrogens. The reaction mechanisms considered are based on the assumption that the third oxidation is initiated by 1 beta-hydrogen atom abstraction. Homolytic cleavage of the C10-C19 bond was modeled for both the 3-keto and 2-en-3-ol forms of the androgen 1-radicals. The addition of a protein nucleophile to the 19-oxo intermediate was also considered, and -OCH3, -SCH3, and -NHCH3 were used to represent the Ser, Cys, and Lys adducts. The transition states were estimated and optimized from the reaction coordinates obtained by constraining and increasing the C10-C19 bond lengths. The enthalpies of activation range from 14 to 21 kcal and are approximately 2 kcal lower for cleavage of the enol form. Given the tendency for AM1 to overestimate activation energies, all reactions may be energetically accessible. Other reactions modeled include a homolytic cleavage reaction from a thioether radical cation and the direct additions of oxygen radical compounds to the carbonyl of the 1-radical-2-en-3-ol-19-oxo androgen. A mechanism is proposed in which the 19-oxo intermediate is subject to initial nucleophilic attack by the protein. Since rotation of the 19-carbonyl can bring the oxygen within 2.1 A of the 2 beta-hydrogen, the formation of a tetrahedral intermediate can occur with concomitant removal of the 2 beta-proton. Enolization activates the C1-position for hydrogen atom abstraction, since the resulting radical is resonance stabilized.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antitumor studies. part 3: design, synthesis, antitumor activity, and molecular docking study of novel 2-methylthio-, 2-amino-, and 2-(N-substituted amino)-10-alkyl-2-deoxo-5-deazaflavins

Various novel 10-alkyl-2-deoxo-2-methylthio-5-deazaflavins have been synthesized by reaction of 6-(N-alkylanilino)-2-methylthiopyrimidin-4(3H)-ones with Vilsmeier reagent. The similar 2-(N-substituted amino) derivatives were prepared by nucleophilic replacement reaction of the 2-methylthio moiety by appropriate amines. The 2-oxo derivatives (i.e., 5-deazaflavins) were obtained by acidic hydrolysis of the 2-methylthio derivatives. The antitumor activities against CCRF-HSB-2 and KB cells and the antiviral activities against HSV-1 and HSV-2 have been investigated in vitro, and many compounds showed promising antitumor activities. Furthermore, AutoDock molecular docking into PTK has been done for lead optimization of these compounds as potential PTK inhibitors. Whereas, the designed 2-deoxo-5-deazaflavins connected with amino acids at the 2-position exhibited the good binding affinities into PTK with more hydrogen bonds.     

The metabolic fate of branched-chain amino acids and 2-oxo acids in rat muscle homogenates and diaphragms

After incubation of muscle preparations with [U-14C]branched-chain amino acids or 2-oxo acids, radioactive metabolites were separated, identified and quantified. Homogenates of rat heart and skeletal muscle incubated with 4-methyl-2-oxopentanoate accumulated isovalerate, 3-hydroxyisovalerate and the corresponding carnitine esters. Incubation with 3-methyl-2-oxobutanoate resulted in the production of isobutyrate, 3-hydroxyisobutyrate and their carnitine esters. Addition of L-carnitine increased the production of the esters. The enzymes 3-methylcrotonyl-CoA carboxylase and 3-hydroxyisobutyric acid dehydrogenase apparently are inactive during incubation of muscle homogenates. With liver homogenates the degradation of both 2-oxo acids was more complete. Rat hemidiaphragms incubated with leucine, valine and isoleucine accumulated the corresponding branched-chain 2-oxo acids, fatty acids and hydroxylated fatty acids. The degradation of valine was markedly limited by the release of these metabolites. Considerable amounts (relatively smaller for valine) of radioactivity were also recovered in CO2 and glutamine and glutamate. Incubations with branched-chain 2-oxo acids gave the same radioactive products, except for glutamine and glutamate. Radioactivity was never found in lactate, pyruvate or alanine. These data indicate that the carbon-chains of amino acids entering the citric acid cycle in muscle, are not used for oxidation or for alanine synthesis, but are converted exclusively to glutamine.     

Potential bile acid metabolites. 17. Synthesis of 2 beta-hydroxylated bile acids

The 2 beta-hydroxylated derivatives of lithocholic, chenodeoxycholic, deoxycholic, and cholic acids were synthesized from the respective parent bile acids by established procedures. The principal reactions involved were (1) bromination of 3-oxo formylated bile acids in N,N-dimethylformamide, (2) rearrangement and substitution of the resulting 4 beta-bromo-3-oxo derivatives to the 2 beta-acetoxy-3-oxo compounds with potassium acetate, and (3) reduction to the 2 beta-acetoxy-3 alpha-hydroxy compounds with tert-butylamine-borane complex. As for the prepared 2 beta-hydroxylated bile acids with a diequatorial trans-glycol structure, proton and carbon-13 nuclear magnetic resonance spectroscopic and gas-liquid chromatographic/mass spectrometric properties are discussed.