Proteins of the cell envelope of a marine pseudomonad, Pseudomonas BAL-31

The protein composition of the envelope fraction of Pseudomonas BAL-31 was studied by polyacrylamide gel electrophoresis. Two major polypeptides of molecular weights 130 000 and 110 000 were found. These two polypeptides, which account for as much as 40–50% of the total protein of the envelope, are associated with the outer membrane. One of these proteins might be a glycoprotein. The inner membrane contains a more heterogeneous collection of smaller polypeptides.     

Outer membrane of Pseudomonas aeruginosa: heat- 2-mercaptoethanol-modifiable proteins.

A number of polyacrylamide gel systems and solubilization procedures were studied to define the number and nature of "major" polypeptide bands in the outer membrane of Pseudomonas aeruginosa. It was shown that five of the eight major outer membrane proteins were "heat modifiable" in that their mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was determined by the solubilization temperature. Four of these heat-modifiable proteins had characteristics similar to protein II of the Escherichia coli outer membrane. Addition of lipopolysaccharide subsequent to solubilization caused reversal of the heat modification. The other heat-modifiable protein, the porin protein F, was unusually stable to sodium dodecyl sulfate. Long periods of boiling in sodium dodecyl sulfate were required to cause conversion to the heat-modified form. This was demonstrated both with outer membrane-associated and purified lipopolysaccharide-depleted protein F. Furthermore, lipopolysaccharide treatment had no effect on the mobility of heat-modified protein F. Thus it is concluded that protein F represents a new class of heat-modifiable protein. It was further demonstrated that the electrophoretic mobility of protein F was modified by 2-mercaptoethanol and that the 2-mercaptoethanol and heat modification of mobility were independent of one another. The optimal conditions for the examination of the outer membrane proteins of P. aeruginosa by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis are discussed.     

Two-Dimensional Polyacrylamide Gel Electrophoresis of Envelope Proteins of Escherichia coli

A method of separating envelope proteins by two-dimensional polyacrylamide gel electrophoresis is described. Escherichia coli envelopes (inner and outer membranes) were prepared by French pressing and washed by repeated centrifugation. Membrane proteins were solubilized with guanidine thiocyanate and were dialyzed against urea prior to two-dimensional electrophoretic analysis. The slab gel apparatus and conditions were similar to the technique developed by Metz and Bogorad (1974) for the separation of ribosomal proteins. This separation occurs in 8 M urea for the first dimension and in 0.2% sodium dodecyl sulfate for the second dimension. The technique separates about 70 different membrane proteins in a highly reproducible fashion according to both intrinsic charge and molecular weight. Some examples of alterations in the membrane protein pattern are demonstrated. These alterations are caused by a mutation affecting a sugar transport system and by growth in the presence of D-fucose, inducer of the transport system. A further example of membrane protein changes introduced by growth at the nonpermissive temperature of a temperature-sensitive cell division mutant is shown. Finally, it is demonstrated that the major outer membrane component of Escherichia coli K-12 contains more than four proteins of similar molecular weight.     

Isolation,solubilization and reaggregation of outer membrane of Escherichia coli

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.     

Purification of protein A, an outer membrane component missing in Escherichia coli K-12 ompA mutants.

Outer membrane materials prepared from an Escherichia coli ompA (tolG) strain do not contain one of the major outer membrane proteins found in ompA+ strains. This protein has been purified in high yield from detergent-solubilized cell envelope material prepared from an ompA+ strain by preparative electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate. The purified protein is homogeneous in three electrophoretic systems, contains 2 mol of reducing sugar/mol of peptide and has alanine as the N-terminal amino acid. The amino acid composition is nearly identical to outer membrane protein II or B purified by others from incompletely solubilized cell envelope material. Thus, the fraction of outer membrane protein II or B that is difficult to solubilize is identical with the more readily solubilized fraction.     

Purification of the colicin I receptor

The colicin I outer membrane receptor was solubilized from the cell envelope of Escherichia coli K12 by extraction with Triton X-100 and purified to homogeneity by a combination of ion exchange and gel filtration chromatography as well as isoelectric focusing. The receptor was isolated as a single polypeptide and retained capacity to form a complex with pure colicin. The apparent molecular weight of the receptor as determined by polyacrylamide gel electrophoresis in sodium dodecy sulfate was 74,000 or 54,000 depending on whether the preparation was boiled or not in sodium dodecyl sulfate, respectively, prior to electrophoresis. Isoelectric focusing of the receptor in the presence of Triton X-100 revealed that the protein was slightly acidic (pI 4.75).     

Fractionation and Analysis of Polypeptides of Euglena gracilis Chloroplasts

Intact Euglena gracilis chloroplasts, purified on gradients of silica sol, were lysed osmotically and fractionated by centrifugation on discontinuous gradients of sucrose into their soluble, envelope membrane, and thylakoid membrane components. The proteins of the different subchloroplast fractions, as well as those of whole chloroplasts, were analyzed by electrophoresis on sodium dodecyl sulfate polyacrylamide gels. The polypeptide profile of each fraction was distinctive and was in general similar to the profile obtained for analogous fractions of the chloroplasts of higher plants.     

Identification and preliminary characterization of Vibrio cholerae outer membrane proteins.

Outer membrane proteins of Vibrio cholerae were purified by sucrose density centrifugation and Triton X-100 extraction at 10 mM Mg2+. The proteins were separated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. V. cholerae outer membrane proteins presented a unique pattern when compared with the patterns of other gram-negative rods. There were 8 to 10 major bands (Mr 94,000 to 27,000), with most of the protein located in band 5 (Mr approximately 45,000), which thus appears to be the major structural protein of the outer membrane. Lipid and carbohydrate were associated with band 6.     

Proteins of rod outer segments of toad retina: binding with calmodulin and with GTP

Proteins of purified rod outer segments from toad retina were analysed by electrophoresis on polyacrylamide gel containing sodium dodecyl sulfate. The binding of proteins with calmodulin and with guanosine triphosphate was studied by electroblotting the proteins resolved by electrophoresis onto nitrocellulose sheets and by incubating the blots with labelled ligands. The results indicate that rod outer segments from toad retina contain nine proteins which bind to calmodulin and one protein, different from transducin, that binds to guanosine triphosphate.     

Analysis of acid extractable bean leaf proteins induced by mercuric chloride treatment and alfalfa mosaic virus infection. Partial purification and characterization

Mercuric chloride treatment of Phaseolus vulgaris (var. ‘Saxa’) leaves, induces the synthesis of four new soluble proteins extractable at pH 2.8. The molecular weights of these proteins were found to be 17 000 for pathogenesis related (PR) 1 and PR 2 proteins, 28 000 for PR 3 protein and 32 000 for PR 4 protein, when determined by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. In Alfalfa Mosaic Virus (AMV)-infected bean leaves only three new soluble proteins were found, corresponding to the mercuric chloride-induced PR 1, PR 3 and PR 4 proteins. The four mercuric chloride-induced proteins were purified by a technique including an ammonium sulfate fractionation and a preparative polyacrylamide gel electrophoresis. Some biochemical and serological properties of these proteins have been studied.     

Membrane proteins PmpG and PmpH are major constituents of Chlamydia trachomatis L2 outer membrane complex

The outer membrane complex of Chlamydia is involved in the initial adherence and ingestion of Chlamydia by the host cell. In order to identify novel proteins in the outer membrane of Chlamydia trachomatis L2, proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. By silver staining of the protein profile, a major protein doublet of 100-110 kDa was detected. In-gel tryptic digestion and matrix-assisted laser desorption/ionization mass spectrometry identified these proteins as the putative outer membrane proteins PmpG and PmpH.     

Demonstration of a missing outer membrane protein in tolG mutants of Escherichia cell

A class of Escherichia coli mutants called tolG are specifically tolerant to bacteriocin JF246. Cell envelopes were prepared from three independent spontaneous E. coli. tolG mutants and the parental strain (tolG⁺). Electrophoresis of these preparations in polyacrylamide gels containing sodium dodecyl sulfate showed that the tolG strains lacked a cell envelope protein found in the tolG⁺ strain. It was estimated that this protein accounted for 10% of the total cell envelope proteins by densitometer tracings of gels stained with Fast Green. Membrane fractionation by isopycnic centrifugation in a sucrose density gradient showed that this protein was located in the outer membrane of tolG⁺ cells. Genetic studies using conjugation, transduction and reversion showed that, in the limited number of recombinants or revertants studied, strains exhibiting the tolerant phenotype lacked the outer membrane protein, whereas the protein was present in bacteriocin-sensitive strains.     

The 24kDa outer envelope membrane protein from spinach chloroplasts: molecular cloning,in vivo expression and import pathway of a protein with unusual properties

The 24 kDa outer envelope membrane protein of spinach chloroplasts (omp24) represents a major constituent of this membrane. Sequences of tryptic and endoprotease Glu-C peptides derived from omp24 allowed the design of oligonucleotides which were used to generate a DNA fragment by polymerase chain reaction using spinach cDNA as template. This fragment served as a probe to screen a cDNA library for a full-length clone of the omp24 coding sequence. The protein predicted from the complete sequence only has 148 amino acids and a molecular mass of 16294 Da. It is an acidic protein (calculated isoelectric point 4.8) with a high content of proline residues. Expression of the coding sequence in Escherichia coli and characterization of the purified recombinant protein produced revealed that the overestimation of its molecular mass by SDS-PAGE (ca. 25 kDa) is due to its abnormal amino acid composition. Despite its rather low hydrophobicity (polarity index 49%), omp24 appears to be deeply embedded in the outer membrane. Insertion of omp24 into the membrane proceeds almost independently of surface receptors or targeting sequence but, in contrast to other known outer envelope membrane proteins, is stimulated by ATP.Abbreviations CNBr cyanogen bromide - IP isoelectric point - NCS N-chlorosuccinimide - NTA nitrilotriacetic acid - omp24 outer envelope membrane protein of spinach chloroplasts - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - SV8 protease Staphylococcus aureus V8 protease (Endoprotease Glu-C) - TPT chloroplast triose phosphate/phosphate translocator     

Lipoprotein from the outer membrane of Escherichia coli: purification, paracrystallization, and some properties of its free form.

In the envelope of Escherichia coli, is a lipoprotein of molecular weight 7,200 as a major envelope protein. This lipoprotein was previously shown to exist in two different forms in the outer membrane of E. coli: the free form and the boundform, which is covalently linked to the peptidoglycau. The free form of the lipoprotein has been purified and paracrystallized by adding acetone to a sodium dodecyl sulfate solution in the presence of magnesium ion. The paracrystals were needle shaped. An electron micrograph of the negatively stained paracrystals showed a highly ordered ultrastructure. The chemical structure of the free form was compared with that of the bound form by (i) the amino acid composition, (ii) the fatty acid composition, and (iii) the peptide analysis after cyanogen bromide cleavage. The alpha-helical content of the free form of the lipoprotein was measured from the circular dichroism spectrum of the lipoprotein in 0.01% sodium dodecyl sulfate and found to be 87%. Using the purified lipoprotein as antigen, antiserum against the free form of the lipoprotein was obtained. Immunoprecipitation of the lipoprotein with the antiserum was found to be very specific, since only the free form of the lipoprotein was found as a major peak when the antiserum was reacted with the whole envelope proteins solubilized in 0.2% sodium dodecyl sulfate, and the immunoprecipitate thus formed was analyzed by polyacrylamide gel electrophoresis.     

Structural Polypeptides of the Granulosis Virus of Plodia interpunctella

Techniques were developed for the isolation and purification of three structural components of Plodia interpunctella granulosis virus: granulin, enveloped nucleocapsids, and nucleocapsids. The polypeptide composition and distribution of protein in each viral component were determined by sodium dodecyl sulfate discontinuous and gradient polyacrylamide slab gel electrophoresis. Enveloped nucleocapsids consisted of 15 structural proteins ranging in molecular weight from 12,600 to 97,300. Five of these proteins, having approximate molecular weights of 17,800, 39,700, 42,400, 48,200, and 97,300, were identified as envelope proteins by surface radioiodination of the enveloped nucleocapsids. Present in purified nucleocapsids were eight polypeptides. The predominant proteins in this structural component had molecular weights of 12,500 and 31,000. Whereas no evidence of polypeptide glycosylation was obtained, six of the viral proteins were observed to be phosphorylated.     

Analysis of pea chloroplast inner and outer envelope membrane proteins by two-dimensional gel electrophoresis and their comparison with stromal proteins

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.     

Molecular characterization of Lactobacillus casei strains

Abstract The monoclonal antibody LA7 was raised against the species-specific Borrelia burgdorferi lipoprotein P22 (= IPLA7), which induces antibody formation in patients with Lyme arthritis. It is composed of 194 amino acids with a calculated molecular mass of 21.8 kDa. Its gene on the linear chromosome is 582 nucleotides in length. The aim of this study was to localize the protein P22 by immune electron microscopy. Immunolabeling of Borrelia burgdorferi with LA7 and an anti-mouse immunogold conjugate proved that P22 is an outer membrane protein. This finding was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis of the outer envelope fraction, which contained 99% of the P22 proteins.     

The lectin binding sites on the membranes of the nuclear envelope, mitochondria and the cell surface of human lymphoblastoid cells

The membranes of the cell surface, the endoplasmic reticulum, outer and inner mitochondrial leaflet and nuclear envelope were isolated from three human lymphoblastoid cell lines. Membrane components were separated by dodecyl sulfate polyacrylamide gel electrophoresis and the gels incubated with the radioiodinated lectins from lentil, castor bean, scarlet runner bean, gorse seed and Roman snail. After gel slicing and counting, the molecular weights of the lectin binding sites were determined. About 20 glycoproteins were identified as constituents of the plasma membrane, a similar glycoprotein distribution was observed in the endoplasmic reticulum. The outer mitochondrial membrane contained some impurities from the plasma membrane, the inner mitochondrial membrane lacked specific lectin receptors. Two prominent glycoproteins with molecular weights of 70 000 and 60 000 were identified with the castor bean lectin in the nuclear envelope.     

Membrane proteins of Rhodopseudomonas spheroides III. Isolation,purification, and characterization of cell envelope proteins

Cell envelopes (cell wall and cell membrane) from aerobically grown cells of Rhodopseudomonas spheroides were isolated and purified by a combination of differential centrifugation and centrifugation through 40% sucrose. Cell envelope protein from aerobically grown cells was resolved by dodecyl sulphate-polyacrylamide gel electrophoresis. Biochemical characterization of selected envelope membrane proteins demonstrated heterogeneity between different protein species. Amino acid analyses of individual proteins revealed between 50–60 mole% nonpolar residues.Envelope membranes derived from anaerobically grown cells were also isolated and purified by a combination of differential centrifugation, column chromatography on Sepharose 2B, and centrifugation in 40% sucrose. The dodecyl sulphate-polyacrylamide gel patterns of anaerobic and aerobic envelope membrane proteins were very similar and the results suggest a common protein structure.     

Investigations on the outer membrane proteins of Salmonella typhimurium.

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.