Chemical Structure of Mating Pheromone Peptides, αsk1 and αsk3 Pheromones,Produced by Mating Type α Cells of Saccharomyces kluyveri

Three peptides, α^sk1, α^sk2 and α^sk3 pheromones, have been isolated as α-mating pheromones of Saccharomyces kluyveri, the primary structure of the main active component, α^sk2 pheromone, having already been determined. The unknown N-terminus of α^sk1 pheromone was elucidated to be 1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid (β-CAR) by mass and NMR spectrometric analyses. Synthetic β-CAR-His-Trp-OH was identical with N-terminal tripeptide fragment obtained from α^sk1 pheromone, and the primary structure of α^sk1 pheromone was determined as β-CAR-His-Trp-Leu-Ser-Phe-Ser-Lys-Gly-Glu-Pro-Met(O)-Tyr-OH. The amino acid sequence of α^sk3 pheromone was determined as H-Trp-His-Trp-Leu-Ser-Phe-Ser-Lys-Gly-Glu-Pro-Met-OH by comparing the enzymatic fragments with those of α^sk2 pheromone.     

Plant Protoplasts Immobilized in Calcium Alginate: A Simple Method of Preparing Fragile Cells for Transmission Electron Microscopy

A simple method for electron microscopic preparation of plant protoplasts is described. The main problems in preparing these fragile protoplasts for electron microscopy have been cell collapse due to steep gradients between protoplasts and fixatives and unacceptable loss of material during the many steps of the procedure. These problems may be solved by immobilization of the protoplasts in calcium alginate beads. The free diffusion properties of this gel prevent steep gradients. The beads also simplify handling and prevent loss of material. Protoplasts isolated from hypocotyls of rape, Brassica napus (var. Niklas), have been used as a model system. Transmission electron microscopy of the immobilized protoplasts osmotically stabilized with glucose demonstrated adequate structural and ultrastructural preservation.     

Immobilized Enzyme Three-Phase Reactors for Oxidation of Cephalosporin C

The enzymatic oxidation of Cephalosporin C (CEPHC) was catalyzed by D-aminoacid oxidase, from the red yeast Trigonopsis variabilis, immobilized on Duolite A365. The study was performed in two different three phase bioreactors, gas-liquid-solid (immobilized enzyme): the fluidized-bed batch reactor, fed continuously with oxygen and discontinuously with CEPHC, and the UF-membrane reactor continuously fed with both substrates. Only the first reactor allowed significant product yield (>70%) while the second was a very useful tool for laboratory investigation of both bioconversion kinetics and enzyme stability.

Optimum reaction temperature was 15d`C for the control of CEPHC spontaneous degradation (roughly 15% in 30 h), and enzyme deactivation (half-life greater than 30 h). Immobilization improved (one order of magnitude longer half-life) enzyme resistance to mechanical stresses induced by liquid stirring and gas bubbling. Roughly 0.04g of CEPHC was adsorbed per gram of enzyme carrier. The limiting step in oxygen transfer was the gas to liquid transport. In order to attain kinetic control of the bioconversion the mildest conditions were atmospheric gas pressure and oxygen flow rate equal to 2 × 10 ²NmL/s per mL of liquid phase.     

Membrane insertion of the FYVE domain is modulated by pH

The FYVE domain associates with phosphatidylinositol 3‐phosphate [PtdIns(3)P] in membranes of early endosomes and penetrates bilayers. Here, we detail principles of membrane anchoring and show that the FYVE domain insertion into PtdIns(3)P‐enriched membranes and membrane‐mimetics is substantially increased in acidic conditions. The EEA1 FYVE domain binds to POPC/POPE/PtdIns(3)P vesicles with a Kd of 49 nM at pH 6.0, however associates ～24 fold weaker at pH 8.0. The decrease in the affinity is primarily due to much faster dissociation of the protein from the bilayers in basic media. Lowering the pH enhances the interaction of the Hrs, RUFY1, Vps27p and WDFY1 FYVE domains with PtdIns(3)P‐containing membranes in vitro and in vivo, indicating that pH‐dependency is a general function of the FYVE finger family. The PtdIns(3)P binding and membrane insertion of the FYVE domain is modulated by the two adjacent His residues of the R(R/K)HHCRXCG signature motif. Mutation of either His residue abolishes the pH‐sensitivity. Both protonation of the His residues and nonspecific electrostatic contacts stabilize the FYVE domain in the lipid‐bound form, promoting its penetration and increasing the membrane residence time. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

木聚糖酶分子结构与重要酶学性质关系的研究进展

木聚糖是一种多聚五碳糖 ,是植物细胞中主要的半纤维素成分。木聚糖酶是可将木聚糖降解成低聚木糖和木糖的水解酶 ,它在饲料、造纸、食品、能源工业和环境科学上有着广阔的应用前景。随着分子生物学、结构生物学的发展及蛋白质工程的应用 ,对木聚糖酶结构和功能的研究不断深入。这里重点阐述与酶的活性、热稳定性、作用pH、等电点、底物亲和性及催化效率等重要性质相关的分子结构研究进展 ,讨论了其进一步的研究发展方向。研究木聚糖酶结构与功能的关系 ,对进一步加深木聚糖酶作用机制的了解、指导木聚糖酶的分子改良有重要意义。     

一种改进的制备染色质的方法

介绍一种改进的制备染色质的方法,采用此方法可减少玻璃匀浆步骤,易于操作、易于重复、产率高,且可以得到不同大小的染色质片段．     

供光学显微镜观察的花粉样品制备的一种简单方法

传统的制备供光学显微镜观察的花粉样品的方法 (中国科学院植物研究所形态室孢粉组 ,1 960 )不但程序复杂 ,而且不同种类的花粉容易混杂。最近 ,我们在进行山茶属(Ｃａｍｅｌｌｉａ)花粉形态的系统研究中总结出一种制备供光镜观察的花粉材料的简单方法 ,现将其过程介绍如下 :( 1 )从标本或新鲜植株上取下花药 ,用冰醋酸浸软后 ,置洁净的凹玻片 (单凹玻片 )上 ,于解剖镜下将花药打开 ,滴上 95%酒精将花粉洗出。( 2 )滴上预先配制好的分解液 (醋酸酐 9份和浓硫酸 1份 ) ,于室温下或 50℃恒温箱里放置 5ｍｉｎ(具体温度和时间因花粉种类而异 ) …     

A Method for Preparing DNA Sequencing Templates Using a DNA-Binding Microplate

A DNA-binding matrix was immobilized on the surface of a 96-well microplate and used for plasmid DNA preparation for DNA sequencing. The same DNA-binding plate was used for bacterial growth, cell lysis, DNA purification, and storage. In a single step using one buffer, bacterial cells were lysed by enzymes, and released DNA was captured on the plate simultaneously. After two wash steps, DNA was eluted and stored in the same plate. Inclusion of phosphates in the culture medium was found to enhance the yield of plasmid significantly. Purified DNA samples were used successfully in DNA sequencing with high consistency and reproducibility. Eleven vectors and nine libraries were tested using this method. In 10 μl sequencing reactions using 3 μl sample and 0.25 μl BigDye Terminator v3.1, the results from a 3730xl sequencer gave a success rate of 90–95% and read-lengths of 700 bases or more. The method is fully automatable and convenient for manual operation as well. It enables reproducible, high-throughput, rapid production of DNA with purity and yields sufficient for high-quality DNA sequencing at a substantially reduced cost.     

Computational evidence for the catalytic mechanism of glutaminyl cyclase. A DFT investigation

The results of a DFT theoretical investigation on the catalytic mechanism of the QC enzyme are presented. A rather large model-system is used. It includes the most important residues that are believed to play a key-role in the catalysis. The computational results show that the rate-determining step of the catalytic process is not the nucleophilic attack leading to the cycle formation (a very easy and fast process with a negligible barrier of 0.8 kcal mol(-1)), but a proton transfer, which is assisted by the Glu201 residue acting as a proton shuttle (general base and general acid). A complex network of hydrogen bonds (involving Asp248 and other residues) contribute to lower the activation barrier for the proton shift which affords the formation of an ammonia molecule bonded to the substrate. The ammonia molecule is a good leaving group which is easily expelled from the substrate in the last step of the catalytic cycle, but remains anchored to the enzyme as a ligand of the zinc cation. The metal plays a key-role in assisting the nucleophilic attack (electrostatic catalysis) since it polarizes the substrate gamma-amide carbonyl group (its electrophilic character increases). Also, the strength of the nucleophilic nitrogen (substrate alpha-amino group) is enhanced by hydrogen bonds involving the Glu201 residue. The computations outline the important role of Trp329 in helping the substrate binding process and stabilizing the cyclization transition state.     

A New Method for Preparing Slide Mounts of Whole Bodies of Microlepidoptera

A new method is described for preparing slide mounts of whole bodies of microlepidoptera to facilitate comparative morphological studies. This method conserves traditional characters of wing pattern while revealing wing venation and other morphological structures of the denuded body. Examples of new characters revealed on slide mounts of whole bodies and photographed with a Confocal Laser Scanning Microscope are given for selected species of Gelechioidea. Also, the historical use of morphological characters for defining taxa of Lepidoptera is briefly reviewed.     

A Fast Method to Estimate Kinetic Constants for Enzyme Inhibitors

We present a method to determine the reaction type and kinetic constants for enzyme inhibitors that decreases the number of experimental assays by at least a factor of five. It is based on a new theoretical formalism in terms of concentrations that dismisses the requirement of estimating initial velocities. Expressions for the time evolution of the concentrations of all the reactants are also given.     

Immobilized pH gradients for isoelectric focusing. III. Preparative separations in highly diluted gels

A further improvement on the preparative aspects of immobilized pH gradients (IPG) (J. Biochem. Biophys. Methods (1983) 8, 135–172) is described, based on the use of soft (highly diluted) polyacrylamide gels. While in conventional IPGs in 5%T gels an upper load limit of 40–45 mg protein/ml gel volume is found, in 2.5%T gels, containing the same amount of Immobiline, as much as 90 mg protein/ml gel can be applied, without overloading effects. This is an extraordinary amount of material to ba carried by a gel phase, and renders IPG by far the leading technique in any electrophoretic fractionation. A new, two-step casting technique, based on the formation of a %T step and a pH plateau around the application trench, is described. A new method for electrophoretic protein recovery from IPG gel strips, based on embedding on low-gelling agarose (37°C), is reported. The physico-chemical properties of highly diluted gels, in relation to their protein loading ability, are evaluated and discussed. It is recommended that diluted gels (e.g. 3.5%T) be used also in analytical runs, since sharper protein zones are obtained, due to the increased charge density on the polymer coil.