共查询到20条相似文献,搜索用时 15 毫秒
1.
We investigated whether the redistribution of surface membrane receptors is associated with rearrangement of integral membrane proteins. Using a newly developed process, which combines histochemical analysis with an immunofluorescence or immuno-electron microscopy-staining technique, we studied the redistribution of two membrane-bound enzymes, 5'-nucleotidase and ATPase, on mouse splenic lymphocytes and B lymphoma cells induced by anti-mouse immunoglobulin antibodies. Labeling and capping of the membrane surface immunoglobulin induced a similar rearrangement of both 5'-nucleotidase and ATPase from uniform distribution at 4 degrees C into 'patches' and caps at 37 degrees C. 相似文献
2.
3.
Ultrastructural localization of erythrocyte cytoskeletal and integral membrane proteins in Plasmodium falciparum-infected erythrocytes 总被引:4,自引:0,他引:4
C T Atkinson M Aikawa G Perry T Fujino V Bennett E A Davidson R J Howard 《European journal of cell biology》1988,45(2):192-199
The distributions of ankyrin, spectrin, band 3, and glycophorin A were examined in Plasmodium falciparum-infected erythrocytes by immunoelectron microscopy to determine whether movement of parasite proteins and membrane vesicles between the parasitophorous vacuole membrane and erythrocyte surface membrane involves internalization of host membrane skeleton proteins. Monospecific rabbit antisera to spectrin, band 3 and ankyrin and a mouse monoclonal antibody to glycophorin A reacted with these erythrocyte proteins in infected and uninfected human erythrocytes by immunoblotting. Cross-reacting malarial proteins were not detected. The rabbit sera also failed to immunoprecipitate [3H]isoleucine labeled malarial proteins from Triton X-100 and sodium dodecyl sulfate (SDS) extracts of infected erythrocytes. These three antibodies as well as the monoclonal antibody to glycophorin A bound to the membrane skeleton of infected and uninfected erythrocytes. The parasitophorous vacuole membrane was devoid of bound antibody, a result indicating that this membrane contains little, if any, of these host membrane proteins. With ring-, trophozoite- and schizont-infected erythrocytes, spectrin, band 3 and glycophorin A were absent from intracellular membranes including Maurer's clefts and other vesicles in the erythrocyte cytoplasm. In contrast, Maurer's clefts were specifically labeled by anti-ankyrin antibody. There was a slight, corresponding decrease in labeling of the membrane skeleton of infected erythrocytes. A second, morphologically distinct population of circular, vesicle-like membranes in the erythrocyte cytoplasm was not labeled with anti-ankyrin antibody. We conclude that membrane movement between the host erythrocyte surface membrane and parasitophorous vacuole membrane involves preferential sorting of ankyrin into a subpopulation of cytoplasmic membranes. 相似文献
4.
5.
Characterization of membrane proteins in detergent solutions. 总被引:56,自引:0,他引:56
6.
Detergent/polymer aqueous two-phase systems are studied as a fast, mild and efficient general separation method for isolation of labile integral membrane proteins. Mechanisms for phase behaviour and protein partitioning of both membrane-bound and hydrophilic proteins have been examined in a large number of detergent/polymer aqueous two-phase systems. Non-ionic detergents such as the Triton series (polyoxyethylene alkyl phenols), alkyl polyoxyethylene ethers (C(m)EO(n)), Tween series (polyoxyethylene sorbitol esters) and alkylglucosides form aqueous two-phase systems in mixtures with hydrophilic polymers, such as PEG or dextran, at low and moderate temperatures. Phase diagrams for these mixtures are shown and phase behaviour is discussed from a thermodynamic model. Membrane proteins, such as bacteriorhodopsin and cholesterol oxidase, were partitioned strongly to the micelle phase, while hydrophilic proteins, BSA and lysozyme, were partitioned to the polymer phase. The partitioning of membrane protein is mainly determined by non-specific hydrophobic interactions between detergent and membrane protein. An increased partitioning of membrane proteins to the micelle phase was found with an increased detergent concentration difference between the phases, lower polymer molecular weight and increased micelle size. Partitioning of hydrophilic proteins is mainly related to excluded volume effects, i.e. increased phase component size made the hydrophilic proteins partition more to the opposite phase. Addition of ionic detergent to the system changed the partitioning of membrane proteins slightly, but had a strong effect on hydrophilic proteins, and can be used for enhanced separation between hydrophilic proteins and membrane protein. 相似文献
7.
Immunoblotting of hydrophobic integral membrane proteins 总被引:4,自引:0,他引:4
For diagnosis and research purposes it is frequently desirable to measure by immunoblotting small amounts of proteins in complex mixtures such as tissue biopsy homogenates. Standard immunoblot procedures that give excellent results for soluble proteins unexpectedly gave low and irreproducible signals with some hydrophobic membrane proteins. We found that this was due to inefficient electrophoretic transfer to nitrocellulose, which could be corrected by modification of the transblot buffer. Hydrophobic integral membrane proteins of peroxisomes as well as other rat and human liver proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose filters. The nitrocellulose-bound proteins were detected both by staining and by immunoblotting with an antiserum against the 22-kDa integral membrane protein of peroxisomes plus 125I-labeled protein A. A modified transblot buffer with 0.7 M glycine and 25 mM Tris (pH 7.7) but no methanol allowed use of a much shorter transfer time and strikingly improved the electrophoretic transfer of membrane proteins such that a peroxisomal integral membrane protein could be easily detected in human liver biopsy homogenates. 相似文献
8.
Integral membrane proteins (IMPs) are essential components of the plasma and organellar membranes of the eukaryotic cell. Non-native IMPs, which can arise as a result of mutations, errors during biosynthesis or cellular stress, can disrupt these membranes and potentially lead to cell death. To protect against this outcome, the cell possesses quality control (QC) systems that detect and dispose of non-native IMPs from cellular membranes. Recent studies suggest that recognition of non-native IMPs by the QC machinery is correlated with the thermodynamic stability of these proteins. Consistent with this, small molecules known as chemical and pharmacological chaperones have been identified that stabilize non-native IMPs and enable them to evade QC. These findings have far-reaching implications for treating human diseases caused by defective IMPs. 相似文献
9.
《Micron and Microscopica Acta》1991,22(4):361-369
This review describes aspects of negative staining of isolated integral membrane proteins. Detergents play a central role in the isolation of membrane proteins and also in their solubility in aqueous solutions. Specimens of mixed micelles of membrane proteins and nonionic detergents can be easily prepared as long as the detergent concentration remains above the critical micellar concentration. Membrane proteins involved in the process of photosynthesis have been taken as examples to illustrate their interaction with different detergents. Upon negative staining, mixed micelles of membrane proteins and detergents show characteristic top and side view projections. On their sides, mixed micelles can easily aggregate into strings. 相似文献
10.
In eukaryotes, numerous complex sub-cellular structures exist. The majority of these are delineated by membranes. Many proteins are trafficked to these in order to be able to carry out their correct physiological function. Assigning the sub-cellular location of a protein is of paramount importance to biologists in the elucidation of its role and in the refinement of knowledge of cellular processes by tracing certain activities to specific organelles. Membrane proteins are a key set of proteins as these form part of the boundary of the organelles and represent many important functions such as transporters, receptors, and trafficking. They are, however, some of the most challenging proteins to work with due to poor solubility, a wide concentration range within the cell and inaccessibility to many of the tools employed in proteomics studies. This review focuses on membrane proteins with particular emphasis on sub-cellular localization in terms of methodologies that can be used to determine the accurate location of membrane proteins to organelles. We also discuss what is known about the membrane protein cohorts of major organelles. 相似文献
11.
Olga Vinogradova Frank Sönnichsen Charles R. Sanders II 《Journal of biomolecular NMR》1998,11(4):381-386
Translational diffusion coefficients and catalytic activities were measured for the integral membrane protein diacylglycerol kinase (DAGK) in a variety of types of detergent micelles. Despite the structural diversity of the detergents examined, the translational diffusion coefficients observed for DAGK spanned a fairly limited range of values: 2.7 to 4.7 (× 10-7cm2/s). No general correlation was observed between the diffusion coefficients for the detergent-DAGK aggregates and the sizes of the corresponding protein-free micelles. These results indicate that the effective molecular weights of the DAGK-detergent aggregates were determined more by the structural properties of the protein than by the properties of the detergents. The catalytic activity of DAGK in detergents having medium-length alkyl chains such as dodecylphosphocholine or decylmaltoside was usually observed to be substantially higher than in short-chain detergents such as octylphosphocholine or octylglucoside. Taken together, the diffusion and activity results indicate that medium-chain detergents are generally preferred for use in NMR studies of complex membrane proteins because they are no worse than short-chained detergents in terms of increasing the effective molecular weight of the protein of interest while they are considerably better at maintaining native-like protein conformation. Among the 10 detergents examined, only sodium dodecylsulfate was observed to be unable to support DAGK activity under any conditions examined, suggesting that this well-known protein denaturant should be used with care in studies of complex membrane proteins. 相似文献
12.
To selectively detect amphiphilic proteins from a mixture of proteins separated by SDS-polyacrylamide gel electrophoresis, the gel was electro-blotted through another polyacrylamide gel containing a non-ionic detergent (NP40) onto a nylon membrane filter. Most soluble proteins of E. coli passed through the detergent-containing gel, whereas a major fraction of the proteins from the cytoplasmic (inner) membrane, including the lactose and melibiose carrier proteins, were trapped in the detergent layer. The major outer membrane proteins, OmpA, OmpF and LamB, partitioned to the detergent layer only when solubilized at low temperature which avoids complete denaturation. This simple procedure, termed "detergent blotting", should have wide application in the study of integral membrane proteins. 相似文献
13.
Topogenic signals in integral membrane proteins 总被引:65,自引:0,他引:65
Integral membrane proteins are characterized by long apolar segments that cross the lipid bilayer. Polar domains flanking these apolar segments have a more balanced amino acid composition, typical for soluble proteins. We show that the apolar segments from three different kinds of membrane-assembly signals do not differ significantly in amino acid content, but that the inside/outside location of the polar domains correlates strongly with their content of arginyl and lysyl residues, not only for bacterial inner-membrane proteins, but also for eukaryotic.proteins from the endoplasmic reticulum, the plasma membrane, the inner mitochondrial membrane, and the chloroplast thylakoid membrane. A positive-inside rule thus seems to apply universally to all integral membrane proteins, with apolar regions targeting for membrane integration and charged residues providing the topological information. 相似文献
14.
Tate CG 《FEBS letters》2001,504(3):94-98
Recent successes in the determination of atomic resolution structures of integral membrane proteins have relied on purifying the proteins from abundant natural sources. In contrast, the majority of mammalian receptors, ion channels and transporters need to be overexpressed to obtain sufficient material for structural studies. This has often proved to be very difficult. Overexpression studies on a wide range of mammalian membrane proteins have shown that a few can be expressed functionally in bacteria, but many others require an insect or mammalian cell host for activity or high level expression. The serotonin transporter, which has been expressed in all the major hosts available, is a good example that has given insights into the problem of overexpressing mammalian membrane proteins for structural studies. 相似文献
15.
The size and detergent binding of membrane proteins. 总被引:32,自引:0,他引:32
S Clarke 《The Journal of biological chemistry》1975,250(14):5459-5469
Sucrose density gradient centrifugation has been used to measure the binding of Triton X-100 above its critical micellar concentration to a variety of purified membrane and non-membrane proteins. In addition, binding studies were done on the three proteins below the critical micellar concentration of detergent to distinguish between the interaction of proteins with detergent monomers and detergent micelles. A procedure is described for the calculation of the molecular weight of these Triton X-100 protein complexes and measurements were made for opsin, plasma low density lipoprotein, the (Na-+ plus K-+)-dependent adenosine triphosphatase, the human red blood cell major sialoglycoprotein (PAS-1) and the human red blood cell minor glycoprotein (bandIII). These proteins behave as monomers or dimers in detergent and bind between 0.28 and 1.12 g of detergent per g of protein. A general method is also present for calculating the molecular size and shape of impure membrane proteins in detergent. Finally, Triton X-100 was shown to replace bound Na dodecyl-SO4 on the minor glycoprotein of the red blood cell. 相似文献
16.
17.
18.
Signals between a cell and its environment are often transmitted through membrane proteins; therefore, many membrane proteins, including G protein-coupled receptors (GPCRs) and ion channels, are important drug targets. Structural information about membrane proteins remains limited owing to challenges in protein expression, purification and the selection of membrane-mimicking systems that will retain protein structure and function. This review describes recent advances in solution NMR applied to the structural study of integral membrane proteins. The examples herein demonstrate that solution NMR spectroscopy will play a unique role not only in structural analysis, but also drug discovery of membrane proteins. 相似文献
19.
《Molecular membrane biology》2013,30(5-8):156-178
AbstractSolid-state NMR is unique for its ability to obtain three-dimensional structures and to measure atomic-resolution structural and dynamic information for membrane proteins in native lipid bilayers. An increasing number and complexity of integral membrane protein structures have been determined by solid-state NMR using two main methods. Oriented sample solid-state NMR uses macroscopically aligned lipid bilayers to obtain orientational restraints that define secondary structure and global fold of embedded peptides and proteins and their orientation and topology in lipid bilayers. Magic angle spinning (MAS) solid-state NMR uses unoriented rapidly spinning samples to obtain distance and torsion angle restraints that define tertiary structure and helix packing arrangements. Details of all current protein structures are described, highlighting developments in experimental strategy and other technological advancements. Some structures originate from combining solid- and solution-state NMR information and some have used solid-state NMR to refine X-ray crystal structures. Solid-state NMR has also validated the structures of proteins determined in different membrane mimetics by solution-state NMR and X-ray crystallography and is therefore complementary to other structural biology techniques. By continuing efforts in identifying membrane protein targets and developing expression, isotope labelling and sample preparation strategies, probe technology, NMR experiments, calculation and modelling methods and combination with other techniques, it should be feasible to determine the structures of many more membrane proteins of biological and biomedical importance using solid-state NMR. This will provide three-dimensional structures and atomic-resolution structural information for characterising ligand and drug interactions, dynamics and molecular mechanisms of membrane proteins under physiological lipid bilayer conditions. 相似文献