Deformation and instability in membrane structure of phospholipid vesicles caused by osmophobic association: mechanical stress model for the mechanism of poly(ethylene glycol)-induced membrane fusion

The mechanism of poly(ethylene glycol)-induced fusion of phospholipid vesicles was studied based on the "osmophobic association" theory which was recently proposed both theoretically [Ito, T., Yamazaki, M., & Ohnishi, S. (1989) Biochemistry 28, 5626-5630] and also experimentally [Yamazaki, M., Ohnishi, S., & Ito, T. (1989) Biochemistry 28, 3710-3715]. Osmophobic association and fusion were detected by measuring the light scattering of the vesicle suspension; the former was detected from the increase in light scattering induced by the addition of PEG, and the latter was from the irreversibility of the increase in light scattering. Threshold concentrations of PEG were required not only for osmophobic association but also for fusion. The threshold concentration for fusion depended on the molecular weight of PEG and also on the electrostatic repulsive interaction between phospholipid vesicles, which was manipulated by the use of vesicles with negative surface charge; increasing the molecular weight of PEG lowered the threshold concentration, and increasing the electrostatic repulsive interaction raised it. In addition, a transient leakage of internal contents from the vesicles was observed at the concentration that caused fusion. When the surface charge of the vesicle was varied, the threshold for fusion coincided with that for osmophobic association, provided that the latter exceeded 22 wt % of PEG 6000. However, when the threshold for osmophobic association was less than 22 wt %, the threshold for fusion remained approximately 22 wt %, irrespective of the difference in the threshold for osmophobic association.(ABSTRACT TRUNCATED AT 250 WORDS)     

Poly(ethylene glycol)-induced shrinkage of Sephadex gel. A model system for quantitative analysis of osmoelastic coupling.

Shrinkage of Sephadex gels caused by addition of a high-molecular weight molecule, poly(ethylene glycol) (PEG) was studied. A quantitative analysis based on the cross-linked network theory by Flory and Tanaka (Tanaka, T. 1978. Phys. Rev. Lett. 40:820-823) showed that the shrinkage is due to a mechanochemical coupling between the elasticity of the network and the osmotic stress arising from preferential exclusion of PEG. These results may provide good evidence for "osmoelastic coupling", the coupling between elasticity of macromolecular structures and osmotic stress, which has been predicted in some biological systems such as phospholipid bilayer membranes (Ito, T., M. Yamazaki, and S. Ohnishi. 1989. Biochemistry. 28:5626-5630; Yamazaki, M., S. Ohnishi, and T. Ito. 1989. Biochemistry. 28:3710-3715) or actin filaments.     

Thermophilic HB8 DNA ligase: effects of polyethylene glycols and polyamines on blunt-end ligation of DNA

NB8 DNA ligase from an extract of Thermus thermophilus HB8 could catalyze blunt-end ligation in the presence of high concentration of polyethylene glycols (PEG) or in the presence of polyamines. In the presence of high molecular weight PEG 20,000, 6,000, or 1,000 (8-28%), the enzyme catalyzed blunt-end intermolecular joining to yield linear oligomers, but no circular DNA forms. But in the presence of low molecular PEG 400, 200 (8-80%), or the monomer, ethylene glycol (16-80%), the circular forms were also detected by intramolecular ligation. In the presence of polyamines, the blunt-end ligation products were linear oligomers and the optimum concentrations were as follows: caldopentamine (0.05 mM), thermine (0.1-0.2 mM), spermine (0.2 mM), thermospermine (0.4 mM), and sperminediol (0.75 mM). Spermidine and putrescine were less capable of producing oligomers. PEG and polyamines elevated the ligation temperature by HB8 DNA ligase. The optimum temperature of blunt-end ligation was about 65 degrees C.     

Synthesis and self-assembly of well-defined poly(amino acid) end-capped poly(ethylene glycol) and poly(2-methyl-2-oxazoline)

Poly(ethylene glycol) (PEG) and poly(2-methyl-2-oxazoline) (PMOx) are water-soluble, biocompatible polymers with stealth hemolytic activities. Poly(amino acid) (PAA) end-capped PEG and PMOx were prepared using amino-terminated derivatives of PEG and PMOx as macroinitiators for the ring-opening polymerization of γ-benzyl protected l-glutamate N-carboxyanhydride and S-benzyloxycarbonyl protected l-cysteine N-carboxyanhydride, respectively, in the presence of urea, at room temperature. The molecular weight of the PAA moiety was kept between M(n) = 2200 and 3000 g mol(-1). PMOx was polymerized by cationic ring-opening polymerization resulting in molecular weights of M(n) = 5000 and 10,000 g mol(-1), and PEG was a commercial product with M(n) = 5000 g mol(-1). Here, we investigate the self-assembly of the resulting amphiphilic block copolymers in water and the effect of the chemical structure of the block copolymers on the solution properties of self-assembled nanostructures. The PEG-block-poly(amino acid), PEG-b-PAA, and PMOx-block-poly(amino acid), PMOx-b-PAA, block copolymers have a narrow and monomodal molecular weight distribution (PDI < 1.3). Their self-assembly in water was studied by dynamic light scattering and fluorescence spectroscopy. In aqueous solution, the block copolymers associate into particles with hydrodynamic radii (R(H)) ranging in size from R(H) 70 to 130 nm, depending on the block copolymer architecture and the polymer molecular weight. Larger R(H) and critical association concentration values were obtained for copolymers containing poly(S-benzyloxycarbonyl-l-cysteine) compared to their poly(γ-benzyl-L-glutamate) analogue. FTIR investigations revealed that the poly(γ-benzyl-L-glutamate) block adopts a helical conformation, while the poly(S-benzyloxycarbonyl-L-cysteine) block exists as β-sheet.     

Polyethylene glycol-induced mammalian cell hybridization: effect of polyethylene glycol molecular weight and concentration.

The effects of polyethylene glycol (PEG) molecular weight and concentration on mammalian cell hybridization were studied. The peak hybridization-inducing activity with all grades of PEG from 400-6000 was found to occur in the concentration range of 50-55%. However, changes in concentration were seen to have different quantitative effects with different grades of PEG. For monolayer fusions, PEG 1000 at 50% seems to be the optimal combination of PEG molecular weight and concentration, in terms of both efficiency of hybridization and relative insensitivity to dilution effects.     

牛血清白蛋白在聚乙二醇/葡聚糖双水相体系中分配特性的研究

采用考马斯亮蓝G250染色法测得室温下BSA在PEG/dextran双水相体系中的分配系数。以BSA在PEG/dextran体系的下相富集为目标,研究了PEG的分子量、浓度、dextran浓度以及所加入中性盐的种类与浓度、体系pH诸因素对其分配特性的影响。实验结果表明,在PEG4000/dextran体系中,采用PEG质量分数9%-dextran质量分数9%的浓度组成,同时在pH=7.0,NaC l浓度为0.2 mol.L-1或pH6.0,NaC l浓度为0.34 mol.L-1的工艺条件下萃取BSA均可达最小分配系数,其值为0.014。     

Novel approach towards recovery of glycosaminoglycans from tannery wastewater

Poly ethylene glycol (PEG)-poly acrylic acid (PAA) based aqueous two-phase system (ATPS) was selected as a practical model to recover glycosaminoglycans (GAGs) from tannery wastewater. The influence of PEG molecular weight, tie line length (TLL), pH, temperature and NaCl concentration on the partition coefficient of glycosaminoglycans from tannery wastewater was studied. Partition coefficient of glycosaminoglycan decreases on increase of PEG molecular weight, NaCl concentration and temperature, whereas it increases with increase of pH. In the PEG-rich phase, increased partitioning of GAGs was observed with increase in TLL. The partitioning of GAGs was better in PEG 4000 at pH 8.0, 20 °C with a yield of 91.50%. This study demonstrates the potential application of ATPS processes for the recovery of GAGs from complex biological suspensions.     

Steric exclusion is the principal source of the preferential hydration of proteins in the presence of polyethylene glycols.

The preferential interactions of bovine serum albumin, lysozyme, chymotrypsinogen, ribonuclease A, and beta-lactoglobulin with polyethylene glycols (PEGs) of molecular weight 200-6,000 have been measured by dialysis equilibrium coupled with high precision densimetry. All the proteins were found to be preferentially hydrated in all the PEGs, and the magnitude of the preferential hydration increased with increasing PEG size for each protein. The change in the chemical potentials of the proteins with the addition of the PEGs had highly positive values, indicating a strong thermodynamic destabilization of the system by the PEGs. A viscosity study of the PEGs showed them to be randomly coiled polymers, as their radii of gyration were related to the molecular weight by Rg = aM0.55. The thickness of the effective shell impenetrable to PEG around protein molecules, calculated from the preferential hydration, was found to vary with PEG molecular weight in similar fashion as the PEG radius of gyration, supporting the proposal (Arakawa, T. & Timasheff, S.N., 1985a, Biochemistry 24, 6756-6762) that the preferential exclusion of PEGs from proteins is due principally to the steric exclusion of PEG from the protein domain, although favorable interactions with protein surface residues, in particular nonpolar ones, may compete with the exclusion. These thermodynamically unfavorable preferential exclusion interactions lead to the action of PEGs as precipitants, although they may destabilize protein structure at higher temperatures.     

Effect of poly(ethylene glycol) on the Ca2+-induced fusion of didodecyl phosphate vesicles

This paper reports a study of the effect of the dehydrating agent poly(ethylene glycol) (PEG) on didodecyl phosphate (DDP) bilayers and on the fusion activity of DDP vesicles as a function of the molecular weight of PEG. PEG 8K in a concentration of 10 wt % does not induce fusion. However, Ca2+-induced fusion is promoted as reflected by a lowering of the Ca2+ threshold concentration. This effect can most likely be attributed to the dehydrating capacity of the polymer. Interestingly, low concentrations (0.1 wt %) of PEG 20 K induce a moderate fusion capacity. At higher concentrations (0.5 wt %) fusion is inhibited, irrespective of the presence of Ca2+. These molecular weight dependent effects can be rationalized by taking into account that the clouding temperature differs for PEGs of different molecular weights. In the case of PEG 20K a microscopic phase separation will occur at the bilayer-water interface because PEG-PEG interactions and presumably PEG-DDP interactions are favored over PEG-water interactions. As a consequence, the DDP vesicle surface becomes covered with PEG 20K, resulting in a steric stabilization of the vesicles. This will impede or prevent, depending on the polymer concentration, the vesicles from approaching each other sufficiently close for fusion to occur.     

Precipitation of S,M, X and Y potato viruses by polyethyleneglycols with different molecular weights

The minimum concentration of polyethyleneglycol (PEG) with molecular weights 4000, 6000, and 15000 necessary for precipitation of S, M, X and Y potato viruses was determined. An excessive amount of PEG causes the precipitation of other protein compounds from potato leaf cell sap. In order to obtain highly purified samples, it is necessary to use just the minimum sufficient amount of PEG. Using the minimum quantity of PEG is, also, advisable from an economical point of view. The minimum concentration of PEG of given molecular weight differs for different potato disease viruses. The concentration of PEG necessary for precipitation of a given potato virus depends on the molecular weight of PEG used—4000, 6000 and 15000. As the molecular weight increases, the concentration of PEG necessary for precipitation decreases.     

Osmoelastic coupling in biological structures: formation of parallel bundles of actin filaments in a crystalline-like structure caused by osmotic stress

A high molecular weight inert molecule, poly(ethylene glycol) (PEG), or a soluble protein, ovalbumin, causes parallel bundles of actin filaments in a crystalline-like structure under physiological conditions of ionic compositions and pH. The bundle formation depends on the molecular weight of PEG, and a larger molecular weight of PEG can make the bundle at a lower concentration. Actin bundle formation has a discrete dependence on the concentration of PEG. The light scattering following PEG-induced bundle formation increased abruptly at 4.5% (w/w) PEG 6000, while at concentrations less than or equal to 4.0% (w/w) no increase was observed. Labeling actin filaments with heavy meromyosin indicated that the polarity of the filament in the bundle is random. The PEG-induced bundle formation depends on the ionic strength of the solutions and also the concentration of the filament, showing that a higher concentration of PEG was required at lower ionic strength or a lower concentration of the filament. The results described above cannot be explained on the basis of the postulation that the direct binding of PEG molecules to the actin filaments may cause bundle formation. Alternatively, the mechanism can be explained reasonably by the theory of osmoelastic coupling based on preferential exclusion of PEG molecules from the filament surface. High molecular weight molecules such as PEG should be preferentially excluded from the region adjacent to the actin filaments (exclusion layer) by steric hindrance, thereby making imbalance of osmolarity between the bulk and the exclusion layer. This imbalance puts an osmotic stress on the actin filament.(ABSTRACT TRUNCATED AT 250 WORDS)     

alpha-Conotoxins, small peptide probes of nicotinic acetylcholine receptors

alpha-Conotoxins, a family of small peptides from the venoms of the Conus marine moluscs, are selective, snake alpha-neurotoxin-competitive antagonists of the nicotinic acetylcholine receptor. A new alpha-conotoxin, SIA, has been purified, sequenced, and synthesized. Cross-linking with bivalent reagents and photoaffinity labeling of the acetylcholine receptor with alpha-conotoxin yield covalent adducts. Surprisingly, cross-linking to other subunits is considerably more efficient than to the alpha subunit. The relative efficiency of photoactivatable cross-linking to different subunits of the receptor is a function of placement of the photoactivatable group on the toxin. Since the structures of alpha-conotoxins can be solved by 2D NMR [see Pardi et al. (1989) Biochemistry 28, 5494-5508; Kobayashi et al. (1989) Biochemistry 28, 4853-4860], this family of toxins should provide a set of new ligands for probing the acetylcholine receptor with considerable precision.     

Geotrichum sp．SYBC WU-3脂肪酶的双水相萃取和酶学性质

初步研究双水相体系对Geotrichum sp．SYBC WU-3脂肪酶的萃取分离效果,选用PEC4000／NaH2 PO4作为戍相系统进行系统研究,考察影响脂肪酶萃取的各种因素（如PEG相对分子质量及质量分数、NaH2PO4质量浓度、pH）,并采用正交实验进一步优化实验条件,确定双水相萃取体系为PEG质量分数为30％、NaH2PO4质量分数为20％、体系pH为6,在此条件下Geotrichum sp．SYBC WU-3脂肪酶经硫酸铵沉淀和双水相萃取两步纯化的纯化倍数达到最大,较Geotrichum sp．SYBC WU-3脂肪酶粗酶纯化了22倍。Geotrichum sp．SYBC WU-3脂肪酶纯酶为低温碱性脂肪酶,最适反应温度为15oC,最适pH为9．5,相对分子质量为3．58×10^4。     

A differential scanning calorimetric study of the thermal unfolding of mutant forms of phage T4 lysozyme.

In continuation of our earlier work on the effects of amino acid replacements on the thermodynamics of the thermal unfolding of T4 lysozyme [Kitamura, S., & Sturtevant, J. M. (1989) Biochemistry 28, 3788-3792; Connelly, P., Ghosaini, L., Hu, C.-Q., Kitamura, S., Tanaka, A., & Sturtevant, J. M. (1991) Biochemistry 30, 1887-1891; Hu, C.-Q., Kitamura, S., Tanaka, A., & Sturtevant, J. M. (1992) Biochemistry 31, 1643-1647], we report here a study by differential scanning calorimetry of the effects of five replacements at Ile3. Four of these replacements, those with Glu, Phe, Pro, and Thr, caused apparent destabilizations, while the replacement by Leu led to a small apparent stabilization. The largest observed destabilization (Ile3Pro) amounted to -3.0 kcal mol-1 in free energy at pH 2.00 and 38.8 degrees C (the denaturational temperature of the wild-type protein at this pH), and the largest stabilization amounted to +1.2 kcal mol-1 at pH 3.00 and 53.6 degrees C.     

Photocurable biodegradable liquid copolymers: synthesis of acrylate-end-capped trimethylene carbonate-based prepolymers, photocuring, and hydrolysis

Various photocurable liquid biodegradable trimethylene carbonate (TMC)-based (co)oligomers were prepared by ring-opening (co)polymerization of TMC with or without L-lactide (LL) using low molecular weight poly(ethylene glycol) (PEG) (mol wt 200, 600, or 1000) or trimethylolpropane (TMP) as an initiator. Resultant (co)oligomers were pastes, viscous liquids, or liquids at room temperature, depending on the monomer composition and monomer/initiator ratio. Liquid (co)oligomers were subsequently end-capped with acrylate groups. Upon visible-light irradiation in the presence of camphorquinone as a radical generator, rapid liquid-to-solid transformation occurred to produce photocured solid. The photocuring yield increased with photoirradiation time, photointensity, and camphorquinone concentration. The photocured polymers derived from low molecular weight PEG (PEG200) and TMP exhibited much reduced hydrolysis potential compared with PEG1000-derived polymers in terms of weight loss, water uptake, and swelling depth. Force-distance curve measurements by nanoindentation using atomic force microscopy clearly showed that Young's moduli of the photocured polymer films decreased with increasing hydrolysis time. Their potential biomedical applications are discussed.     

Polymer fractionation in aqueous two-phase polymer systems.

We consider the effects of the addition of poly(ethylene glycol) (PEG) of different molecular weights to aqueous two-phase system of PEG 8000 and dextran 500. The first purpose of this study was to determine the molecular weight partitioning of the polymers themselves so that, for example, aqueous two-phase separations using affinity ligands can be improved. The second purpose was to examine whether this molecular weight partitioning could be predicted by using solution thermodynamic models so that it would be possible to optimize affinity partitioning without extensive laboratory work. Experimentally, we find that, by increasing the PEG concentration of any molecular weight in the feed, the high molecular weight PEG concentration in the dextran-rich phase is reduced. This observation can be used to reduce the loss of expensive ligated PEG used in affinity partitioning. Further, there is generally good agreement between our experimental data and the predictions of a solution thermodynamic model.     

Osmoelastic coupling in biological structures: decrease in membrane fluidity and osmophobic association of phospholipid vesicles in response to osmotic stress

Poly(ethylene glycol)- (PEG-) induced change in membrane fluidity and aggregation of phospholipid vesicles were studied. A threshold concentration of PEG was required to induce the aggregation. This concentration increased with a decrease in the molecular weight of PEG, e.g., from 5% (w/w) with PEG 6000 (PEG with an average molecular weight of 7500) to more than 30% (w/w) with PEG 200. The aggregation was reversible upon dilution of PEG if the initial PEG concentration was smaller than a certain value, e.g., 22% (w/w) for PEG 6000. Addition of PEG caused a decrease in membrane fluidity of the vesicles detected by fluorescence anisotropy of diphenylhexatriene and by electron spin resonance of a spin-labeled fatty acid. The anisotropy change of diphenylhexatriene fluidity change had an inflection point at approximately 5% (w/w) of PEG 6000, which might suggest that the aggregation would make the decrease of membrane fluidity smaller. Transfer of lipid molecules between phospholipid vesicles was enhanced by the PEG-induced aggregation. The enhancement occurred not only upon direct addition of PEG to the suspending medium, but also upon dialysis of the vesicle suspension against a high concentration of PEG. All these features are consistent with osmoelastic coupling in the phospholipid membranes and the subsequent osmophobic association of the vesicles. The imbalance of osmolarity between the region adjacent to the vesicle surface (exclusion layer) and the bulk aqueous phase, which results from the preferential exclusion of PEG from the exclusion layer in the case of direct addition of PEG, exerts an osmotic stress on the vesicles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanism of photoinhibition of photosynthetic water oxidation by Cl- depletion and F- substitution: oxidation of a protein residue

New evidence on the chloride requirement for photosynthetic O2 evolution has indicated that Cl- facilitates oxidation of the manganese cluster by the photosystem II (PSII) Tyr-Z+ radical. Illumination above 250 K of spinach PSII centers which are inhibited in O2 evolution by either Cl- depletion or F- substitution produces a new EPR signal which has magnetic characteristics similar to one recently discovered in samples inhibited by depletion of Ca2+ only [Boussac et al. (1989) Biochemistry 28, 8984; Sivaraja et al. (1989) Biochemistry 28, 9459]. The physiological roles of Cl- and Ca2+ in water oxidation are thus linked. The characteristics include a nearly isotropic g = 2.00 +/- 0.005, a symmetric line shape with line width = 16 +/- 2 mT, almost stoichiometric spin concentration relative to Try-D+ = 0.6 +/- 0.3 spin/PSII, very rapid spin relaxation at all temperatures measured down to 6 K, and an undetectable change in magnetic susceptibility upon formation (less than 1 mu B2). The signal appears to originate from a spin doublet (radical) in magnetic dipolar contact with a transition-metal ion, most probably a photooxidized protein residue within 10 A of the Mn cluster (Mn-proximal radical). It is distinct from the three other protein-bound radical-type electron donors found in the PSII reaction center: Tyr-D+, Tyr-Z+, and C+. This signal photoaccumulates to a stable level under continuous illumination at 270 K and decays only after illumination stops.(ABSTRACT TRUNCATED AT 250 WORDS)     

One of the major sulphated proteins secreted by rat hepatocytes contains low-sulphated chondroitin sulphate.

When isolated hepatocytes are incubated with 35SO4(2-), a specific set of secretory proteins is labelled. One of these proteins is electrophoretically heterogeneous, with an apparent molecular mass of 35-45 kDa [Marcks von Würtemberg & Fries (1989) Biochemistry 28, 4088-4093]. Here we report that treatment with chondroitinase ABC converted the broad electrophoretic band of this protein, with a 50-60% loss of radioactivity, into a relatively homogeneous band with a molecular mass of 28 kDa. Size determination by gel chromatography of the protein's oligosaccharide chain (released by alkali treatment) indicated that it contained about 40 hexose units. Similar analysis of the enzyme-resistant oligosaccharide chain remaining linked to the protein after chondroitinase ABC treatment indicated a size of between six and eight hexose units. These observations suggest that the protein's oligosaccharide chain carries only three or four sulphate groups, of which one or two are located close to the polypeptide chain. Consistent with this hypothesis, the free oligosaccharide behaved like a low-sulphated glycosaminoglycan upon ion-exchange chromatography.     

Interaction of poly(ethylene-glycols) with air-water interfaces and lipid monolayers: investigations on surface pressure and surface potential.

We have characterized the surface activity of different-sized poly(ethylene-glycols) (PEG; M(r) 200-100,000 Da) in the presence or absence of lipid monolayers and over a wide range of bulk PEG concentrations (10(-8)-10% w/v). Measurements of the surface potential and surface pressure demonstrate that PEGs interact with the air-water and lipid-water interfaces. Without lipid, PEG added either to the subphase or to the air-water interface forms relatively stable monolayers. Except for very low molecular weight polymers (PEGs < 1000 Da), low concentrations of PEG in the subphase (between 10(-5) and 10(-4)% w/v) increase the surface potential from zero (with respect to the potential of a pure air-water interface) to a plateau value of approximately 440 mV. At much higher polymer concentrations, > 10(-1)% (w/v), depending on the molecular weight of the PEG and corresponding to the concentration at which the polymers in solution are likely to overlap, the surface potential decreases. High concentrations of PEG in the subphase cause a similar decrease in the surface potential of densely packed lipid monolayers spread from either diphytanoyl phosphatidylcholine (DPhPC), dipalmitoyl phosphatidylcholine (DPPC), or dioleoyl phosphatidylserine (DOPS). Adding PEG as a monolayer at the air-water interface also affects the surface activity of DPhPC or DPPC monolayers. At low lipid concentration, the surface pressure and potential are determined by the polymer. For intermediate lipid concentrations, the surface pressure-area and surface potential-area isotherms show that the effects due to lipid and PEG are not always additive and that the polymer's effect is distinct for the two lipids. When PEG-lipid-mixed monolayers are compressed to surface pressures greater than the collapse pressure for a PEG monolayer, the surface pressure-area and surface potential-area isotherms approach that of the lipid alone, suggesting that for this experimental condition PEG is expelled from the interface.