Increased Stability and Catalytic Efficiency of Yeast Hexokinase Upon Interaction with Zwitterionic Micelles. Kinetics and Conformational Studies

The effect of ligands (glucose, ATP and Mg2+) and zwitterionic micellesof lysophosphatidylcholine (LPC) or N-hexadecyl-N,N-dimethyl-3-ammoniumpropanesulfonate (HPS) in the yeast hexokinase (HK) stability was studied at35°C. The thermal inactivation kinetics followed one-exponentialdecay. The effect of ligands on protecting the enzyme against inactivationfollowed the order: glucose>glucose/Mg2+>ATP/Mg2+Mg2+bufferonly. Both LPC and HPS micelles increased the enzyme stability only whenthe incubation medium contained glucose or glucose/Mg2+,suggesting that the protein conformation is a key prerequisite for theenzyme-micelle interaction to take place. This enzyme-micelle interactionresulted in an increased catalytic efficiency (with a decrease in Km forATP and increase in Vmax as well as in changes on the tertiary (intrinsicfluorescence) structure of the yeast hexokinase.     

Synthesis of Alkyl Glycosides Catalyzed by β-Glycosidases in a System of Reverse Micelles

A basic possibility of enzymic synthesis of alkyl glycosides in a system of the Aerosol-OT (AOT) reverse micelles was studied. Octyl -D-galactopyranoside and octyl -D-glucopyranoside were synthesized from the corresponding sugars (lactose or glucose) and octyl alcohol under catalysis with glycolytic enzymes, -galactosidase and -glucosidase, respectively. The transglycosylation/hydrolysis ratio was shifted toward transglycosylation by using octyl alcohol, one of the substrates, as an organic solvent. The alkyl glycosides were thus obtained in one step from a hydrophilic mono- or disaccharide and a hydrophobic aliphatic alcohol. The direction of the reaction was shown to depend on the pH of aqueous solution solubilized in reverse micelles. The maximum yields were 45% and 40% for octyl galactoside and octyl glucoside, respectively; they markedly exceeded the yields of enzymic syntheses in a two-phase system reported previously.     

高分子药用控释材料研究进展

本文综述了高分子药用控释材料的性能、分类及其应用;并介绍了其发展方向。     

Molecular simulation study on sorption and diffusion processes in polymeric pervaporation membrane materials

Since MD simulations of membrane separation processes of aqueous/organic solutions lead to reasonable results that are in good agreement with experimental investigations an approach has been made to apply this method also for organic/organic membrane separation. In this connection the separation of a model feed mixture of 20 wt% benzene and 80 wt% n-heptane with dense amorphous polymer membranes was simulated utilising molecular modelling techniques. Special modifications of standard polymers such as polymethacrylates and polysiloxanes were investigated. It could be demonstrated that MD simulations of polymer–feed interface models can reproduce at least qualitatively important experimental results from related membrane pervaporation processes. These models are therefore suited in principle to obtain a better insight in the atomistic mechanisms of pervaporation. In addition, the knowledge about the underlying diffusion mechanism could be improved.     

Structural analysis of telechelic polymer solution using dissipative particle dynamics simulations

A telechelic polymer is an amphiphilic polymer that can form micellar structures when dissolved in water. A telechelic polymer solution shows viscoelastic behaviour owing to the formation of characteristic networks, i.e. loops, bridges and dangling chains. For industrial purposes, telechelic polymers have many applications as thickening agents, such as in paints and cosmetics. Thus, it is desirable to predict and control the rheological properties of telechelic polymers. However, detailed studies at the molecular level have not yet been performed. In this study, I use the dissipative particle dynamics (DPD) method to investigate the relationship between the characteristic structural properties and the molecular structure in telechelic polymer solutions. I show that the morphology of telechelic polymer solutions depends on the concentration and chain length, the distribution of the end-to-end distance, the mean square end-to-end distance, the mean square radius of gyration and the time-averaged mean square displacement. Although an effect of entanglement is important for properties of polymer melts, the polymer chain composed of DPD particles cannot reproduce it. Therefore, I compare telechelic polymer solutions with and without the segmental repulsive potential (SRP), which can simulate the effect of entanglement in DPD simulations. The results indicate that it is necessary to include the SRP in DPD simulations to correctly analyse the behaviour of telechelic polymer solutions.     

Endothelins specifically recognize lysophosphatidylcholine micelles

Lysophosphatidylcholine (LPC), a major phospholipid component of oxidized low‐density lipoprotein (ox‐LDL), is implicated in numerous inflammatory diseases, including atherosclerosis. Here, to clarify the relationship between bioactive endothelins (ETs) (which are considered to be potent proinflammatory mediators) and LPC/ox‐LDL, we investigated the interaction between ETs and LPC/ox‐LDL by fluorescence spectroscopy and western blotting. Tryptophan fluorescence measurements revealed ETs specifically interacted with LPC at concentrations that exceeded the critical micelle concentration (CMC). The tryptophan residue in ETs was not likely to be involved directly in the interaction between ETs and LPC micelles. Tryptophan fluorescence quenching revealed tryptophan residue in ETs where LPC concentrations were below the CMC may be buried deeply in the peptide or may interact with other amino acid residues, whereas tryptophan residue in ETs in the presence of LPC at concentrations exceeding the CMC was exposed outside of the peptide. Furthermore, ETs bind to ox‐LDL in a concentration‐dependent manner. These results strongly suggest that ox‐LDL contains micelle‐rich LPCs and that ETs specifically interact with the bioactive LPC micelles. Further study of the interaction between ETs and LPC micelles contained in ox‐LDL will provide important information on the development and progression of many inflammatory diseases, including atherosclerosis. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Triton X-114 phase separation in the isolation and purification of mouse liver microsomal membrane proteins

Integral membrane proteins (IMPs) mediate several cellular functions including cell adhesion, ion and nutrient transport, and cell signalling. IMPs are typically hard to isolate and purify due to their hydrophobic nature and low cellular abundance, however, microsomes are small lipid vesicles rich in IMPs, which form spontaneously when cells are mechanically disrupted. In this study, we have employed mouse liver microsomes as a model for optimising a method for IMP isolation and characterisation. Microsomes were collected by differential centrifugation, purified with sodium carbonate, and subjected to GeLC–MS/MS analysis. A total of 1124 proteins were identified in the microsome fraction, with 47% (524/1124) predicted by TMHMM to contain at least one transmembrane domain (TMD). The ability of phase partitioning using the detergent Triton X-114 (TX-114) to further enrich for membrane proteins was evaluated. Microsomes were subjected to successive rounds of solubility-based phase separation, with proteins partitioning into the aqueous phase, detergent phase, or TX-114-insoluble pellet fraction. GeLC–MS/MS analysis of the three TX-114 fractions identified 1212 proteins, of which 146 were not detected in the un-fractionated microsome sample. Conspicuously, IMPs partitioned to the detergent phase, with 56% (435/770) of proteins identified in that fraction containing at least one TMD. GO Slim characterisation of the microsome proteome revealed enrichment of proteins from the endoplasmic reticulum, mitochondria, Golgi apparatus, endosome, and cytoplasm. Further, enzymes including monooxygenases were well represented with 35 cytochrome P450 identifications (CYPs 1A2, 2A5, 2A12, 2B10, 2C29, 2C37, 2C39, 2C44, 2C50, 2C54. 2C67, 2C68, 2C70, 2D10, 2D11, 2D22, 2D26, 2D9, 2E1, 2F2, 2J5, 2U1, 3A11, 3A13, 3A25, 4A10, 4A12A, 4A12B, 4F13, 4F14, 4F15, 4V3, 51,7B1, and 8B1). Evaluation of biological processes showed enrichment of proteins involved in fatty acid biosynthesis and elongation, as well as steroid synthesis. In addition, transport proteins including 24 members of the Rab family of GTPases were identified. Comparison of this dataset with the current mouse liver microsome proteome contributes an additional 648 protein identifications, of which 50% (326/648) contain at least one TMD.     

Self-assembly of mixtures of a dendrimer and lipids: effects of hydrophobicity and electrostatics

Self-assemblies of mixtures of a G7 dendrimer and dimyristoylphosphatidylcholine (DMPC) lipids were simulated using the coarse-grained force fields. A single G7 dendrimer, which consists of either a hydrophilic or a hydrophobic interior, was simulated with the randomly distributed zwitterionic DMPC or anionic lipids. For the dendrimer with hydrophilic interior, its mixture with anionic lipids self-assembles to the dendrimer-encasing liposome, but the one with zwitterionic lipids does not. The liposome diameter agrees with experiment, which is smaller than the typical liposome without dendrimer. This indicates that the strong electrostatic interactions between dendrimer terminals and lipid phosphates induce high curvature of the bilayer, leading to such a small liposome. For the dendrimer with hydrophobic interior, lipids penetrate the dendrimer interior because of the hydrophobic interactions between the dendrimer interior and lipid tails, leading to the dendrimer–lipid micelle with increased dendrimer size. These simulation findings agree qualitatively with the experimentally proposed liposome and micelle models, and indicate that these proposed conformations of the dendrimer–lipid complexes are significantly modulated by dendrimer hydrophobicity and lipid charge.     

Improved detection of botulinum neurotoxin type A in stool by mass spectrometry

A novel electrochemical method, termed flash chronopotentiometry (FCP), is used to develop a rapid and sensitive method for detecting protease activities. In this method, an appropriate current pulse is applied across a polycation-selective polymer membrane to induce a strong flux of the polycationic peptides from the sample phase into the organic membrane of the electrode. During this current pulse, the cell potential (EMF) is monitored continuously, and is a function of the polypeptide concentration. The imposed current causes a local depletion of the polypeptide at the sample/membrane interface, which yields a drastic potential change in the observed chronopotentiogram at a characteristic time, called the transition time (τ). For a given magnitude of current, the square root of τ is directly proportional to the concentration of the polypeptide. Proteases cleave polypeptides into smaller fragments that are not favorably extracted into the membrane of the sensor. Therefore, a decrease in the transition time is observed during the proteolysis process. The degree of change in the transition time can be correlated to protease activity. To demonstrate this approach, the activities of trypsin and α-chymotrypsin are detected using protamine and synthetic polycationic oligopeptides that possess specific cleavage sites that are recognized by these proteases.     

On the Acaciin

Synthesis of N-2,2,3,3-tetrafluoropropionyl-amino acids (abbreviated as tetrafluoropropionyl- or Tfp-amino acids) by the reaction of amino acids with tetrafluoropropionyl anhydride was described, Racemization was observed to occur to a considerable extent during the process of the introduction of this group. Optically active Tfp-amino acids were able to be prepared free from racemization by transesterification of methyl tetrafluoropropionate with amino acid tert-butyl esters, followed by treatment of the resulting Tfp-amino acid tert-butyl esters with trifluoroacetic acid. Some Tfp-dipeptide esters were prepared from the corresponding dipeptide tert-butyl esters in this way. This group was cleavable by mild alkaline hydrolysis or by reduction with sodium borohydrde as well as the Tfa-group.