Qualitative difference between “bulb” membranes and other vacuolar membranes

“Bulb” is a mobile and complex structure appearing in vacuolar membrane of plant cell. We recently reported new fluorescent marker lines for bulbs and bulb-less mutants. We tried multicolor visualization of vacuolar membrane to show distinct segregation of bulb-positive protein (γTIP or AtVAM3) and bulb-negative protein (AtRab75). Unexpectedly, GFP-AtRab75 resulted to localize in bulb under the condition of co-expression with TagRFP-AtVAM3. The signal intensities of GFP-AtRab75 and TagRFP-AtVAM3 were quantified and compared. The result indicates that TagRFP-AtVAM3 is concentrated in bulb than GFP-AtRab75.     

Comparison of protein and lipid composition of the human platelet α-granule membranes and glycerol lysis membranes

Platelet glycerol lysis membranes and α-granule membranes were compared with respect to protein and lipid composition. Crossed immunoelectrophoresis using antibodies against whole platelets, and sodium dodecyl sulphate polyacrylamide gel electrophoresis, revealed the presence of the glycoproteins IIb and IIIa, myosin and an antigen termed G4 in both membrane fractions. The glycoproteins Ia, Ib and IIIb, in addition to β₂-microglobulin and actin, appeared specific for the glycerol lysis membranes, whereas two antigens, termed G8 and G18, were observed only in the α-granule membranes. The localization of glycoprotein IIa was inconclusive. Comparison with the surface-located proteins revealed that the glycerol lysis membranes represented a reasonable approximation to a plasma membrane preparation. Radioactively labelled immunoprecipitates obtained after crossed immunoelectrophoresis of ¹²⁵I-labelled platelets were cut out and applied to sodium dodecyl sulphate electrophoresis on polyacrylamide slab gels. Autoradiography of the dried gels revealed that antigen G4 represented a protein with an average molecular weight of 146 000 in its unreduced state and 132 000 in its reduced state. Antigen G18 represented a protein of molecular weight 130 000–135 000 in the reduced as well as unreduced state. Quantitation of protein and lipids showed that the α-granule membranes contained about one-third as much cholesterol and 2-times as much protein in relation to phospholipids as compared to the glycerol lysis membranes. No significant difference between the two membrane preparations was found as regards the composition of their phospholipids.     

5′-Nucleotidases of retinal rod membranes

5′-Nucleotidase (EC 3.1.3.5) was solubilized from rod membranes with Ammonyx LO and purified by chromatographic methods. A highly sensitive radioassay was developed. The purified enzyme behaved as a homogeneous protein of 75,000 daltons in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and as a protein of 79,000 in gel filtration. Thus, the enzyme does not contain subunits. The K_m values obtained were 1.3 μm for 5′-AMP and 2.3 μm for 5′-GMP. The enzyme was inhibited by concanavalin A, wheat germ agglutinin, and Ricinus communis agglutinin. Rabbit muscle G-actin formed a complex with the enzyme and inhibited its activity. The catalytic site of the enzyme was localized on the internal surface of the disk which, in terms of membrane sidedness, corresponds to the cell surface. A soluble 5′-nucleotidase was extracted from rod membranes with Tris buffer (pH 8.0) containing EGTA in the dark; less enzyme was extracted if the membranes had been exposed to light or incubated with Ca²⁺. The extracted enzyme was partially purified. The enzyme was unstable and lost 50% of its activity in 3 days at 3 °C. The K_m values were 1.3 μm for 5′-AMP and 2.3 μm for 5′-GMP. The enzyme was inhibited by G-actin. A role for the soluble enzyme in the regulation of 5′-GMP in the rod outer segment was suggested.     

Adsorption of γ-Aminobutyric acid to phosphatidylserine membranes

The interaction of the negatively-charged phosphatidylserine (PS) and γ-Aminobutyric acid (GABA) is examined in black lipid membranes (BLM) and inverse micelles. GABA does not permeate through PS membranes and, in concentrations of 10^?5-10^?4 M, it reduces the negative potential at the membrane-aqueous solution interface. The effect is owing to the adsorption of the GABA cationic species and the consequent decrease of the negative surface charge density of the membrane. When the intrinsic pH of the membrane-solution interface is considered, the Gouy-Chapman-Stern theory describes the GABA screening effect and makes it possible to calculate the GABA-PS binding constant. This value is compared with that obtained measuring the partition of¹⁴C-GABA between an organic phase containing PS and the aqueous solution. The results presented strongly suggest that the electrostatic force plays a major role in GABA-PS interaction.     

Is a fluid-mosaic model of biological membranes fully relevant? Studies on lipid organization in model and biological membranes

The basic concept of the fluid-mosaic model of Singer and Nicolson, an essential point of which is that the membrane proteins are floating in a sea of excess lipid molecules organized in the lipid bilayer, may be misleading in understanding the movement of membrane components in biological membranes that show distinct domain structure. It seems that the lipid bilayer is an active factor in forming the membrane structure, and the lipid composition is responsible for the presence of domains in the membrane. The main role in the process of domain formation is played by cholesterol and sphingolipids. The results presented here show that in a binary mixture of cholesterol and unsaturated phospholipids, cholesterol is segregated out from the bulk unsaturated liquid-crystalline phase. This forms cholesterol-enriched domains or clustered cholesterol domains due to the lateral nonconformability between the rigid planar ring structure of cholesterol and the rigid bend of the unsaturated alkyl chain at double bond position. These cholesterol-enriched domains may be stabilized by the presence of saturated alkyl chains of sphingomyelin or glycosphingolipids, and also by specific proteins which selectively locate in these domains and stabilize them as a result of protein-protein interaction. Such lipid domains are called "rafts" and have been shown to be responsible both for signal transduction to and from the cell and for protein sorting. We also looked at whether polar carotenoids, compounds showing some similarities to cholesterol and affecting membrane properties in a similar way, would also promote domain formation and locate preferentially in one of the lipid phases. Our preliminary data show that in the presence of cholesterol, lutein (a polar carotenoid) may segregate out from saturated lipid regions (liquid-ordered phase) and accumulate in the regions rich in unsaturated phospholipids forming carotenoid-rich domains there. Conventional and pulse EPR (electron paramagnetic resonance) spin labeling techniques were employed to assess the molecular organization and dynamics of the raft-constituent molecules and of the raft itself in the membrane.     

Interactions between amyloid β peptide and lipid membranes

The presence of amyloid plaques in the brain is a typical characteristic of Alzheimer's disease (AD). Amyloid plaques are formed from the deposits of aggregated amyloid β peptide (Aβ). The toxicity induced by Aβ aggregates is correlated with Aβ-membrane interactions. The mutual influences between aggregation and membranes are complicated and unclear. In recent years advanced experiments and findings are emerging to give us more detailed information on Aβ-membrane interactions. In this review, we mainly focus on the Aβ-membrane interactions and membrane-induced Aβ structures. The mechanism of Aβ-membrane interactions is also summarized, which provides insights into the prevention and treatment of AD.     

Alzheimer Aβ peptide interactions with lipid membranes

Fibrillar aggregates of misfolded amyloid proteins are involved in a variety of diseases such as Alzheimer disease (AD), type 2 diabetes, Parkinson, Huntington and prion-related diseases. In the case of AD amyloid β (Aβ) peptides, the toxicity of amyloid oligomers and larger fibrillar aggregates is related to perturbing the biological function of the adjacent cellular membrane. We used atomistic molecular dynamics (MD) simulations of Aβ_9–40 fibrillar oligomers modeled as protofilament segments, including lipid bilayers and explicit water molecules, to probe the first steps in the mechanism of Aβ-membrane interactions. Our study identified the electrostatic interaction between charged peptide residues and the lipid headgroups as the principal driving force that can modulate the further penetration of the C-termini of amyloid fibrils or fibrillar oligomers into the hydrophobic region of lipid membranes. These findings advance our understanding of the detailed molecular mechanisms and the effects related to Aβ-membrane interactions, and suggest a polymorphic structural character of amyloid ion channels embedded in lipid bilayers. While inter-peptide hydrogen bonds leading to the formation of β-strands may still play a stabilizing role in amyloid channel structures, these may also present a significant helical content in peptide regions (e.g., termini) that are subject to direct interactions with lipids rather than with neighboring Aβ peptides.     

Membrane pores—From biology to track-etched membranes

Flow of ions through narrow pores, either induced in biological membranes or created in synthetic membrane filters, exhibits, under appropriate conditions: 1) rapid switching of ion current between high and low conducting states; 2) selectivity between different ions; 3) inhibition by protons or divalent cations with an order of efficacy usually H⁺ >Zn²⁺>Ca²⁺ >Mg²⁺. It seems reasonable to conclude that these common properties arise from a common cause-the nature of the flow of ions close to a charged surface.     

Immobilization of β-galactosidase on modified polypropilene membranes

A new immobilized system: β-galactosidase-modified polypropylene membrane was created. It was obtained 13 different carriers by chemical modification of polypropylene membranes by two stages. The first stage is treatment with K(2)Cr(2)O(7) to receive carboxylic groups on membrane surface. The second stage is treatment with different modified agents ethylendiamine, hexamethylenediamine, hydrazine dihydrochloride, hydroxylamine, o-phenylenediamine, p-phenylenediamine, N,N'-dibenzyl ethylenediamine diacetate to receive amino groups. The quantity of the amino groups, carboxylic groups and the degree of hydrophilicity of unmodified and modified polypropilene membranes were determined. β-Galactosidase was chemically immobilized on the obtained carries by glutaraldehyde. The highest relative activity of immobilized enzyme was recorded at membrane modified with 10% hexamethylenediamine (Membrane 5) - 92.77%. The properties of immobilized β-galactosidase on different modified membranes - pH optimum, temperature optimum, pH stability and thermal stability were investigated and compared with those of free enzyme. The storage stability of all immobilized systems was studied. It was found that the most stable system is immobilized enzyme on Membrane 5. The system has kept 90% of its initial activity at 300th day (pH=6.8; 4°C). The stability of the free and immobilized β-galactosidase on the modified membrane 5 with 10% HMDA in aqueous solutions of alcohols - mono-, diol and triol was studied. The kinetics of enzymatic reaction of free and immobilized β-galactosidase on the modified membrane 5 at 20°C and 40°C and at the optimal pH for both forms of the enzyme were investigated. It was concluded that the modified agent - hexamethylenediamine, with long aliphatic chain ensures the best immobilized β-galactosidase system.     

Effect ofγ-irradiated carbohydrates on isolated synaptic membranes

The effect of-irradiated solutions of carbohydrates, mainly glucose, upon Na⁺, K⁺-ATPase and lipid peroxidation in rat brain synaptosomal membranes was studied. The membrane damage by irradiated glucose was enhanced in the presence of Fe²⁺ and was diminished when a free-radical scavenger (BHT) or metal chelators (EDTA, EGTA) were present. It is suggested that a key element in the free-radical membrane damage by irradiated carbohydrates is an Fe²⁺-complex of some species of the radiolysis products. Participation of radiotoxins of carbohydrate origin in radiobiological effects is discussed.     

Assembly of β-barrel proteins into bacterial outer membranes

Membrane proteins with a β-barrel topology are found in the outer membranes of Gram-negative bacteria and in the plastids and mitochondria of eukaryotic cells. The assembly of these membrane proteins depends on a protein folding reaction (to create the barrel) and an insertion reaction (to integrate the barrel within the outer membrane). Experimental approaches using biophysics and biochemistry are detailing the steps in the assembly pathway, while genetics and bioinformatics have revealed a sophisticated production line of cellular components that catalyze the assembly pathway in vivo. This includes the modular BAM complex, several molecular chaperones and the translocation and assembly module (the TAM). Recent screens also suggest that further components of the pathway might remain to be discovered. We review what is known about the process of β-barrel protein assembly into membranes, and the components of the β-barrel assembly machinery. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.     

Does VDAC insert into membranes in random orientation?

It is widely accepted that voltage-dependent anion-selective channel (VDAC) inserts into planar lipid bilayers in a random orientation. This is in contrast to the well-documented oriented insertion of various channel-forming proteins. Because of the potential importance of this issue, we have examined the orientation of VDAC inserted in membranes. The time constants of the VDAC-current relaxation in response to applied positive and negative voltage pulses were used to characterize the channel orientation.We have found that VDAC channels can be separated into two groups according to differences in the time constant ratio. The difference in time constant ratio between the two main groups of VDAC channels was quantitative, and not qualitative as would be expected for opposite topologies. This finding allows us to hypothesize that both groups of VDAC channels possess a qualitatively similar asymmetry with respect to the localization of voltage-gated domains and, consequently, with respect to its entire molecular structure. The probability of having each type of VDAC channel conformation is predetermined by the protein structure in aqueous solution.A striking resemblance between asymmetry in voltage sensitivity at the single-channel and multi-channel levels was also demonstrated. The first inserted channel seems to direct subsequent insertions of channels with a similar conformation.     

Dimeric structures of α-synuclein bind preferentially to lipid membranes

There is substantial evidence which implicates α-synuclein and its ability to aggregate and bind vesicle membranes as critical factors in the development of Parkinson's disease. In order to investigate the interaction between α-synuclein wild type (Wt) and its familial mutants, A53T and A30P with lipid membranes, we developed a novel lipid binding assay using surface enhanced laser desorption/ionisation-time of flight-mass spectrometry (SELDI-TOF MS). Wt and A53T exhibited similar lipid binding profiles; monomeric species and dimers bound with high relative affinity to the lipid surface, the latter of which exhibited preferential binding. Wt and A53T trimers and tetramers were also detected on the lipid surface. A30P exhibited a unique lipid binding profile; monomeric A30P bound with a low relative affinity, however, the dimeric species of A30P exhibited a higher binding ability. Larger order A30P oligomers were not detected on the lipid surface. Tapping mode atomic force microscopy (AFM) imaging was conducted to further examine the α-synuclein-lipid interaction. AFM analysis revealed Wt and its familial mutants can penetrate lipid membranes or disrupt the lipid and bind the hydrophobic alkyl self-assembled monolayer (SAM) used to form the lipid layer. The profile of these studied proteins revealed the presence of ‘small features’ consistent with the presence of monomeric and dimeric forms of the protein. These data collectively indicate that the dimeric species of Wt and its mutants can bind and cause membrane perturbations.     

Incorporation of lipids into cellular membranes—A spin-label assay

A spin-label method is described for the quantitative assay of lipid incorporation into biological membranes, using computer difference spectroscopy. The incorporation of spin-labeled sphingomyelin into synaptic plasma membranes from calf brain has been studied as a function of sonication time. The spin-label ESR spectra are able to distinguish labeled sphingomyelin which is integrated into the membrane, from the unincorporated label, even if the latter cosediments with the membranes. Spectral subtraction has been used to quantitate the degree of incorporation. The percentage of incorporation increases with increasing sonication time and also with incubation after sonication. The extent of degradation of tritium-labeled sphingomyelin by the neutral sphingomyelinase present in the membrane closely correlates with the dependence of the incorporation of the spin-labeled sphingomyelin on sonication time. This illustrates the utility of the method in the study of membrane-bound, lipid-metabolizing enzymes.