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1.
Cytoplasmic (inner) and outer membranes of Escherichia coli K-12 were isolated with fair separation from each other, and their chemical, biological and morphological properties were compared. The outer membrane isolated was composed of protein, phospholipid and lipopolysaccharide as major high molecular weight components in a ratio of 100:82:34 (by wt), and was solubilized in 1% sodium dodecyl sulfate without any sediments. In polyacrylamide disc gel electrophorsis with the sodium dodecyl sulfate-solubilized outer membrane, six proteins were found to be major. Removal of sodium dodecyl sulfate from the sodium dodecyl sulfate-solubilized outer membrane by dialysis induced a self-assembly to form a membrane structure which has similar properties in chemical composition, density and morphology to those of the original outer membrane.  相似文献   

2.
Proteins from the outer membrane of Escherichia coli were studied on a ureadodecyl sulfate polyacrylamide gel by electrophoresis. A polyacrylamide gel containing sodium dodecyl sulfate and urea gave an excellent resolution of outer membrane proteins. Seventeen protein bands were reproducibly observed on a gel. By use of Sephadex G-200, DEAE-cellulose and polyacrylamide gel, eight proteins were purified to near homogeneity. Five of them were found to be heat-modifiable proteins. The behavior of these purified proteins was studied on a polyacrylamide gel under three different electrophoretic conditions, which had been used for the analysis of cell envelope proteins. Thus correspondence was made between these purified proteins and envelope proteins reported by other investigators.  相似文献   

3.
The 30 S ribosomal subunits of Escherichia coli were treated with methyl-4-mercaptobutyrimidate and oxidized to promote the formation of intermolecular disulfate bonds between neighboring proteins. Attention was focused on protein dimers, which were partially purified either by stepwise extraction of the 30 S particle with LiCl or by polyacrylamide/urea gel electrophoresis of the total crosslinked protein. Protein fractions were then analyzed by polyacrylamide/ sodium dodecyl sulfate diagonal gel electrophoresis. Final identification of the components of crosslinked protein pairs, indicated by molecular weight analysis, was accomplished by two-dimensional polyacrylamide/urea gel electrophoresis. The identification of 21 protein pairs is presented, 14 of which have not been reported previously.  相似文献   

4.
Protein compositions of the inner and outer membranes of Escherichia coli K-12 have been analyzed by two-dimensional gel electrophoresis in which proteins are separated according to apparent isoelectric point (first dimension) and to apparent molecular weight (second dimension). Membrane proteins except for a pair of major outer membrane proteins (proteins Ia and Ib) were found to be solubilized effectively by lysis buffer containing urea, Triton X-100, ampholines and 2-mercaptoethanol. The latter two proteins could be solubilized after precipitation of membrane fraction with trichloroacetic acid; they formed a pair of spots at an acidic region on the electropherogram. Another major protein of the outer membrane, protein II, was also identified. Most of the inner and outer membrane proteins were shown to be focused at a pH range between 4 and 6.5. Specific protein patterns characteristic for both the inner and outer membranes could thous be visualized by the present system. At least 120 and 50 protein species were detected for the inner and outer membranes, respectively.  相似文献   

5.
1.
1. The proteins of the outer membrane of Escherichia coli were characterized by polyacrylamide gel electrophoresis and their molecular weights determined.  相似文献   

6.
Cytoplasmic and outer membranes of Caulobacter crescentus were separated by isopycnic sucrose gradient centrifugation into two peaks with buoyant densities 1.22 and 1.14 g/cm3. These peaks were identified as outer and cytoplasmic membranes by the enrichment of malate dehydrogenase and NADH oxidase in the lower density peak and the presence of flagellin, a cell surface protein, in the heavier peak. The identity of the heavier peak as outer membrane was confirmed by labeling of cells with diazotized [35S]sulfanilic acid, a reagent that does not penetrate intact cells. Under these conditions only outer membrane proteins were substituted by the sulfanilic acid. The distribution of proteins between the cytoplasmic and outer membranes were examined by the analysis of [35S]methionine-labeled membranes by SDS-polyacrylamide and two-dimensional gel electrophoresis. These results showed that the inner and outer membranes contain approximately equal numbers of proteins, and that the distribution of these proteins between the two layers is highly asymmetric. Although many of the proteins could be assigned to one or the other membrane fraction, a number of the outer membrane proteins in the 32 000–100 000 molecular weight range frequently contaminate the inner membrane fractions. The implications of these results for membrane isolation and separation in C. crescentus are discussed.  相似文献   

7.
Analysis of inner and outer pea (Pisum sativum var. Laxtons Progress No. 9) chloroplast envelope membranes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that, although the two membranes have distinct polypeptide compositions, there are several comigrating polypeptides in the two membrane fractions. To determine whether these comigrating polypeptides were identical by criteria other than molecular weight, the membrane proteins were analyzed by two-dimensional gel electrophoresis. The results demonstrated that an 86-kilodalton band found in both membranes represents at least two different polypeptides, one an outer membrane protein and the other an inner membrane protein. Several other polypeptide bands found in both membranes appear to be of stromal origin. Two of these polypeptides were shown to be the large and small subunits of ribulose 1,5-bisphosphate carboxylase. The large subunit was identified by two-dimensional electrophoresis of envelope membranes to which stromal proteins were added. Additionally, the large and small subunits of ribulose 1,5-bisphosphate carboxylase were immunologically identified using an electrophoretic transfer procedure coupled with an enzyme-linked immunosorbent assay. Various treatments, including sonication, resulted in no significant loss of the stromal polypeptides from the outer envelope membranes. Based on these results, it is suggested that the stromal proteins are not simply bound to the outer surface of the vesicles.  相似文献   

8.
S Kabir 《Microbios》1977,20(79):47-62
The number, nature and organization of the outer membrane proteins of Salmonella typhimurium have not yet been resolved. Therefore these proteins were isolated using a concentrated solution of guanidine hydrochloride and studied using different analytical techniques. Upon chromatography on Sephadex G-200 four fractions were obtained. Only the fraction containing a protein of molecular weight 13,000 produced immunoprecipitation reactions with the antisera raised against the whole bacteria. On polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate, 7 major proteins were found, with molecular weights between 13,000 and 43,000. Isoelectric focusing on 4.6% polyacrylamide gels resolved the outer membrane proteins into 10 bands with apparent isoelectric points between 5.0 and 8.4. Finally these proteins could be further resolved into as many as 50 spots where a two-dimensional electrophoresis was carried out with isoelectric focusing in the first dimension, and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate in the second dimension. These results demonstrated that the outer membrane proteins of S. typhimurium are extremely heterogeneous. To investigate the mode of organization of lipopolysaccharides in the outer membrane, the membrane proteins were separated by the liquid isoelectric focusing technique. Lipopolysaccharides were primarily found to be associated with a protein of isoelectric point 7.8.  相似文献   

9.
ATP-binding cassette (ABC) transporters are integral membrane proteins that actively transport molecules across cell membranes. In Escherichia coli they consist primarily of import systems that involve in addition to the ABC transporter itself a substrate binding protein and outer membrane receptors or porins, and a number of transporters with varied functions. Recent crystal structures of a number of ATPase domains, substrate binding proteins, and full-length transporters have given new insight in the molecular basis of transport. Bioinformatics approaches allow an approximate identification of all ABC transporters in E. coli and their relation to other known transporters. Computational approaches involving modeling and simulation are beginning to yield insight into the dynamics of the transporters. We summarize the function of the known ABC transporters in E. coli and mechanistic insights from structural and computational studies.  相似文献   

10.
Freeze etching showed that the loss of each of the major outer membrane proteins b, c or d in mutants of Escherichia coli K12 does not influence the morphology of fracture faces of the outer membrane.Mutants that possess a heptose-deficient lipopolysaccharide and which in addition are deficient in one or more major outer membrane proteins exhibit a reduction in the number of intramembranous particles of the outer membrane.Moreover it was shown that lipid phase transitions induce a lateral lipid protein separation in the outer membrane, similar to that found in the cytoplasmic membrane.  相似文献   

11.
Membrane vesicles released by Escherichia coli O157:H7 into culture medium were purified and analyzed for protein and DNA content. Electron micrographs revealed vesicles that are spherical, range in size from 20 to 100 nm, and have a complete bilayer. Analysis of vesicle protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrates vesicles that contain many proteins with molecular sizes similar to outer membrane proteins and a number of cellular proteins. Immunoblot (Western) analysis of vesicles suggests the presence of cell antigens. Treatment of vesicles with exogenous DNase hydrolyzed surface-associated DNA; PCR demonstrated that vesicles contain DNA encoding the virulence genes eae, stx1 and stx2, and uidA, which encodes for β-galactosidase. Immunoblot analysis of intact and lysed, proteinase K-treated vesicles demonstrate that Shiga toxins 1 and 2 are contained within vesicles. These results suggest that vesicles contain toxic material and transfer experiments demonstrate that vesicles can deliver genetic material to other gram-negative organisms.  相似文献   

12.
1. Stimulation of the Escherichia coli ATPase activity by urea and trypsin shows that the ATPase activity both in the membrane-bound and the solubilized form is partly masked.2. A protein, inhibiting the ATPase activity of Escherichia coli, can be isolated by sodium dodecyl sulphate polyacrylamide gel electrophoresis of purified ATPase. The inhibitor was identified with the smallest of the subunits of E. coli ATPase.3. The molecular weight of the ATPase inhibitor is about 10 000, as determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis and deduced from the amino acid composition.4. The inhibitory action is independent of pH, ionic strength or the presence of Mg2+ or ATP.5. The ATPase inhibitor is heat-stable, insensitive to urea but very sensitive to trypsin degradation.6. The Escherichia coli ATPase inhibitor does not inhibit the mitochondrial or the chloroplast ATPase.  相似文献   

13.
The proteins of the outer membrane of Neisseria gonorrhoeae play an important role in the serotyping system defined by K. H. Johnston et al. (J. Exp. Med. 143:741–758, 1976). This study attempted to delineate the molecular arrangement of the major proteins of the outer membrane of the gonococcus by using three approaches. First, natural protein-protein relationships were demonstrated by symmetrical, two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Second, proteins exposed on the surface of outer membrane vesicles were cross-linked by using the bifunctional reagents dimethyl-3,3′-dithiobispropionimidate and dithiobis[succinimidyl propionate]. Third, specific antigen-antibody interactions on the surface of membrane vesicles were analyzed by radioautographic techniques. The major proteins of the outer membrane of the gonococcus were defined, and a nomenclature was devised to take into account the effects of heat and reducing agents on the resolution of these proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Results of cross-linking experiments strongly suggest that two of the major proteins of the gonococcal outer membrane (proteins 1 and 3) form a hydrophobically associated trimeric unit in situ which can be stabilized by selective cross-linking reagents. Results substantiated that these proteins are responsible for imparting serotypic specificity.  相似文献   

14.
The Mu phage virion contains tail-spike proteins beneath the baseplate, which it uses to adsorb to the outer membrane of Escherichia coli during the infection process. The tail spikes are composed of gene product 45 (gp45), which contains 197 amino acid residues. In this study, we purified and characterized both the full-length and the C-terminal domains of recombinant gp45 to identify the functional and structural domains. Limited proteolysis resulted in a Ser64-Gln197 sequence, which was composed of a stable C-terminal domain. Analytical ultracentrifugation of the recombinant C-terminal domain (gp45-C) indicated that the molecular weight of gp45-C was about 58 kDa and formed a trimeric protomer in solution. Coprecipitation experiments and a quartz crystal microbalance (QCM) demonstrated that gp45-C irreversibly binds to the E. coli membrane. These results indicate that gp45 shows behaviors similar to tail-spike proteins of other phages; however, gp45 did not show significant sequence homology with the other phage tail-spike structures that have been identified.  相似文献   

15.
Two types of proteins are discussed in their role of facilitating the transport of maltose and sn-glycerol-3-phosphate in E. coli. The first protein is the receptor for phage δ, known to be an outer membrane protein. By facilitating the diffusion of maltose and the higher maltodextrins through the outer membrane the effect of the δ receptor is to decrease the Km of the transport system without influencing the Vmax of substrate flux. The second protein is a periplasmic protein that is induced by growth on glycerol and is essential for transport of sn-glycerol-3-phosphate in whole cells but not in membrane vesicles. This protein has solely been identified by the use of a two-dimensional polyacrylamide gel electrophoresis of periplasmic proteins in wild-type and mutants defective in sn-glycerol-3-phosphate transport.  相似文献   

16.
The expression of cloned bacteriophage phi X174 lysis gene E was analyzed in minicells of Escherichia coli using two-dimensional gel electrophoresis. Beside the 10-11-kDa protein-E, at least two additional protein bands were detected, associated with the inner membrane, which showed the same isoelectric point as E. To clarify whether these proteins were E-specific, two different antibodies directed against a beta-galactosidase-E' hybrid protein and a synthetic oligopeptide corresponding to the C-terminal end of protein-E were raised. Immunoadsorption studies with anti-peptide-specific antibodies resulted in the detection of protein-E as well as in the detection of proteins of higher molecular weight. Two of these protein bands were positively recognized by anti beta-galactosidase-E' antibodies. The latter protein bands had the same molecular weight as the putative protein-E bands detected by two-dimensional gel electrophoresis indicating that these bands represent protein-E-specific oligomers. These data support the idea that an E-specific oligomeric structure penetrating the inner and outer membrane of E. coli is formed during the lytic action of protein-E.  相似文献   

17.
We used SDS-polyacrylamide gel electrophoresis to investigate the outer membrane proteins (OMPs) band composition of 19 Escherichia coli K1 strains that have spontaneously lost the ability to form K1 polysaccharide capsule (E. coli K1?) and demonstrated different degrees of susceptibility to the bactericidal action of normal human serum. Presented results showed that there were differences between E. coli K1? strains in OMPs expressing capacity. The analysis performed on OMPs has not revealed a direct association between the different OMPs band composition and the susceptibility of these strains to the serum.  相似文献   

18.
A high molecular weight protein aggregate, which agglutinates yeast cells, human epithelial cells and mouse lymphocytes, was isolated from extracts of Escherichia coli by differential centrifugation and gel filtration. The agglutination is specifically inhibited by d-mannose and its derivatives, the best inhibitor being p-nitrophenyl α-d-mannoside. Sodium dodecyl sulfate gel electrophoresis showed that the lectin consists of protein subunits with identical Mr of ~36500. The amino acid composition of the purified lectin is different from that reported for the type I pili protein, the K99 antigen and the major outer membrane protein Ia of E. coli. The protein appears to be located on the bacterial surface, and is probably involved in the mannose-specific adherence of E. coli to eukaryotic cells.  相似文献   

19.
An assessment study was carried out to evaluate the performance of the low-angle laser light scattering technique combined with high-performance gel chromatography in the presence of a nonionic surfactant, octaethyleneglycol n-dodecyl ether, precision differential refractometry and ultraviolet photometry. It was found that the combined technique is highly promising as a method for the determination of the molecular weight of a membrane protein solubilized by the surfactant. For trial, molecular weights of the following membrane proteins of Escherichia coli, both solubilized in oligomeric forms, were measured; porin that forms the transmembrane diffusion pore in the outer membrane, and lambda-receptor protein that facilitates the diffusion of maltose-maltodextrins across the outer membrane. The result obtained indicates that both porin and lambda-receptor protein exist as trimers in the surfactant solution.  相似文献   

20.
Ruthenium complexes with similar octahedral structures but different intrinsic inductive properties significantly influence the total cellular protein distributions, which may affect different metabolic pathways. A systematic study of the relationship between ruthenium complexes and Escherichia coli was undertaken, using two-dimensional gel electrophoresis analysis and the identification of various proteins by mass data mining. Based on the low similarities (< 40%) between the total protein distributions, the inductive properties of the ruthenium complexes are relevant to the formation of the protein-Ru interaction in addition to the Ru-DNA interaction. Two major protein functions in E. coli BL21 that were reduced by compound 1 were oxidoreductases and transporters, corresponding to 29% and 25% of the 24 down-regulated proteins. The main biological processes of the proteins down-regulated by compound 1 were related to carbohydrate reactions, including in transport, tricarboxylic acid (TCA) cycle, glycolysis, and gluconeogenesis. All four ruthenium complexes shared similar up-regulated proteins, including clpB and kpyk1, and down-regulated similar proteins, including ompA and ybbN. This result supports that the presence of Ru-protein interactions is a major factor affecting bacteria growth, and particularly transport and carbohydrate-related reactions.  相似文献   

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