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1.
Cross-linking experiments with the MONOfunctional imidoester methyl-acetimidate, in the pH range 7.0 - 8.0, on rat liver nucleosomes generate a cross-linking pattern almost identical with the one observed for much longer BIfunctional reagents (e.g. dimethylsuberimidate). Combined cross-linking and trypsin digestion experiments suggest that all or at least the great majority of this cross-linking occurs on trypsin digestible segments (or "tails") of the histones. The formation of oligomers over such extremely short distances and especially the observation of an H3 homodimer suggests a very close proximity of half-nucleosomes. 相似文献
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Dimethyl adipimidate was used to cross-link the polypeptides within hemoglobin, haptoglobin, and hemoglobin-haptoglobin complex. Cross-linked hemoglobin retained considerable ability to bind haptoglobin, although the amounts bound were reduced and the haptoglobin reaction could be used to fractionate the modified hemoglobin. With cross-links limited to intramolecular sites, hemoglobin showed four bands on polyacrylamide gel electrophoresis in sodium dodecyl sulfate, identified, with reference to the subunit polypeptides, as monomer, dimer, trimer, and tetramer. The dimer region consisted of at least two separable species. When hemoglobin-haptoglobin complex was cross-linked, a band of hemoglobin dimer was present, which demonstrates that at least two hemoglobin subunits have a close spatial relation when bound to haptoglobin. Some comparisons with adipimidate-reacted hemoglobin were made using malonimidate and suberimidate and some marked differences were noted. 相似文献
4.
《The Journal of cell biology》1981,89(2):261-266
Vasopressin increases the water permeability of the luminal membrane of the toad bladder epithelial cell. This change in permeability correlates with the occurrence in luminal membranes of intramembrane particle aggregates, which may be the sites for transmembrane water flow. Withdrawal of vasopressin is ordinarily associated with a rapid reduction of water flow to baseline values and a simultaneous disappearance of the particle aggregates. The bifunctional imidoesters dithiobispropionimidate (DTBP) and dimethylsuberimidate (DMS), which cross-link amino groups in membrane proteins and lipids, slow the return of water flow to baseline after vasopressin withdrawal. Cross- linking is maximal at pH 10, and is reduced as pH is lowered. Freeze- fracture studies show persistence of luminal membrane particle aggregates in cross-linked bladders and a reduction in their frequency as water flow diminishes. Fusion of aggregate-containing cytoplasmic tubular membrane structures with the luminal membrane is also maintained by the imidoesters. Reductive cleavage of the central S-S bond of DTBP by beta-mercaptoethanol reverses cross-linking, permitting resumption of the rapid disappearance of the vasopressin effect. Bladders that have undergone DTBP cross-linking and beta- mercaptoethanol reduction respond to a second stimulation by vasopressin. Thus, the imidoesters provide a physiologic and reversible means of stabilizing normally rapid membrane events. 相似文献
5.
竹红菌甲素对红细胞膜蛋白及膜磷脂的光敏损伤 总被引:4,自引:2,他引:4
In this paper, using human erythrocyte membrane, the photodamage of Hypocrellin A to membrane protein and phospholipid was studied by measuring the lipid peroxidation, the damage of phospholipid, the change of protein secondary structure, the endogenous fluorescence change and SDS-polyacrylamide gel electrophoresis analysis. These results showed that illumination of erythrocyte membrane in presence of Hypocrellin A can cause lipid peroxidation producing fluorescence adduct and MDA, decomposing in phospholipid composition in which PE and PS were more sensitive than others. Meanwhile, the secondary structure of membrane protein was destroyed and endogenous fluorescence decreased. The photodamage on phospholipid and spectrin occurred more seriously in the case they were embedded in membrane than they were in isolated form. So we suggest that they are interactions existing between proteins and phospholipids to enhance the damage on protein and phospholipid during the HA-sensitized photodamage on membrane. 相似文献
6.
Kewei Li Wenpeng Gu Junrong Liang Yuchun Xiao Haiyan Qiu Haoshu Yang Xin Wang Huaiqi Jing 《BMC genomics》2014,15(1)
Background
Yersinia enterocolitica outer membrane protein A (OmpA) is one of the major outer membrane proteins with high immunogenicity. We performed the polymorphism analysis for the outer membrane protein A and putative outer membrane protein A (p-ompA) family protein gene of 318 Y. enterocolitica strains.Results
The data showed all the pathogenic strains and biotype 1A strains harboring ystB gene carried both ompA and p-ompA genes; parts of the biotype 1A strains not harboring ystB gene carried either ompA or p-ompA gene. In non-pathogenic strains (biotype 1A), distribution of the two genes and ystB were highly correlated, showing genetic polymorphism. The pathogenic and non-pathogenic, highly and weakly pathogenic strains were divided into different groups based on sequence analysis of two genes. Although the variations of the sequences, the translated proteins and predicted secondary or tertiary structures of OmpA and P-OmpA were similar.Conclusions
OmpA and p-ompA gene were highly conserved for pathogenic Y. enterocolitica. The distributions of two genes were correlated with ystB for biotype 1A strains. The polymorphism analysis results of the two genes probably due to different bio-serotypes of the strains, and reflected the dissemination of different bio-serotype clones of Y. enterocolitica. 相似文献7.
In Escherichia coli, the major cytoplasmic domain (C6) of the polytopic membrane protein lactose permease (LacY) is exposed to the opposite side of the membrane from a neighboring periplasmic domain (P7). However, these domains are both exposed on the periplasmic side of the membrane in a mutant of E.coli lacking phosphatidylethanolamine (PE) wherein LacY only mediates facilitated transport. When purified LacY was reconstituted into liposomes lacking PE or phosphatidylcholine (PC), C6 and P7 were on the same side of the bilayer. In liposomes containing PE or PC, C6 and P7 were on opposite sides of the bilayer. Only the presence of PE in the liposomes restored active transport function of LacY as opposed to restoration of only facilitated transport function in the absence of PE. These results were the same for LacY purified from PE-containing or PE-lacking cells, and are consistent with the topology and function of LacY assembled in vivo. Therefore, irrespective of the mechanism of membrane insertion, the subdomain topological orientation and function of LacY are determined primarily by membrane phospholipid composition. 相似文献
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The full-length cDNA of a phospholipid transfer protein (PLTP) was isolated from Aspergillus oryzae by a RACE-PCR procedure using degenerated primer pool selected from the N-terminal sequence of the purified phosphatidylinositol/phosphatidylglycerol transfer protein (PG/PI-TP). The cDNA encodes a 173 amino acid protein of 18823 Da. The deduced amino acid sequence from position 38 to 67 is 100% identical to the N-terminal sequence (first 30 amino acids) of the purified PG/PI-TP. This amino acid sequence is preceded by a leader peptide of 37 amino acids which is predicted to be composed of a signal peptide of 21 amino acids followed by an extra-sequence of 16 amino acids, or a membrane anchor protein signal (amino acid 5-29). This strongly suggests that the PG/PI-TP is a targeted protein. The deduced mature protein is 138 amino acids long with a predicted molecular mass of 14933 Da. Comparison of the deduced PG/PI-TP sequence with other polypeptide sequences available in databases revealed a homology with a protein deduced from an open reading frame coding for an unknown protein in Saccharomyces cerevisiae (36% identity and 57% similarity). Apart from this homology, the PG/PI-TP is unique and specific to the filamentous fungi on the basis of comparison of PLTP protein sequences. Northern blot analysis of RNA isolated from A. oryzae cultures grown on glucose or glucose supplemented with phospholipids suggests that the PG/PI-TP is transcribed by only one RNA species and allows us to show that expression of the protein is regulated at the messenger RNA level. 相似文献
9.
Identification of a putative membrane receptor for the bioactive phospholipid, lysophosphatidic acid. 总被引:10,自引:0,他引:10
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R L van der Bend J Brunner K Jalink E J van Corven W H Moolenaar W J van Blitterswijk 《The EMBO journal》1992,11(7):2495-2501
Lysophosphatidic acid (LPA) is a naturally occurring phospholipid with hormone- and growth factor-like activities. Exogenous LPA stimulates GTP-dependent phosphoinositide hydrolysis and inhibits adenylate cyclase in its target cells, but the site of action of LPA is unknown. We now report the identification by photoaffinity labeling of a putative LPA membrane receptor in various LPA-responsive cell types. A 32P-labeled LPA analogue containing a photoreactive fatty acid, [32P]diazirine-LPA, labels a membrane protein of apparent molecular mass of 38-40 kDa in various cell types, including neuronal cells, brain homogenates, carcinoma cells, leukemic cells and normal fibroblasts. Labeling of the 38-40 kDa protein is competitively inhibited by unlabeled 1-oleoyl-LPA (IC50 approximately 10 nM), but not by other phospholipids. Specific labeling is not detected in rat liver membranes or in human neutrophils, which are physiologically unresponsive to LPA. Suramin, an inhibitor of both early and late events in the action of LPA, completely inhibits the binding of photoreactive LPA. We suggest that the 38-40 kDa protein represents a specific LPA cell surface receptor mediating at least part of the multiple cellular responses to LPA. 相似文献
10.
Cross-linking of glycolipids in erythrocyte ghost membrane 总被引:2,自引:0,他引:2
T H Ji 《The Journal of biological chemistry》1974,249(24):7841-7847
11.
Cross-linking analysis of yeast mitochondrial outer membrane 总被引:2,自引:0,他引:2
By enrichment of contact sites between the two mitochondrial boundary membranes it has been shown that this fraction contained a high activity of glutathione transferase and hexokinase which was bound to the outer membrane pore protein (Ohlendieck, K. et al. (1986) Biochim. Biophys. Acta 860, 672-689). Therefore, an interaction between the three proteins in the contact sites has been suggested. Cross-linking experiments with isolated outer membrane of yeast mitochondria show that glutathione transferase and the pore protein are already associated in the free outer membrane. Porin appeared to adopt four different oligomeric complexes in the membrane, including interactions with a 14 kDa polypeptide, which has glutathione transferase activity. The latter polypeptide could be phosphorylated by intrinsic or extrinsic protein kinases, while the porin itself was not phosphorylated. Yeast hexokinase, when bound to the outer membrane, was able to cross-link to the pore protein. 相似文献
12.
Connell E Giniatullina A Lai-Kee-Him J Tavare R Ferrari E Roseman A Cojoc D Brisson AR Davletov B 《Journal of molecular biology》2008,380(1):42-50
Synaptotagmins are vesicular proteins implicated in many membrane trafficking events. They are highly conserved in evolution and the mammalian family contains 16 isoforms. We now show that the tandem C2 domains of several calcium-sensitive synaptotagmin isoforms tested, including Drosophila synaptotagmin, rapidly cross-link phospholipid membranes. In contrast to the tandem structure, individual C2 domains failed to trigger membrane cross-linking in several novel assays. Large-scale liposomal aggregation driven by tandem C2 domains in response to calcium was confirmed by the following techniques: turbidity assay, dynamic light-scattering and both confocal and negative stain electron microscopy. Firm cross-linking of membranes was evident from laser trap experiments. High-resolution cryo-electron microscopy revealed that membrane cross-linking by tandem C2 domains results in a constant distance of ∼9 nm between the apposed membranes. Our findings show the conserved nature of this important property of synaptotagmin, demonstrate the significance of the tandem C2 domain structure and provide a plausible explanation for the accelerating effect of synaptotagmins on membrane fusion. 相似文献
13.
Effect of oxidative stress on membrane phospholipid and protein organization in human erythrocytes 总被引:1,自引:0,他引:1
A Arduini A Stern S Storto M Belfiglio G Mancinelli R Scurti G Federici 《Archives of biochemistry and biophysics》1989,273(1):112-120
Membrane phospholipid and protein organization was studied in intact human erythrocytes exposed to phenylhydrazine, an oxidative agent inducer. The evaluation of the membrane phospholipid and protein organization was carried out in terms of asymmetric distribution across the membrane bilayer for the phospholipids, and in terms of accessibility of cleavable sites present on the outer membrane surface for the proteins. Treatment of phenylhydrazine-exposed erythrocytes either with bee venom phospholipase A2 or with trinitrobenzenesulfonic acid indicated that phosphatidylserine (PS), which is the only phospholipid not formally present on the outer leaflet of the membrane, was translocated to the outer surface of the cell membrane. The extent of this phenomenon was directly proportional to the concentration of the oxidant having a peak value at 0.1 mM. Phosphatidylcholine and phosphatidylethanolamine conserved their original distribution across the erythrocyte membrane throughout the study. The oxidant, at a dose which did not induce any modification of the sodium dodecyl sulfate-polyacrylamide gel electrophoresis cytoskeleton membrane protein pattern, did not provoke any alteration of the membrane protein surface architecture, although the translocation of PS to the membrane outer leaflet in intact erythrocytes was present. 相似文献
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Cross-linking analysis of the two forms of protein I, a major outer membrane protein of Escherichia coli.
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The two forms of protein I were cross-linked to molecules of the same species, even when both were present simultaneously. This suggests that they form separate multimers in the outer membrane. 相似文献
16.
Co-ordination between membrane phospholipid synthesis and accelerated biosynthesis of cytoplasmic ribonucleic acid and protein 总被引:3,自引:9,他引:3
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J. R. Tata 《The Biochemical journal》1970,116(4):617-630
1. The rate of synthesis of membrane phospholipid was studied in rat liver and seminal vesicles by following the incorporation of [(32)P]orthophosphate, [(14)C]choline and [(14)C]glycerol. Particular emphasis was laid on the endoplasmic reticulum, which was fractionated into smooth microsomal membranes, heavy rough membranes, light rough membranes and free polyribosomes. 2. Phospholipid labelling patterns suggested a heterogeneity in the synthesis and turnover of the different lipid moieties of smooth and rough endoplasmic membranes. The major phospholipids, phosphatidylcholine and phosphatidylethanolamine, were labelled relatively rapidly with (32)P over a short period of time whereas incorporation of radioisotope into the minor phospholipids, sphingomyelin, lysolecithin and phosphatidylinositol proceeded slowly but over a longer period of time. 3. The incorporation of orotic acid into RNA and labelled amino acids into protein of the four submicrosomal fractions was also studied. 4. Rapid growth of the liver was induced by the administration of growth hormone and tri-iodothyronine to hypophysectomized and thyroidectomized rats and by partial hepatectomy. Growth of seminal vesicles of castrated rats was stimulated with testosterone propionate. 5. The rate of labelling of membrane phospholipids was enhanced in all major subcellular particulate fractions (nuclear, mitochondrial and microsomal) during induced growth. However, it was in the rough endoplasmic reticulum that the accumulation of phospholipids, RNA and protein was most marked. The effect of hormone administration was also to accelerate preferentially the labelling with (32)P of sphingomyelin relative to that of phosphatidylcholine or phosphatidylethanolamine. 6. Time-course analyses showed that, in all four growth systems studied, the enhancement of the rate of membrane phospholipid synthesis coincided with the rather abrupt increase in the synthesis of RNA and protein of the rough endoplasmic reticulum. Growth hormone and tri-iodothyronine administered to hypophysectomized rats had additive effects in all the biosynthetic processes. The latent period of action of each hormone was maintained so that two waves of proliferation of endoplasmic reticulum occurred if the hormones were administered simultaneously. 7. It is concluded that there is some mechanism in the cell that tightly co-ordinates the formation of membranes, especially those of the endoplasmic reticulum, when an increased demand is made for protein synthesis. 相似文献
17.
Allen SJ Curran AR Templer RH Meijberg W Booth PJ 《Journal of molecular biology》2004,342(4):1279-1291
We report a detailed kinetic study of the folding of an alpha-helical membrane protein in a lipid bilayer environment. SDS denatured bacteriorhodopsin was folded directly into phosphatidylcholine lipid vesicles by stopped-flow mixing. The folding kinetics were monitored with millisecond time resolution by time-resolving changes in protein fluorescence as well as in the absorption of the retinal chromophore. The kinetics were similar to those previously reported for folding bacteriorhodopsin in detergent or lipid micelles, except for the presence of an additional apoprotein intermediate. We suggest this intermediate is a result of the greater internal two-dimensional pressure present in these lipid vesicles as compared to micelles. These results lay the groundwork for future studies aimed at understanding the mechanistic origin of the effect of lipid bilayer properties on protein folding. Furthermore, the use of biologically relevant phosphatidylcholine lipids, together with a straightforward rapid mixing process to initiate the folding reaction, means the method is generally applicable, and thus paves the way for an improved understanding of the in vitro folding of transmembrane alpha-helical proteins. 相似文献
18.
Synaptophysin binds to physophilin, a putative synaptic plasma membrane protein 总被引:4,自引:2,他引:4
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《The Journal of cell biology》1990,111(5):2041-2052
We have developed procedures for detecting synaptic vesicle-binding proteins by using glutaraldehyde-fixed or native vesicle fractions as absorbent matrices. Both adsorbents identify a prominent synaptic vesicle-binding protein of 36 kD in rat brain synaptosomes and mouse brain primary cultures. The binding of this protein to synaptic vesicles is competed by synaptophysin, a major integral membrane protein of synaptic vesicles, with half-maximal inhibition seen between 10(-8) and 10(-7) M synaptophysin. Because of its affinity for synaptophysin, we named the 36-kD synaptic vesicle-binding protein physophilin (psi nu sigma alpha, greek = bubble, vesicle; psi iota lambda os, greek = friend). Physophilin exhibits an isoelectric point of approximately 7.8, a Stokes radius of 6.6 nm, and an apparent sedimentation coefficient of 5.6 S, pointing to an oligomeric structure of this protein. It is present in synaptic plasma membranes prepared from synaptosomes but not in synaptic vesicles. In solubilization experiments, physophilin behaves as an integral membrane protein. Thus, a putative synaptic plasma membrane protein exhibits a specific interaction with one of the major membrane proteins of synaptic vesicles. This interaction may play a role in docking and/or fusion of synaptic vesicles to the presynaptic plasma membrane. 相似文献
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A Kumar S R Gudi S M Gokhale V Bhakuni C M Gupta 《Biochimica et biophysica acta》1990,1030(2):269-278
Rhesus monkey erythrocytes were subjected to heating at 50 degrees C for 5-15 min, and the heat-induced effects on the membrane structure were ascertained by analysing the membrane phospholipid organization and membrane skeleton dynamics and interactions in the heated cells. Membrane skeleton dynamics and interactions were determined by measuring the Tris-induced dissociation of the Triton-insoluble membrane skeleton (Triton shells), the spectrin-actin extractability at low ionic strength, spectrin self-association and spectrin binding to normal monkey erythrocyte membrane inside-out vesicles (IOVs). The Tris-induced Triton shell dissociation and spectrin-actin extractability were markedly decreased by the erythrocyte heating. Also, the binding of the heated erythrocyte membrane spectrin-actin with the IOVs was much smaller than that observed with the normal erythrocyte spectrin-actin. Further, the spectrin structure was extensively modified in the heated cells, as compared to the normal erythrocytes. Transbilayer phospholipid organization was ascertained by employing bee venom and pancreatic phospholipases A2, fluorescamine, and Merocyanine 540 as the external membrane probes. The amounts of aminophospholipids hydrolysed by phospholipases A2 or labeled by fluorescamine in intact erythrocytes considerably increased after subjecting them to heating at 50 degrees C for 15 min. Also, the fluorescent dye Merocyanine 540 readily stained the 15-min-heated cells but not the fresh erythrocytes. Unlike these findings, the extent of aminophospholipid hydrolysis in 5-min-heated cells by phospholipases A2 depended on the incubation time. While no change in the membrane phospholipid organization could be detected in 10 min, prolonged incubations led to the increased aminophospholipid hydrolysis. Similarly, fluorescamine failed to detect any change in the transbilayer phospholipid distribution soon after the 5 min heating, but it labeled greater amounts of aminophospholipids in the 5-min-heated cells, as compared to normal cells, after incubating them for 4 h at 37 degrees C. These results have been discussed to analyse the role of membrane skeleton in maintaining the erythrocyte membrane phospholipid asymmetry. It has been concluded that both the ATP-dependent aminophospholipid pump and membrane bilayer-skeleton interactions are required to maintain the transbilayer phospholipid asymmetry in native erythrocyte membrane. 相似文献