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.     

NaK-dependent conformation change of proteins of excitable membranes

The K⁺, Na⁺ and Ca²⁺ form of excitable membranes of rat brain were investigated by infrared, ORD and CD spectroscopy. It is shown that with the K⁺ form the conformatio of relatively large parts of the membrane proteins occurs as an antiparallel β structure. No β structure is found with the Na⁺ and Ca²⁺ form. In the presence of these ions the proteins are largely helical. This suggests that during the action potential, membrane proteins change their conformation depending on the cations shifted.     

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.     

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.     

α-Synuclein interacts differently with membranes mimicking the inner and outer leaflets of neuronal membranes

The toxicity of α-synuclein (α-syn), the amyloidogenic protein responsible for Parkinson's disease, is likely related to its interaction with the asymmetric neuronal membrane. α-Syn exists as cytoplasmatic and as extracellular protein as well. To shed light on the different interactions occurring at the different α-syn localizations, we have here modelled the external and internal membrane leaflets of the neuronal membrane with two complex lipid mixtures, characterized by phase coexistence and with negative charge confined to either the ordered or the disordered phase, respectively. To this purpose, we selected a five-component (DOPC/SM/DOPE/DOPS/chol) and a four-component (DOPC/SM/GM1/chol) lipid mixtures, which contained the main membrane lipid constituents and exhibited a phase separation with formation of ordered domains. We have compared the action of α-syn in monomeric form and at different concentrations (1 nM, 40 nM, and 200 nM) with respect to lipid systems with different composition and shape by AFM, QCM-D, and vesicle leakage experiments. The experiments coherently showed a higher stability of the membranes composed by the internal leaflet mixture to the interaction with α-syn. Damage to membranes made of the external leaflet mixture was detected in a concentration-dependent manner. Interestingly, the membrane damage was related to the fluidity of the lipid domains and not to the presence of negatively charged lipids.     

Lipid modifications of proteins – slipping in and out of membranes

Protein lipid modification, once thought to act as a stable membrane anchor for soluble proteins, is now attracting more widespread attention for its emerging role in diverse signaling pathways and regulatory mechanisms. Most multicellular organisms have recruited specific types of lipids and a suite of unique enzymes to catalyze the modification of a select number of proteins, many of which are evolutionarily conserved in plants, animals and fungi. Each of the three known types of lipid modification – palmitoylation, myristylation and prenylation – allows cells to target proteins to the plasma membrane, as well as to other subcellular compartments. Among the lipid modifications, protein prenylation might also function as a relay between cytoplasmic isoprene biosynthesis and regulatory pathways that control cell cycle and growth. Molecular and genetic studies of an Arabidopsis mutant that lacks farnesyl transferase suggest that the enzyme has a role in abscisic acid signaling during seed germination and in the stomata. It is becoming clear that lipid modifications are not just fat for the protein, but part of a highly conserved intricate network that plays a role in coordinating complex cellular functions.     

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.     

Dynamics of viscoelastic spherical membranes—the balloon model of the alveolus

Double-valued pressure-volume relationships in dynamic conditions for spherical membranes, modelling the lung alveoli, were obtained at small deformations. This hysteretic behavior was considered to be produced by at least three independent mechanisms: (1) the lung parenchyma exhibits viscoelastic properties; (2) the lung surface film, independent of the tissue, exhibits viscoelastic properties and (3) the pressure acting on the inner membrane surface depends on the rate of the alveolus volume change, due to the air viscous resistance in the bronchial tree. In each case, the maximum volume change, the hysteresis loop area, the tilt angle of the hysteresis loop and the relaxation time of the system were calculated. The results show pronounced hysteresis at normal breathing due to the air viscous resistance and smaller one due to the tissue and surface viscoelastic properties. In quasistatic conditions the values of the surface viscoelasticity and the tissue viscoelasticity effects are comparable or different, depending on the concrete external conditions. Comparison with the available experimental data is discussed in detail.     

The effect of bupivacaine·HCl on the physical properties of neuronal membranes

Fluorescent probe techniques were used to evaluate the effect of bupivacaine.HCl on the physical properties (transbilayer asymmetric lateral and rotational mobilities, annular lipid fluidity and protein distribution) of synaptosomal plasma membrane vesicles (SPMVs) isolated from bovine cerebral cortex. An experimental procedure was used based on selective quenching of both 1,3-di(1-pyrenyl)propane (Py-3-Py) and 1,6-diphenyl-1,3,5-hexatriene (DPH) by trinitrophenyl groups, and radiationless energy transfer (RET) from the tryptophans of membrane proteins to Py-3-Py. Bupivacaine.HCl increased the bulk lateral and rotational mobilities, and annular lipid fluidity in SPMVs lipid bilayers, and had a greater fluidizing effect on the inner monolayer than that of the outer monolayer. The magnitude of increasing effect on annular lipid fluidity in SPMVs lipid bilayer induced by bupivacaine.HCl was significantly far greater than magnitude of increasing effect of the drug on the lateral and rotational mobilities of bulk SPMVs lipid bilayer. It also caused membrane proteins to cluster. These effects of bupivacaine.HCl on neuronal membranes may be responsible for some, though not all, of the local anesthetic actions of bupivacaine.HCl.     

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.     

Interaction of α-synuclein with vesicles that mimic mitochondrial membranes

α-Synuclein, an intrinsically-disordered protein associated with Parkinson's disease, interacts with mitochondria, but the details of this interaction are unknown. We probed the interaction of α-synuclein and its A30P variant with lipid vesicles by using fluorescence anisotropy and (19)F nuclear magnetic resonance. Both proteins interact strongly with large unilamellar vesicles whose composition is similar to that of the inner mitochondrial membrane, which contains cardiolipin. However, the proteins have no affinity for vesicles mimicking the outer mitochondrial membrane, which lacks cardiolipin. The (19)F data show that the interaction involves α-synuclein's N-terminal region. These data indicate that the middle of the N-terminal region, which contains the KAKEGVVAAAE repeats, is involved in binding, probably via electrostatic interactions between the lysines and cardiolipin. We also found that the strength of α-synuclein binding depends on the nature of the cardiolipin acyl side chains. Eliminating one double bond increases affinity, while complete saturation dramatically decreases affinity. Increasing the temperature increases the binding of wild-type, but not the A30P variant. The data are interpreted in terms of the properties of the protein, cardiolipin demixing within the vesicles upon binding of α-synuclein, and packing density. The results advance our understanding of α-synuclein's interaction with mitochondrial membranes.     

Molecular interactions of Alzheimer's Aβ protofilaments with lipid membranes

Amyloid fibrils and peptide oligomers play central roles in the pathology of Alzheimer's disease, type 2 diabetes, Parkinson's disease, Huntington's disease, and prion-related disease. Here, we investigate the molecular interactions between preformed amyloid β (Aβ) molecular protofilaments and lipid bilayer membranes, in the presence of explicit water molecules, using computational models and all-atom molecular dynamics. These interactions play an important role in the stability and function of both Aβ fibrils and the adjacent cellular membrane. Taking advantage of the symmetry-related and directional properties of the protofilaments, we build models that cover several relative protofilament-membrane orientations. Our molecular dynamics simulations reveal the relative contributions of different structural elements to the dynamics and stability of Aβ protofilament segments near membranes, and the first steps in the mechanism of fibril-membrane interactions. During this process, we observe a significant alteration of the side-chain contact pattern in protofilaments, although a fraction of the characteristic β-sheet content is preserved. As a major driving force, we identify the electrostatic interactions between Aβ charged side chains, including E22, D23, and K28, and lipid headgroups. Together with hydrogen bonding with atoms from lipid headgroups, these interactions can facilitate the penetration of hydrophobic C-terminal amino acids through the lipid headgroup region, which can finally lead both to further loss of the initial fibril structure and to local membrane-thinning effects. Our results may guide new experiments that could test the extent to which the structural features of water-formed amyloid fibrils are preserved, lost, or reshaped by membrane-mediated interactions.     

Interaction of transmembrane-spanning segments of the α2-adrenergic receptor with model membranes

Adrenergic receptors are integral membrane proteins involved in cellular signalling that belong to the G protein-coupled receptors. Synthetic peptides resembling the putative transmembrane (TM) segments TM4, TM6 and TM7, of the human α2-adrenergic receptor subtype C10 (P08913) and defined lipid vesicles were used to assess protein-lipid interactions that might be relevant to receptor structure/function. P6 peptide contains the hydrophobic core of TM6 plus the N-terminal hydrophilic motif REKR, while peptides P4 and P7 contained just the hydrophobic stretches of TM4 and TM7, respectively. All the peptides increase their helical tendency at moderate concentrations of TFE (30–50%) and in presence of 1,2-dielaidoyl-sn-glycero-3-phosphatidylethanolamine (DEPE) lipids. However, only P6 displays up to 19% of α-helix in the presence of just the DEPE lipids, evidences a transmembrane orientation and stabilizes the Lα lipid phase. Conversely, P4 and P7 peptides form only stable β-sheet structures in DEPE and favour the non-lamellar, inverted hexagonal (H_II) phase of DEPE by lowering its phase transition temperature. This study highlights the potential of using synthetic peptides derived from the amino acid sequence in the native proteins as templates to understand the behaviour of the transmembrane segments and underline the importance of interfacial anchoring interactions to meet hydrophobic matching requirements and define membrane organization.     

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.     

Mitochondria: One of the origins for autophagosomal membranes?

Macroautophagy is a transport pathway to the lysosome/vacuole that contributes to the degradation of numerous intracellular components. Despite the recent advances achieved in the understanding of the molecular mechanism underlying macroautophagy, the membrane origin of autophagosomes, the hallmark of this process is still a mystery. It has been suggested that mitochondria may be one of the lipid sources for autophagosome formation and that possibly this organelle provides the phosphatidylethanolamine (PE) that covalently links to the members of the ubiquitin-like Atg8/microtubule-associated protein 1 light chain 3 (LC3) protein family. These lipidated proteins are inserted into the outer and inner surface of autophagosomes and are essential for the biogenesis of these large double-membrane vesicles. However, because PE is an integral component of all cellular membranes, designing appropriate experiments to determine the origin of the autophagosomal PE is not easy. In this review, we discuss the idea that mitochondria provide the pool of PE necessary for the autophagosome biogenesis and we propose some possible experimental approaches aimed to explore this possibility.