Action of polyene antibiotics on ribosome binding with Candida albicans membranes]

The effect of treatment of the protoplasts and cell membranes of C. albicans with polyenic entibiotics on formation of the ribosomal-membrane complex was studied in vitro. It was shown that amphotericin B and nystatin had no effect on this process. Significant suppression of the ribosome binding with the membranes was observed only in vivo in the presence of levorin. The role of the structural changes occurring in the membranes on formation of the polyen-sterol complex, as well as the role of the lipid components of the membrane in binding of the ribosomes is discussed.     

Transbilayer organization of membrane cholesterol at low concentrations: Implications in health and disease

Cholesterol is an essential and representative lipid in higher eukaryotic cellular membranes and is often found distributed nonrandomly in domains in biological membranes. A large body of literature exists on the organization of cholesterol in plasma membranes or membranes with high cholesterol content. However, very little is known about organization of cholesterol in membranes containing low amounts of cholesterol such as the endoplasmic reticulum or inner mitochondrial membranes. In this review, we have traced the discovery and subsequent development of the concept of transbilayer cholesterol dimers (domains) in membranes at low concentrations. We have further discussed the role of membrane curvature and thickness on the transbilayer organization of cholesterol. Interestingly, this type of cholesterol organization could be relevant in cellular sorting and trafficking, and in pathological conditions.     

Fusion in phospholipid spherical membranes. I. Effect of temperature and lysolecithin.

A study concerning membrane contact and fusion phenomena was made for phospholipid spherical bilayer systems with respect to temperature. Specific temperatures were obtained for the spherical bilayer membranes of phosphatidyl choline (PC) and phosphatidyl serine (PS) which indicated a greater degree of membrane fusion and were designated Tf (the fusion temperature -- PC: 43 degrees C, PS: 38 degrees C). These temperatures were reduced by about 10 degrees C for the membranes incorporated with 20% lysophosphatidyl choline. The results of the contact and fusion observed in the spherical membranes are compared and discussed with the conductance characteristics of the PC and PS planar bilayer membranes as well as dissolution study on the phospholipid monolayers formed at the air/water interface with respect to temperature. Also, a possible molecular mechanism of membrane fusion is discussed in terms of the fluidity and instability of the membrane.     

Structure and Function of Chloroplast Membrane XI. The Effects of Linolenic Acid on the Structure and the Absorption and Fluorescence Spectra of Wheat Chloroplast Membranes as well as Regulations by MgCl2

The effects of various concentrations of linolenic acid on the structure and the absorption and fluorescence spectra of wheat chloroplast membranes were studied. Linolenic acid increases the absorption peaks in both red and blue regions of the chloroplast membranes. The degree of increase in the absorption peaks is proportional to the concentration within a range of concentration. Linolenic acid increased the fluorescence yield of F685 and F738 of chloroplast membranes. Electron microscopical studies revealed that the increases were mainly due to the disappearance of grana stacks and the unfolding of thylakoid membranes. The causes of effects of linolenic acid on the absorption and fluorescence spectra and the structure of chloroplast membranes as well as the reversion and regulation by MgCl2 were discussed.     

Influence of the microenvironment on the activity of enzymes immobilized on Teflon membranes grafted by γ-radiation

The effect of the microenvironment and immobilization method on the activity of immobilized β-galactosidase was investigated. Immobilization was done on Teflon membranes grafted with different acrylic monomers by γ-radiation and activated by two different coupling agents through the functional groups of the grafted monomers. 2-Hydroxyethyl methacrylate (HEMA) and methacrylic acid (MAA) were grafted on the membrane, and 1,6-hexamethylenediamine (HMDA) was used as a spacer. Glutaraldehyde or cyanuric chloride were used as coupling agents to bind the enzyme to the membrane. Four different catalytic membranes were obtained using the same solid support. Direct comparison between the isothermal behaviour of the biocatalyst in its free and immobilized form was carried out. In particular the dependence of the isothermal activity on the temperature and pH was studied and the kinetic parameters determined. The influence of the microenvironment on the observed activity of the four membranes was evidenced and discussed. The way of improving the yield of these catalytic membranes is discussed also.     

William Trager: An Appreciation

SYNOPSIS. Additional information on host interactions with trypanosomatid membranes was obtained from studies of a monomorphic strain of Trypanosoma brucei harvested at peak parasitemia from intact and lethally irradiated rats. Pellets of trypanosomes were fixed briefly in glutaraldehyde and processed for thin section electron microscopy or freeze-cleave replicas. Observations of sectioned material facilitated orientation and comparison of details seen in replicas. Fracture faces of cell body and flagellar membranes as well as 3-dimensional views of the nuclear membrane were studied. Cell body membranes of 80% of the organisms from intact rats contained random arrays of intramembranous particles (IMP). Aggregated clusters of particles appeared on the fracture faces of 20% of the trypanosomes. Some of these membranes had nonrandomly distributed particles aligned in distinct rows on the outer fracture face of both cell body and flagellum. Many inner face fractures of the cell body membranes had a particle arrangement similar to the longitudinal alignment of cytoskeletal microtubules. No aggregated particle distribution was seen in membranes of trypanosomes harvested from lethally irradiated rats. Replicas of trypanosome pellets also had plasmanemes as a series of attached, empty, coated membrane vesicles. These structures were found in close association with, as well as widely separated from the parasites. The shedding of these vesicles and the variation of particles in cell body membranes are discussed in light of antibody-induced architectural and antigenic changes in surface properties of trypanosomatids. The convex face of the inner membrane of the nucleus also is covered with randomly arrayed particles. More IMP were observed on the inner than on the outer nuclear membranes. Images of nuclear pores were also seen. The importance of these structures in drug and developmental studies of trypanosomes is discussed. On fracture faces of the flagellar membrane there were miniature maculae adherentes, unique to the inner fracture face and occurring only at regions of membrane apposition between cell body and flagellum. Each cluster of particles exposed by the freeze-cleave method corresponds to an electron-dense plaque seen in thin section images. However, because of a unique fracture pattern, these plaques were not revealed on the apposing body membranes, as illustrated in thin sectioned organisms.     

Mechanosensitivity of cell membranes

Physical and biophysical mechanisms of mechano-sensitivity of cell membranes are reviewed. The possible roles of the lipid matrix and of the cytoskeleton in membrane mechanoreception are discussed. Techniques for generation of static strains and dynamic curvatures of membrane patches are considered. A unified model for stress-activated and stress-inactivated ion channels under static strains is described. A review of work on stress-sensitive pores in lipid-peptide model membranes is presented. The possible role of flexoelectricity in mechano-electric transduction, e.g. in auditory receptors is discussed. Studies of flexoelectricity in model lipid membranes, lipid-peptide membranes and natural membranes containing ion channels are reviewed. Finally, possible applications in molecular electronics of mechanosensors employing some of the recognized principles of mechano-electric transduction in natural membranes are discussed.Abbreviations BLM Layer lipid membrane - SAC stress-activated channel - SIC stress-inactivated channel - MCYST microcystin-LR - DPhL diphytanoyl lecithin - CME condenser microphone effect Dedicated to Professor Alexander Derzhanski on the occasion of his 60th birthday Correspondence to: A. G. Petrov     

Characterization, enzymatic and lectin properties of isolated membranes from Phaseolus aureus.

Cellular membranes were prepared from the non-extending part of dark grown hypocotyls of Phaseolus aureus. The relative effectiveness of continuous and discontinuous sucrose gradient centrifugation for the separation of membranes was investigated. Characteristic densities of membranes were determined by the localization of enzyme activities on continuous sucrose gradients: NADH-cytochrome c-reductase for endoplasmic reticulum, beta-1-3-glucan synthetase for plasma-membrane and IDPase for dictyosomes. The difficulties involved in the application of ATPase and IDPase as specific membrane markers are discussed. Negative staining of isolated fractions indicated that intact dictyosomes could be prepared from this tissue without the use of chemical fixatives in the homogenization medium. Extraction of isolated membranes showed that carbohydrate-binding proteins (lectins) were present both in an easily removable and in a more strongly bound form. In vivo incorporation of D-[U-14C]glucose and subsequent isolation and solubilization of the different membranes showed that sugar-containing polymers could be released without hydrolytic techniques and were present in the equivalent extracts that exhibited lectin activity. The possibility of lectin-polysaccharide complexes in endoplasmic reticulum and dictyosomes and their involvement in the synthesis and transport of secretory substances by the membranes is discussed.     

Production of low-lactose milk by means of nonisothermal bioreactors

The effect of the immobilization time on the activity of immobilized beta-galactosidase from K. lactis was investigated. Six biocatalytic membranes, different only for the time of the enzyme immobilization, were obtained by using nylon membranes grafted with glycidyl methacrylate (GMA) and activated by hexamethylenediamine (HMDA) and glutaraldehyde (Glu), used as spacer and coupling agent, respectively. Comparison between the isothermal and nonisothermal yield of these biocatalytic membranes was carried out in the process of lactose hydrolysis in milk. All of the results, reported as a function of the immobilization time, have evidenced the influence of our variable parameter on the activity of the catalytic membranes. The membrane giving highest yield under isothermal and nonisothermal conditions was that obtained with 2 h of immobilization time. The industrial application of these membranes has been discussed in terms of percentage reduction of the production times.     

A polysome-membrane binding system from rat liver. I. Basic characterization of the binding system.

A simple reaction system was developed to examine the binding of polysomes to membranes of the endoplasmic reticulum and to investigate the fate of ribosomes and nascent chains during protein synthesis in vitro. The system conssited of Sephadex G-25 treated post-mitochondrial fraction prepared from rat liver (Sephadex-PM) as a source of membranes, and radioactive free polysomes prepared from another rat liver. The following results were obtained. 1. Nascent chains on free polysomes labeled in vivo were transferred to membranes in vitro. The process required protein synthesis. 2. This reaction occurred in two steps: a) Binding of the free polysomes to membranes in the absence of protein synthesis. b) Release of ribosomes, leaving nascent chains on the membranes, requiring protein syntehsis. 3. A portion of the ribosomes found on membranes in vivi (membrane-bound ribosomes) was also released from the membranes during incubation in vitro, leaving their nascent chains on the membranes. The significance of the transfer of nascent chains from free polysomes to membranes in vitro is discussed in the light of known polysome-membrane interaction in vivo.     

Spin label reduction kinetics,a procedure to study the effect of drugs on membrane permeability: The effects of monosodium urate,dimethyl sulfoxide and amphotericin B

A method is presented to study the effect of drugs on membrane permeability. It is based on the reduction of a spin label trapped in the internal aqueous compartment(s) of membranes by ascorbate ions added to the bulk aqueous phase. The decay of the electron spin resonance signal of the spin label as a function of time gives an indication of the effect of added agents on the permeability of membranes. To demonstrate the technique, the effect on model membranes of egg phosphatidylcholine of the gout-implicated compound monosodium urate, the aprotic solvent dimethyl sulfoxide and the polyene antibiotic amphotericin B were examined. Monosodium urate did not affect the permeability, casting doubt on a proposed mechanism whereby the agent disrupts the membranes via hydrogen bonding. Dimethyl sulfoxide promoted a gradual increase in rate of solute passage across cholesterol-containing model membranes. Amphotericin B had pronounced effect on the permeability of cholesterol-containing membranes, causing nearly total loss of paramagnetism immediately after addition. Some aspects of the mechanism of action of the drugs are discussed as well as the advantages and disadvantages of the method. The experiments also allow the evaluation of the effect of surface charge and cholesterol on the dimensions of model membranes.     

Dehydration of an ethanol/water azeotrope by novel organic-inorganic hybrid membranes based on quaternized chitosan and tetraethoxysilane

To control swelling of quaternized chitosan (q-Chito) membranes, mixtures of q-Chito as an organic component and tetraethoxysilane (TEOS) as an inorganic component were prepared using the sol-gel reaction, and novel q-Chito/TEOS hybrid membranes were formed. In the separation of an ethanol/water azeotrope by pervaporation, the effect of TEOS content on the water/ethanol selectivity of q-Chito/TEOS hybrid membranes was investigated. Hybrid membranes containing up to 45 mol % TEOS exhibited higher water/ethanol selectivity than the q-Chito membrane. This resulted from depressed swelling of the membranes by formation of a cross-linked structure. However, introduction of excess TEOS led to greater swelling of the hybrid membranes. Therefore, the water/ethanol selectivity of the hybrid membranes containing more than 45 mol % TEOS was lower than that of the q-Chito membrane. The relationship between the structure of q-Chito/TEOS hybrid membranes and their permeation and separation characteristics during pervaporation of an ethanol/water azeotrope is discussed in detail.     

Protein composition of cell and forespore membranes of Bacillus subtilis

A sporulation mutant of Bacillus subtilis 168 was isolated and characterized. The mutant, designated SB-23, releases viable forespores at the end of the developmental period. Forespores were isolated on linear Renografin gradients and used as a source of forespore membranes. The protein composition of forespore membranes was found to differ from the protein composition of vegetative cell membranes by discgel electrophoresis. The results are discussed in relationship to morphological and physiological differentiation during bacterial sporulation.     

Rhodopsin and other proteins in artificial lipid membranes

Some basic aspects of incorporation of hydrophobic peptides and proteins in artificial lipid membranes are discussed. As examples valinomycin as a carrier model and gramicidin A as a channel former in lipid vesicles and in planar lipid membranes are presented.In the second part of the lecture some examples of incorporation of membrane proteins into lipid vesicles and planar lipid membranes are reported. The interaction with artificial lipid membranes of the Ca⁺ ATPase from the sarcoplasmic reticulum, of Rhodopsin, and of Bacteriorhodopsin is presented.Presented at the EMBO-Workshop on Transduction Mechanism of Photoreceptors, Jülich, Germany, October 4–8, 1976     

ESR studies on the effect of cholesterol on chlorpromazine interaction with saturated and unsaturated liposome membranes

In this study, the effects of chlorpromazine (CPZ) on lipid order and motion in saturated (DMPC, DMPG) and unsaturated (SOPC) liposome membranes were investigated by electron spin resonance (ESR) spin labeling technique. We have shown that above the main phase transition temperature of membrane lipids (T(M)), CPZ slightly increases lipid order in membranes without cholesterol, whereas below T(M) it has a strong opposite effect. Addition of 30 mol% of cholesterol into DMPC and SOPC membranes changes significantly the CPZ effects both above and below T(M). Additionally, above T(M), the ordering effect of CPZ on pure SOPC membrane is stronger at pH 7.4 than at pH 9.0, whereas below T(M), as well as in the presence of cholesterol, pH does not seem to play a role in CPZ effect on both membranes. Because of the strong influence of membrane composition on CPZ effect on membranes, the use of cholesterol as a marker of CPZ photosensitized reactions has been discussed.     

Studies with Composite Membranes: I. Preparation and Measurement of Impedances

Simple and composite membranes have been prepared from 2% collodion solutions containing different amounts of polystyrenesulfonic acid (PSSA). Various membrane parameters such as water content, electrolyte uptake, exchange capacity, and permselectivity of these membranes have been determined. The resistance and capacitance of simple membranes have been measured as functions of both external electrolyte concentration and internal fixed charge density. The impedance characteristics of composite membranes also have been determined and discussed in terms of the resistance and capacitance characteristics of simple membranes from which the composite structures have been formed.     

The computer as a laboratory for the physical chemistry of membranes

A mini-review is given of some recent advances in the use of computer-simulation approaches to the study of physico-chemical properties of lipid bilayers and biological membranes. The simulations are based on microscopic molecular interaction models as well as random-surface models of fluid membranes. Particular emphasis is put on those properties that are controlled by the many-particle character of the lamellar membrane, i.e. correlations and fluctuations in density, composition and large-scale conformational structure. It is discussed how dynamic membrane heterogeneity arises and how it is affected by various molecular species interacting with membranes, such as cholesterol, drugs, insecticides, as well as polypeptides and integral membrane proteins. The influence of bending rigidity and osmotic-pressure gradients on large-scale membrane conformation and topology is described.     

Rhodopsin and other proteins in artificial lipid membranes.

Some basic aspects of incorporation of hydrophobic peptides and proteins in artificial lipid membranes are discussed. As examples valinomycin as a carrier model and gramicidin A as a channel former in lipid vesicles and in planar lipid membranes are presented. In the second part of the lecture some examples of incorporation of membrane proteins into lipid vesicles and planar lipid membranes are reported. The interaction with artificial lipid membranes of the Ca++ ATPase from the sarcoplasmic reticulum, of Rhodopsin, and of Bacteriorhodopsin is presented.     

Mass spectrometric characterization of proteins transferred from polyacrylamide gels to membrane filters

In the 1990s, a technique was developed to transfer proteins from electrophoresis gels onto poly(vinylidene difluoride) (PVDF) membranes, digest the proteins on the membranes with proteases such as trypsin and analyze the resulting peptides on the membranes directly by mass spectrometry to identify the proteins. This technique, based on gel electrophoresis, is particularly useful for analyzing protein isoforms, splicing variants and post-translationally modified proteins. Previously, the low ionization efficiency of peptides immobilized on the membranes often rendered this technique useless. However, this technique has been improved by the use of PVDF membranes with a small pore size, which has enabled highly efficient and effective electroblotting and mass spectrometric analyses. Here, the advantage of this technique is discussed.     

山莨菪碱对人红细胞膜蛋白及膜脂运动的影响

本文采用自旋标记顺磁共振波谱技术,研究了山茛菪碱对人红细胞膜蛋白和膜脂运动的影响.结果表明:用马来酰亚胺标记的人红细胞膜,加入山茛菪碱后,其顺磁共振波谱中强、弱固定化作用谱的峰值比增大,膜蛋白的运动受到限制.山茛菪碱对红细胞膜脂的作用部位主要在极性头部,并影响膜脂的流动性.本文还对山茛菪碱与红细胞膜作用的可能机制进行了讨论.