The Structure of Ammonium,D,L—Tartrate

1 INTRODUCTION The enantiomers separated by a co- crystallization method using an appropriate chiral as separating reagent has attracted many attentions in the past years[1]. Recently we are interested in enantiomeric discrimination by chiral amino acid. When racemic D,L-tartaric acid was tried to be separated by the L-glutamine in a hot aqueous solution, the well shaped crystals were obtained. The determination of melting point (220℃) and density (1.66 g穋m－3) showed it is…     

MM and QM/MM Modeling of Threonyl-tRNA Synthetase: Model Testing and Simulations

Aminoacyl-tRNA synthetases are centrally important enzymes in protein synthesis. We have investigated threonyl-tRNA synthetase from E. coli, complexed with reactants, using molecular mechanics and combined quantum mechanical/molecular mechanical (QM/MM) techniques. These modeling methods have the potential to provide molecular level understanding of enzyme catalytic processes. Modeling of this enzyme presents a number of challenges. The procedure of system preparation and testing is described in detail. For example, the number of metal ions at the active site, and their positions, were investigated. Molecular dynamics simulations suggest that the system is most stable when it contains only one magnesium ion, and the zinc ion is removed. Two different QM/MM methods were tested in models based on the findings of MM molecular dynamics simulations. AM1/CHARMM calculations resulted in unrealistic structures for the phosphates in this system. This is apparently due to an error of AM1. PM3/CHARMM calculations proved to be more suitable for this enzyme system. These results will provide a useful basis for future modeling investigations of the enzyme mechanism and dynamics.     

Purification and partial characterization of CMP-Neu5Ac synthetase from rat brain

N-Acetyl-neuraminic acid cytidylyltransferase (EC 2.7.7.43) (CMP-Neu5Ac synthetase), which catalyzes the formation of cytidine-5′-monophospho-N-acetyl-neuraminic acid (CMP-Neu5Ac) from cytidine-5′-triphosphate (CTP) and N-acetyl-neuraminic acid (Neu5Ac), was purified from rat brains aged 8-9 days, which presented the highest specific activity, and partially characterized. Partial protein fractionation in the crude extract was achieved by using 40-60% ammonium sulphate. Subsequently, CMP-Neu5Ac synthetase was purified by column chromatography on Sephacryl S-200 (gel filtration), Yellow-86-Agarose (affinity) and Phenyl-Sepharose (hydrophobic affinity). The pure enzyme had a specific activity of 3.6555 U/mg of protein and was purified 1662-fold, with an 18% yield. The purified CMP-Neu5Ac synthetase had a molecular weight of about 46 ± 1 kDa. Its purity was confirmed by sodium dodecyl sulphate and polyacrylamide gel electrophoresis (SDS-PAGE) and high-performance liquid chromatography (HPLC). The active enzyme chromatographed on a gel filtration column at 190 kDa, suggesting it exists in its native form as a tetramer. The greatest activity of enzyme was observed a temperature of 40 °C for a period of 45 min of incubation, revealing a certain thermal stability. The enzyme was found to remain stable in the pH range 8.5-9.5 at 40 °C, specifically at pH 9.0 for a 45 min incubation period. The enzyme was blocked by thiol-modifying reagents and such heavy metal cations as Mn²⁺, Cu²⁺, Sn²⁺, Co²⁺, Zn²⁺ and Hg²⁺, but was not inhibited by thiol-containing reagents like reduced glutathione (GSH), mercaptoethanol and cysteine. Finally, in the presence of 0.01 M of dithiothreitol (DTT) or 0.06 M of NaF, the enzyme showed activity losses of approximately 20 and 17%, respectively.     

Glucose and glutamine metabolism of a murine B-lymphocyte hybridoma grown in batch culture

The energy metabolism of a mammalian cell line grown in vitro was analyzed by substrate consumption rates and metabolic flux measurements. The data allowed the determination of the relative importance of the pathways of glucose and glutamine metabolism to the energy requirements of the cell. Changes in the substrate concentrations during culture contributed to the changing catalytic activities of key enzymes, which were determined.

Immobilization of prostaglandin synthetase by hydrophobic adsorption

In this article, the immobilization of prostaglandin synthetase onn-alkyl or aryl amino-agar beads by hydrophobic adsorption is reported. The effects of different hydrophobic groups in the agar beads, pH of buffer, concentration of salts on the adsorption of prostaglandin synthetase, and the properties of immobilized prostaglandin synthetase were also studied. The results showed that 20–35 mg of microsome containing PG synthetase (protein content 8–15 mg) could be adsorbed on each gram ofn-dodecylamino-agar beads after suction drying the gel in the buffer of pH 5.5 (containing 0.5 mol/L KC1), 0.1 mol/L citric-phosphate at 4‡C. The remaining immobilized enzyme activity was over 80%. The optimum pH of immobilized PG synthetase is 8.0, similar to that of the native enzymes. The thermostability of immobilized PG synthetase in the buffer containing 0.5 mol/L KC1 was increased. Immobilized PG synthetase was used as a catalyst of synthesis of prostaglandin E₁. The preservation of activity after 10 working cycles was 86.2%.     

Fragmentation reactions of protonated peptides containing glutamine or glutamic acid

A variety of protonated dipeptides and tripeptides containing glutamic acid or glutamine were prepared by electrospray ionization or by fast atom bombardment ionization and their fragmentation pathways elucidated using metastable ion studies, energy-resolved mass spectrometry and triple-stage mass spectrometry (MS(3)) experiments. Additional mechanistic information was obtained by exchanging the labile hydrogens for deuterium. Protonated H-Gln-Gly-OH fragments by loss of NH(3) and loss of H(2)O in metastable ion fragmentation; under collision-induced dissociation (CID) conditions loss of H-Gly-OH + CO from the [MH - NH(3)](+) ion forms the base peak C(4)H(6)NO(+) (m/z 84). Protonated dipeptides with an alpha-linkage, H-Glu-Xxx-OH, are characterized by elimination of H(2)O and by elimination of H-Xxx-OH plus CO to form the glutamic acid immonium ion of m/z 102. By contrast, protonated dipeptides with a gamma-linkage, H-Glu(Xxx-OH)-OH, do not show elimination of H(2)O or formation of m/z 102 but rather show elimination of NH(3), particularly in metastable ion fragmentation, and elimination of H-Xxx-OH to form m/z 130. Both the alpha- and gamma-dipeptides show formation of [H-Xxx-OH]H(+), with this reaction channel increasing in importance as the proton affinity (PA) of H-Xxx-OH increases. The characteristic loss of H(2)O and formation of m/z 102 are observed for the protonated alpha-tripeptide H-Glu-Gly-Phe-OH whereas the protonated gamma-tripeptide H-Glu(Gly-Gly-OH)-OH shows loss of NH(3) and formation of m/z 130 as observed for dipeptides with the gamma-linkage. Both tripeptides show abundant formation of the y(2)' ion under CID conditions, presumably because a stable anhydride neutral structure can be formed. Under metastable ion conditions protonated dipeptides of structure H-Xxx-Glu-OH show abundant elimination of H(2)O whereas those of structure H-Xxx-Gln-OH show abundant elimination of NH(3). The importance of these reaction channels is much reduced under CID conditions, the major fragmentation mode being cleavage of the amide bond to form either the a(1) ion or the y(1)' ion. Particularly when Xxx = Gly, under CID conditions the initial loss of NH(3) from the glutamine containing dipeptide is followed by elimination of a second NH(3) while the initial loss of H(2)O from the glutamic acid dipeptide is followed by elimination of NH(3). Isotopic labelling shows that predominantly labile hydrogens are lost in both steps. Although both [H-Gly-Glu-Gly-OH]H(+) and [H-Gly-Gln-Gly-OH]H(+) fragment mainly to form b(2) and a(2) ions, the latter also shows elimination of NH(3) plus a glycine residue and formation of protonated glycinamide. Isotopic labelling shows extensive mixing of labile and carbon-bonded hydrogens in the formation of protonated glycinamide.     

天冬酰胺合成酶B抑制剂高效液相色谱筛选方法的建立与应用

建立了一种快速、高效测定天冬酰胺合成酶B(AS-B)酶活性的反相高效液相色谱法(RP-HPLC)。酶反应体系中的氨基酸经2,4-二硝基氟苯(DNFB)柱前衍生,通过RP-HPLC测定酶反应体系前后底物及产物的变化来分析酶的活性。采用的色谱柱为Agilent C18柱(250 mm×4.6 mm,5 μm),以50 mmol/L醋酸钠缓冲液(pH 6.2)-乙腈(体积比为15:85)为流动相,流速为1.0 mL/min,柱温为30 ℃,检测波长365 nm,于6 min内实现了各组分的基线分离。通过该方法测定反应动力学参数进行AS-B的抑制定量分析。将已知AS-B抑制剂L-谷氨酸-γ-甲酯作用于酶反应体系,测得的抑制剂的抑制常数与文献值相接近,证明该方法可用于AS-B抑制剂的筛选。     

水稻线粒体tRNATrp种属特异性元件研究

为了研究水稻线粒体tRNA^Trp的种属特异性元件,在野生型水稻线粒体tRNA^Trp的基础上,设计并完成了3种向人tRNA^Trp的突变,体外转录并用枯草杆菌和人这两种不同种属来源的色氨酰-tRNA合成酶（TrpRS）测定了这些tRNA^Trp分子的氨酰化活力（Kcat／KM）．结果表明,与野生型水稻线粒体tRNA^Trp相比,3个突变体被人TrpRS氨酰化的活力分别提高了354、407和803倍,其中以PMPH3（水稻线粒体tRNA^Trp的氨基酸接受茎的C2-G71和G3-C70都突变为人tRNA^Trp的氨基酸接受茎的相应部位）的氨酰化活力改变最大．而3个突变体对B．subtilis TrpRS氨酰化活力有进一步负影响,氨酰化活力微弱．说明水稻线粒体tRNA^Trp氨基酸接受茎上的第2个碱基对C2-G71和第3个碱基对G3-C70在人色氨酰-tRNA合成酶识别过程中有着极为重要的作用,是水稻线粒体tRNA^Trp的种属特异性元件．     

LIMITED TRYPTIC DIGESTION OF LEUCYL-tRNA SYNTHETASE AND CHARACTERIZATION OF ITS ACTIVE FRAGMENT

Leucyl-tRNA synthetase (LeuRS, EC 6.1.1.4) from E. coli underwent limitedproteolysis by trypsin which cut off 6K peptide and converted the intact LeuRS into a 96K fragment. The truncated enzyme retained the PPi exchange activity with the same kinetic parameters as those of native LeuRS but lost the tRNA~(Leu)charging, binding and other tRNA~(Leu)-related activities. N-terminus analysis showed that the 6K peptide was located at the C-terminus of LeuRS. This small part played a crucial role in tRNA~(Leu) binding. Our results suggest that the two activities, PPi exchange and tRNA charging are independent of each other and correspond to different structural regions of LeuRS. The C-terminal region might be the tRNA~(Leu)binding site of LeuRS.     

合成聚肽嵌段共聚物PHEG-PBLG结构的核磁共振分析

通过L-谷氨酸苄酯和三光气反应制备谷氨酸苄酯的N-羧酸酐,以聚羟乙谷氨酸酰胺为大分子引发剂,投入不同配比的单体/引发剂（A/I）,共聚得到两亲性嵌段共聚物聚羟乙谷氨酰胺-聚谷氨酸苄酯（PHEG-PBLG）;通过核磁共振技术分析表征该合成聚肽的结构组成、分子量范围及螺旋结构;通过体外酶解实验研究了共聚物的降解情况. 研究结果表明：该合成聚肽化合物为两亲性嵌段聚合物：当聚合物中亲水链段PHEG分子量为9000,A/I为75∶1, 100∶1, 150∶1, 200∶1时,相应的共聚物的分子量分别为：1.27×10⁴,1.75×10⁴, 1.9×10⁴, 3.60×10⁴;该合成聚肽含有α-螺旋结构, 随着TFA的加入,该聚肽的结构由α-旋构象转化为随机盘绕构象.