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1.
Pneumolysin, a virulence factor from the human pathogen Streptococcus pneumoniae, is a water-soluble protein which forms ring-shaped oligomeric structures upon binding to cholesterol-containing lipid membranes. It induces vesicle aggregation, membrane pore formation and withdrawal of lipid material into non-bilayer proteolipid complexes. Solid-state magic angle spinning and wideline static NMR, together with freeze-fracture electron microscopy, are used to characterize the phase changes in fully hydrated cholesterol-containing lipid membranes induced by the addition ofpneumolysin. A structural model for the proteolipid complexes is proposed where a 30-50-meric pneumolysin ring lines the inside of a lipid torus. Cholesterol is found to be essential to the fusogenic action of pneumolysin. 相似文献
2.
Bonev BB Gilbert RJ Andrew PW Byron O Watts A 《The Journal of biological chemistry》2001,276(8):5714-5719
Pneumolysin, a major virulence factor of the human pathogen Streptococcus pneumoniae, is a soluble protein that disrupts cholesterol-containing membranes of cells by forming ring-shaped oligomers. Magic angle spinning and wideline static (31)P NMR have been used in combination with freeze-fracture electron microscopy to investigate the effect of pneumolysin on fully hydrated model membranes containing cholesterol and phosphatidylcholine and dicetyl phosphate (10:10:1 molar ratio). NMR spectra show that the interaction of pneumolysin with cholesterol-containing liposomes results in the formation of a nonbilayer phospholipid phase and vesicle aggregation. The amount of the nonbilayer phase increases with increasing protein concentration. Freeze-fracture electron microscopy indicates the coexistence of aggregated vesicles and free ring-shaped structures in the presence of pneumolysin. On the basis of their size and analysis of the NMR spectra it is concluded that the rings are pneumolysin oligomers (containing 30-50 monomers) complexed with lipid (each with 840-1400 lipids). The lifetime of the phospholipid in either bilayer-associated complexes or free pneumolysin-lipid complexes is > 15 ms. It is further concluded that the effect of pneumolysin on lipid membranes is a complex combination of pore formation within the bilayer, extraction of lipid into free oligomeric complexes, aggregation and fusion of liposomes, and the destabilization of membranes leading to formation of small vesicles. 相似文献
3.
N A Denisova 《The International journal of biochemistry》1990,22(5):439-442
1. The phospholipid composition of the main proteolipid complexes of the nervous system was studied in myelin and synaptosomal membranes from brains of representatives of various vertebrate classes. 2. The relative content of acid phospholipids was much higher in proteolipid complexes from myelin and synaptosomal membranes of all vertebrates studied as compared to their content in the initial lipid extract (28-80% and 11-20% of total phospholipid content, respectively). 3. The relative content of acid phospholipids in proteolipid complexes of myelin membranes was much lower in brain of fishes and amphibia as compared to higher vertebrates. 4. The main acid phospholipids of proteolipid complexes was phosphatidylserine, phosphatidic acid being characteristic for myelin proteolipids and diphosphatidyl glycerol for synaptosomal proteolipids of all vertebrates studied. 相似文献
4.
1. Fatty acid composition of five main phospholipids of vertebrate brain myelin and synaptosomal proteolipids and membranes was studied. 2. Higher content of monoenoic and lower content of saturated and polyenoic fatty acids was found to be characteristic of phospholipids from myelin and myelin proteolipids as compared to phospholipids from synaptosomal proteolipids and membranes of vertebrates (from fishes to mammalians). Fatty acid composition of phospholipids of proteolipid complexes and of the membranes, from which they were isolated, were found to be similar in various species studied. 3. Microviscosity was found to be higher in myelin as compared to synaptosomal membranes of frog Rana temporaria and in rabbit Lepus cuniculus. It appears to be due to the difference in proteolipid content and in lipid composition of myelin and synaptosomal membranes. 相似文献
5.
Proteolipid complex of Folch-Lees has been obtained and purified from the myelin and synaptosomes of the brain of the frog Rana temporaria and hen Gallus domesticus. Relative content of this proteolipid and glycolipids in the myelin is almost twice higher, whereas that of phospholipids--1 1/2 times lower than in the synaptosomal membranes of the same animal. Protein content of this complex is higher for myelin than for synaptosomal membranes; opposite relation was found with respect to phospholipid content. Within this complex, lipids are presented mainly by phospholipids, especially by acid ones which amount to 30-60%. Proteolipid complexes fro the myelin and synaptosomes differ from each other by their lipid component. Myelin proteolipid complex contains mainly phosphatidylserine and phosphatid acid, whereas synaptosomal one--phosphatidylserine and diphosphatediglycerol. No significant differences were found in fatty acid composition of phospholipids from proteolipid complex from myelin and synaptosomes as compared to this composition in the initial membranes. 相似文献
6.
R P Morozova V A Kokunin I A Nikolenko N V Kanivets M V Stefanov 《Ukrainski? biokhimicheski? zhurnal》1988,60(3):60-64
The lipid and proteolipid composition of endoplasmic reticulum smooth membranes from rat enterocytes in normalcy and under D-hypovitaminosis is studied to show the Ca2+-binding with these components. These membranes in vitamin D-deficiency are established to differ in the cholesterol content. Proteolipids 1 and 2 are isolated from the lipid fraction of membranes. Their amino acid composition is determined. The vitamin D-dependent binding of Ca by the proteolipid fraction is shown. 相似文献
7.
Negative staining of purified spinach dicyclohexylcarbodiimide (DCCD) sensitive ATPase revealed a population of 110 Å subunits attached by stalks to short string-like aggregates. The interpretation of these data is that 110 Å CF1 are attached by stalks to an aggregate of CF0.The CF1-CF0 complex was incorporated into phospholipid vesicles; freezefracture analysis of this preparation revealed a homogeneous population of particles spanning the lipid bilayer; these averaged 96 Å in diameter. The DCCD binding proteolipid (apparent molecular weight 7500), an integral component of CF0, was isolated from membranes by butanol extraction and was incorporated rated into phospholipid vesicles. Freeze-fracture analysis of the DCCD-binding proteolipid/vesicle preparation revealed a population of particles averaging 83 Å in diameter suggesting that the DCCD-binding proteolipid self-associates in lipid to form a stable complex. This complex may be required for proton transport across chloroplast membranes in vivo. The size difference between CF0 and DCCD-proteolipid freeze-fracture particles may be related to differences in polypeptide composition of the two complexes. 相似文献
8.
Pneumolysin is a member of the cholesterol-dependent cytolysin (CDC) family of pore-forming proteins that are produced as water-soluble monomers or dimers, bind to target membranes and oligomerize into large ring-shaped assemblies comprising approximately 40 subunits and approximately 30 nm across. This pre-pore assembly then refolds to punch a large hole in the lipid bilayer. However, in addition to forming large pores, pneumolysin and other CDCs form smaller lesions characterized by low electrical conductance. Owing to the observation of arc-like (rather than full-ring) oligomers by electron microscopy, it has been hypothesized that smaller oligomers explain smaller functional pores. To investigate whether this is the case, we performed cryo-electron tomography of pneumolysin oligomers on model lipid membranes. We then used sub-tomogram classification and averaging to determine representative membrane-bound low-resolution structures and identified pre-pores versus pores by the presence of membrane within the oligomeric curve. We found pre-pore and pore forms of both complete (ring) and incomplete (arc) oligomers and conclude that arc-shaped oligomeric assemblies of pneumolysin can form pores. As the CDCs are evolutionarily related to the membrane attack complex/perforin family of proteins, which also form variably sized pores, our findings are of relevance to that class of proteins as well. 相似文献
9.
Interactions between lipid-anchored and transmembrane proteins are relevant to the intracellular membrane sorting of glycosyl phosphatidylinositol-linked proteins. We have studied the interaction of a spin-labeled biotinyl diacyl phospholipid, with and without specifically bound avidin, with the myelin proteolipid protein (or the DM-20 isoform) reconstituted in dimyristoylphosphatidylcholine. Tetrameric avidin bound to the N-biotinyl lipid headgroup is a surface-anchored protein, and the myelin proteolipid is an integral protein containing four transmembrane helices. The electron spin resonance (ESR) spectrum of N-biotinyl phosphatidylethanolamine spin-labeled at the C-14 position of the sn-2 chain consists of two components in fluid-phase membranes of dimyristoylphosphatidylcholine containing the proteolipid. In the absence of avidin, this is characteristic of lipid-protein interactions with integral transmembrane proteins. The more motionally restricted component represents the lipid population in direct contact with the intramembranous surface of the integral protein, and the more mobile component corresponds to the bulk fluid lipid environment of the bilayer. In the presence of avidin, the biotin-lipid chains have reduced mobility because of the binding to avidin, even in the absence of the proteolipid [Swamy, M. J., and Marsh, D. (1997) Biochemistry 36, 7403-7407]. In the presence of the proteolipid, the major fraction of the avidin-anchored chains is further restricted in its mobility by interaction with the transmembrane protein. At a biotin-lipid concentration of 1 mol %, approximately 80% of the avidin-linked chains are restricted in membranes with a phosphatidylcholine:proteolipid molar ratio of 37:1. This relatively high stoichiometry of interaction can be explained when allowance is made for the closest interaction distance between the lipid-anchored avidin tetramer and the transmembrane proteolipid hexamer, without any specific interaction between the two types of membrane-associated proteins. The interaction is essentially one of steric exclusion, but the lipid chains are rendered more sensitive to interaction with the integral protein by being linked to avidin, even though they are removed from the immediate intramembrane protein-lipid interface. This could have implications for the tendency of lipid-anchored chains to associate with membrane domains with reduced lipid mobility. 相似文献
10.
Marsh D Horváth LI Swamy MJ Mantripragada S Kleinschmidt JH 《Molecular membrane biology》2002,19(4):247-255
Studies of lipid-protein interactions in double-reconstituted systems involving both integral and peripheral or lipid-anchored proteins are reviewed. Membranes of dimyristoyl phosphatidylglycerol containing either myelin proteolipid protein or cytochrome c oxidase were studied. The partner peripheral proteins bound to these membranes were myelin basic protein or cytochrome c, respectively. In addition, the interactions between the myelin proteolipid protein and avidin that was membrane-anchored by binding to N-biotinyl phosphatidylethanolamine were studied in dimyristoyl phosphatidylcholine membranes. Steric exclusion plays a significant role when sizes of the peripheral protein and transmembrane domain of the integral protein are comparable. Even so, the effects on avidin-linked lipids are different from those induced by myelin basic protein on freely diffusible lipids, both interacting with the myelin proteolipid protein. Both the former and the cytochrome c/cytochrome oxidase couple evidence a propagation of lipid perturbation out from the intramembrane protein interface that could be a basis for formation of microdomains. 相似文献
11.
Purification of the chloroplast-membrane dicyclohexylcarbodi-imide-binding proteolipid by ion-exchange chromatography.
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An efficient, mild and rapid procedure is reported for the separation of the dicyclohexyl-carbodi-imide-binding protein of chloroplast membranes from endogenous lipid components. By the use of ion-exchange chromatography the chloroplast proteolipid can be successfully separated from the major part of chlorophyll and other membrane lipids while being retained in a butan-1-ol milieu. 相似文献
12.
A lipid extract with a composition similar to that of myelin was used to prepare liposomes and proteoliposomes containing the Folch-Lees proteolipid apoprotein. Freeze-fracture replicas of the proteoliposomes were prepared to demonstrate the presence of intramembrane protein particles in the fracture faces of the lipid bilayer. Experiments with 45CaCl2 showed that a steady calcium movement occurs across liposomal membranes, approaching equilibrium between intra- and extravesicular spaces. The most significant finding was that Mg-ATP, ATP analogues, and other nucleotides depressed significantly the calcium fluxes in proteoliposomes, having no effect on liposomes that lacked the proteolipid protein. It is suggested that this intrinsic protein, interacting with nucleotides and endogenous lipids, could be involved in the regulation of calcium levels in myelin by means of a conformational change mechanism. These observations could lead to implications concerning the pathophysiology of myelin. 相似文献
13.
Optical anisotropy in lipid bilayer membranes: coupled plasmon-waveguide resonance measurements of molecular orientation, polarizability, and shape
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The birefringence and linear dichroism of anisotropic thin films such as proteolipid membranes are related to molecular properties such as polarizability, shape, and orientation. Coupled plasmon-waveguide resonance (CPWR) spectroscopy is shown in the present work to provide a convenient means of evaluating these parameters in a single lipid bilayer. This is illustrated by using 1-10 mol % of an acyl chain chromophore-labeled phosphatidylcholine (PC) incorporated into a solid-supported PC bilayer deposited onto a hydrated silica surface. CPWR measurements were made of refractive index and extinction coefficient anisotropies with two exciting light wavelengths, one of which is absorbed by the chromophore and one of which is not. These results were used to calculate longitudinal and transverse molecular polarizabilities, the orientational order parameter and average angle between the longitudinal axis of the lipid molecule and the membrane normal, and the molecular shape factors of the lipid molecules. The values thereby obtained are in excellent agreement with parameters determined by other techniques, and provide a powerful tool for analyzing lipid-protein, protein-protein, and protein-ligand interactions in proteolipid films. 相似文献
14.
Proteolipid of adenosinetriphosphatase from yeast mitochondria forms proton-selective channels in planar lipid bilayers 总被引:5,自引:0,他引:5
Proteolipid isolated from yeast mitochondrial adenosinetriphosphatase by butanol extraction is reincorporated into lipid vesicles from which planar membranes are formed. The proteolipid permits electric conductance through the membrane. This conductance occurs through membrane channels which are highly selective for protons. Proton channels in the membrane are directly observed at high proton concentrations in the aqueous phases. Channels open and close independently from each other; their open-state conductances and lifetimes are monodisperse but influenced by the applied voltage (12 pS and 3 s, respectively, at pH 2.2 and 100 mV). Proton channels do not occur in single proteolipid molecules; the conducting structure consists of at least two polypeptide chains since channels form in a (reversible) bimolecular reaction of nonconducting forms of proteolipid. The number of proton channels at a constant proteolipid concentration changes in sharp transitions and by orders of magnitudes upon critical changes of membrane composition and pH. These transitions are caused by transitions of proteolipid organization in the membrane from a dispersed state (equilibrium between channel-forming "dimers" and a large pool of "monomers") to a state of almost complete aggregation of proteolipid which stabilizes large proton-conducting structures (probably associates of channel-forming dimers). This self-association of isolated proteolipid into structures containing proton-selective channels suggests that the six proteolipids in the adenosinetriphosphatase complex exist as a self-associating entity containing most likely three proton channels. 相似文献
15.
The microwave saturation properties of various spin-labeled lipids in reconstituted complexes of the myelin proteolipid protein with dimyristoyl phosphatidylcholine have been studied both by conventional and saturation transfer electron spin resonance (ESR) spectroscopy. In the fluid phase, the conventional ESR spectra consist of a fluid and a motionally restricted (i.e., protein-associated) component, whose relative proportions can be determined by spectral subtractions and depend on the selectivity of the particular spin-labeled lipid for the protein. At 4 degrees C when the bulk lipid is in the gel phase, the integrated intensity of the saturation transfer ESR spectra displays a linear dependence on the fraction of motionally restricted lipid that is deduced from the conventional ESR spectra in the fluid phase, indicating the presence of distinct populations of free and protein-interacting lipid with no exchange between them on the saturation transfer ESR time scale in the gel phase. At 30 degrees C when the bulk lipid is in the fluid phase, the saturation transfer integral displays a nonlinear dependence on the fraction of motionally restricted lipid, consistent with exchange between the two lipid populations on the saturation transfer ESR time scale in the fluid phase. For lipid spin labels with different selectivities for the protein in complexes of fixed lipid/protein ratio, the data in the fluid phase are consistent with a constant (diffusion-controlled) on-rate for exchange at the lipid-protein interface. Values ranging between 1 and 9 x 10(6) s-1 are estimated for the intrinsic off-rates for exchange of spin-labeled stearic acid and phosphatidylcholine, respectively, at 30 degrees C. Conventional continuous wave saturation experiments lead to similar conclusions regarding the lipid exchange rates in the fluid and gel phases of the lipid/protein recombinants. The ESR saturation studies therefore demonstrate exchange on the time scale of the nitroxide spin-lattice relaxation at the lipid-protein interface of myelin proteolipid/dimyristoyl phosphatidylcholine complexes in the fluid phase but not in the gel phase. 相似文献
16.
Puertollano R Menéndez M Alonso MA 《Biochemical and biophysical research communications》1999,266(2):330-333
The MAL proteolipid, an integral membrane protein with selective residence in glycolipid- and cholesterol-enriched membrane (GEM) microdomains, has recently been identified as being an element of the integral protein machinery necessary for apical transport in MDCK cells. With the use of a recombinant baculovirus, we have expressed and purified polyhistidine-tagged MAL to determine whether MAL has special lipid requirements for becoming incorporated into membranes. In contrast with caveolin-1, a component of GEMs that requires cholesterol for its integration into artificial membranes, MAL incorporation took place with dimyristoylphosphatidylcholine as the only lipid component. The presence of cholesterol, sphingomyelin, or galactocerebrosides did not affect the efficiency of this process. These results indicated that MAL is compatible with membranes containing either only phospholipids or also glycolipids and cholesterol and are consistent with the reported requirement of a sorting event for the specific targeting of MAL to GEM microdomains. 相似文献
17.
The objective of this study was to determine whether in vitro calcification of human aorta is proteolipid dependent. Homogenates were prepared from tissue with no gross pathologic manifestations. Samples were examined for calcifiability in a metastable calcium phosphate solution before and after lipid extraction. Fractions of the extracted lipid were similarly examined. The tissue calcified before but not after lipid extraction. Calcifiability was restricted to the proteolipid portion of the lipid extract. Under the conditions employed, therefore, proteolipid is required for calcification of human aorta, in vitro. 相似文献
18.
Folding of the mitochondrial proton adenosinetriphosphatase proteolipid channel in phospholipid vesicles 总被引:8,自引:0,他引:8
The mitochondrial H+-ATPase proteolipid from Neurospora crassa was incorporated into small unilamellar dimyristoylphosphatidylcholine vesicles and its conformation determined by circular dichroism spectroscopy (CD). While the largely alpha-helical conformation is relatively independent of the method of incorporation into vesicles, i.e., rehydration, detergent dialysis, or detergent dilution, the proteolipid conformation was significantly different in detergent micelles and in organic solvents. Only very slight changes in the CD spectrum were observed upon binding of the H+-ATPase inhibitor dicyclohexylcarbodiimide to the proteolipid in vesicles, thus suggesting that the inhibitor acts either by blocking the channel or by masking an essential charge group, rather by than causing an overall conformational change in the channel. Additionally, very similar CD spectra were obtained for vesicles with different lipid/protein mole ratios, indicating either that no substantial conformational differences exist between monomer and multimers or that monomers self-associate to form stable complexes during incorporation into vesicles. This study has provided a physical basis for model-building studies of the proteolipid channel structure. 相似文献
19.
A protein, the mediatophore, has been purified from Torpedo electric organ presynaptic plasma membranes. This protein mediates the release of acetylcholine through artificial membranes when activated by calcium and is made up of 15-kDa proteolipid subunits. After immunization with purified delipidated mediatophore, monoclonal antibodies binding to the 15-kDa proteolipid band on Western blots of purified mediatophore were selected. A 15-kDa proteolipid antigen was also detected in cholinergic synaptic vesicles. Using an immunological assay, it was estimated that presynaptic plasma membranes and synaptic vesicles contain similar proportions of 15-kDa proteolipid antigen. Detection by immunofluorescence in the electric organ showed that only nerve endings were labeled. In electric lobes, the staining was associated with intracellular membranes of the electroneuron cell bodies and in axons. Nerve endings at Torpedo neuromuscular junctions were also labeled with anti-15-kDa proteolipid monoclonal antibodies. 相似文献
20.
S A Zabelinski? L F Pomazanskaia E V Chirkovskaia 《Zhurnal evoliutsionno? biokhimii i fiziologii》1984,20(3):239-245
The Folch-Lees proteolipid complexes of different purity (crude proteolipids and relative pure proteolipids) were isolated from vertebrate brain: mammalia (Macaca irus, Macaca rhesus and white rat), birds (Columbia livia), reptilia (Testudo horsfieldi), amphibia (Rana temporaria) and fishes (Salmo irideus). The proteolipid complexes were isolated by emulsion-centrifugation method. The content of proteolipid protein (mg/g w. w.) correlates with the level of phylogenetic development of the animals studied. It is the highest in monkey brain (10.5 and 8.6 mg/g) and the lowest in fish brain (2.2 mg/g). The yield of proteolipids from the brains of animals studied shows the same pattern. Crude proteolipids of mammalia, birds and reptiles contain 40-50% of protein and 60-50% of lipids. The content of phospholipids is about 40%. Proteolipids of amphibia and fish brain contain less protein--about 30%. In the conditions of mild purification, the protein content in mammalia, birds and reptilia makes up about 70% and lipid content--about 30-35%. The crude and purified proteolipids in all the animals studied (as compared with the original lipid extracts from which they were isolated) are enriched in acid phospholipids: phosphatidyl serine, phosphatidyl inositol and diphosphatidyl glycerol. Acid phospholipids in total lipid extract make up 10-20% of total phospholipids, in crude proteolipids 16-32 and in purified proteolipids--56-75%. There are no marked differences between fatty acid composition of phospholipids in proteolipids and in the same phospholipids isolated from total lipid extract. 相似文献