An improved procedure for polypeptide analysis of radiolabeled antigens resolved by crossed immunoelectrophoresis and its application to the study of inner and outer membranes of <i>Escherichia coli</i>

Owen, Philip

doi:10.1002/elps.1150070103

Cited by 9 publications

(8 citation statements)

References 40 publications

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“…It could also be argued that the complex is an artifact caused by noncovalent association following detergent solubilization. If this is the case, the association is remarkably specific, since a comprehensive analysis of membrane antigens resolved by CIE for E. coli (23,25) has failed to detect any evidence for random association of polypeptides following detergent solubilization. Cross-linking experiments are presently being undertaken to resolve this issue.…”

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confidence: 99%

“…To establish that antigen 43 is indeed a novel protein complex, it is necessary to rule out a relationship between the constituent polypeptides and other documented E. coli protein antigens which possess similar molecular weights and properties and which are capable of an association with outer membrane fractions, e.g., flagellin and soluble common protein antigen (monomer Mr, about 60,000 in both instances [1,25]), the BtuB protein (Mr, 60,000 [29]), the ToIC protein (Mr, 55,000 [17]), and the heat-modifiable OmpA protein (15) (ii) Common protein antigen. This contaminating antigen generated a CIE immunoprecipitate (4 Fig.…”

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confidence: 99%

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Identification and partial characterization of a novel bipartite protein antigen associated with the outer membrane of Escherichia coli

Owen¹,

Caffrey²,

Josefsson³

1987

J Bacteriol

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A study by crossed immunoelectrophoresis performed in conjunction with precipitate excision and polypeptide analysis identified a new antigen complex in the envelope of Escherichia coli ML308-225. This antigen corresponds to antigen 43 in the crossed immunoelectrophoresis profile of membrane vesicles (P. Owen and H. R. Kaback, Proc. Natl. Acad. Sci. USA 75:3148-3152, 1978). Immunoprecipitation experiments conducted with specific antiserum revealed that the complex was expressed on the cell surface and that it contained, in equal stoichiometry, two chemically distinct polypeptides termed a and (Mrs of 60,000 and 53,000, respectively). The polypeptide was heat modifiable, displaying an apparent Mr of 37,000 when solubilized at temperatures below 70°C. Analysis of fractions obtained following cell disruption, isopycnic centrifugation, and detergent extraction indicated that both a and polypeptides were components of the outer membrane. The two polypeptides were not linked by disulfide bonds, and neither was peptidoglycan associated. The complex contained no detectable lipopolysaccharide, enzyme activity, fatty acyl groups, or other cofactors. Neither correlated with E. coli proteins of similar molecular weight which had previously been shown to be associated with the outer membrane. Antibodies were raised to individual a and I1 polypeptides. Each of these sera was shown to be subunit specific when tested against denatured membrane proteins. In contrast, each immunoglobulin preparation coprecipitated both a and polypeptides when tested against undenatured proteins derived from Triton X-100-treated membranes. The results reveal the presence of a novel bipartite protein antigen in the outer membrane of E. coli.The outer membrane of Escherichia coli is known to contain a number of major protein species (1, 15). Several of these are porins involved in the passive accumulation of small hydrophilic solutes (2,19). Others, such as the ironregulated outer membrane proteins and the vitamin B12 receptor, are involved in the TonB-dependent uptake of more specific solutes (4). Other major proteins of the E. coli outer membrane include the Braun lipoprotein (the Lpp protein), peptidoglycan-associated lipoprotein, and the ompA gene product. In part at least, these appear to play a structural role for the cell envelope (1, 15). Several other minor proteins and enzymes have also been identified and studied (10,17,20,35). In addition to the above components, there exist in the outer membratne several additional proteins about which considerably less is known, e.g., protein III, an 83-kilodalton iron-regulated protein, and several proteins in the 60-to 40-kilodalton range (15,26).In an attempt to identify and characterize the major antigens of the cell envelope of aerobically grown E. coli, a crossed immunoelectrophoresis (CIE) reference profile has been established in which over half of the 50 or so resolved antigens have been identified in functional terms or partially characterized (for reviews, see references 21 to 23).

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confidence: 99%

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Identification and partial characterization of a novel bipartite protein antigen associated with the outer membrane of Escherichia coli

Owen¹,

Caffrey²,

Josefsson³

1987

J Bacteriol

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“…7, lane 4 (and also evident in Fig. 6C and 8), is a consequence of lysozyme digestion of the peptidoglycan layer and reflects lipoprotein molecules covalently linked to differing numbers of murein subunits (43,57).…”

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confidence: 75%

“…Thus, marker antigens for the outer membrane, such as the Lpp, OmpF/C, and OmpA proteins, were found to peak as anticipated at a buoyant density (p) of about 1.24 g/ml. In addition, inner membrane markers, typified by succinate dehydrogenase and the 50-kilodalton polypeptide antigen 40 (43), fractionated predominantly with sedimentable material possessing a buoyant density (p = 1.17 g/ml) expected for plasma membranes ( Fig. 5A and B (Fig.…”

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confidence: 94%

“…By using a variety of reagents and approaches, including purified antigens, monospecific antisera, mutants, charge-shift effects, enzyme (zymogram) stains, analysis for characteristic cofactors, and subunit composition, we have succeeded in identifying in functional terms (or in partially characterizing) about half of these antigens (39,41,43). The most dominant protein antigens of the outer membrane include the Braun lipoprotein, the OmpF/C proteins, and the OmpA protein (42,43). Of those apparently associated with the inner membrane, three of the most prominent are the proton-translocating ATPase, the succinate dehydrogenase complex, and an unidentified immunogen designated antigen 47 (9,10,46).…”

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Identification, immunochemical characterization, and purification of a major lipoprotein antigen associated with the inner (cytoplasmic) membrane of Escherichia coli

Doherty¹,

Yamada²,

Caffrey³

et al. 1986

J Bacteriol

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A major antigenic constituent of the inner membrane of Escherichia coli ML308-225 was identified as a 28.5-kilodalton lipoprotein containing covalently bound glycerol and palmitate. This lipoprotein corresponded to antigen 47 in the crossed immunoelectrophoresis profile of membrane vesicles (P. Owen and H. R. Kaback, Proc. Natl. Acad. Sci. USA 75:3148-3152, 1978) and to new lipoprotein 4 described for E. coli B by Ichihara et al. (S. Ichihara, H. Hussain, and S. Mizushima, J. Biol. Chem. 256:3125-3129, 1980). Experiments involving isopycnic centrifugation of spheroplast envelopes indicated that antigen 47 was enriched in cytoplasmic membrane subfractions of low density. The protein did not manifest an obvious association with peptidoglycan of the types displayed by the bound form of the Braun (Lpp) lipoprotein, the 21-kilodalton peptidoglycan-associated lipoprotein, or the ompFIC gene products. Antibodies specific for antigen 47 were used to demonstrate that the molecule was immunologically distinct from both the Braun lipoprotein and the peptidoglycan-associated lipoprotein of E. coli. Antigens of similar molecular mass to and cross-reacting with antigen 47 were present in the envelopes of eight type species of the Enterobacteriaceae. A protocol for the purification of antigen 47, based upon its solubility in a chloroform-methanol-water mixture, was developed.The covalent attachment of lipid to proteins was first suggested over 30 years ago by Folch and Lees (16). Since that time, an increasing number of membrane proteins of both eucaryotic and procaryotic origin have been reported to contain covalently bound lipid (12,23,29,47,49,52). Common features of most procaryotic lipoproteins studied to date appear to be an N-acyl diglyceride cysteine at the N terminus of the molecule (18,25,35) and a posttranslational modification event involving a signal peptidase which can be selectively inhibited by the antibiotic globomycin (22,23,60). In gram-positive bacteria, the covalent association of membrane-bound penicillinases with lipid is well documented (25,34,35,37,53). In Escherichia coli, the envelope is known to contain a number of lipoproteins (4,23,30). Of these, the most dominant and best characterized are associated with the outer membrane system and include the Braun (Lpp) lipoprotein, the peptidoglycan-associated lipoprotein (PAL), and the traT gene product (4,20,30,31,48). The presence of several other (minor) envelope-associated lipoproteins (termed new lipoproteins or NLPs) has also been documented (23), and there is evidence that some of these, viz., NLP3, NLP4, and possibly NLP6, may reside in the inner membrane of E. coli (23, 32).Reports emanating from this laboratory over the course of the last 10 years (for reviews, see references 39-41) have been concerned with an analysis of the numerous antigens associated with the envelope of E. coli (50, 54). Our understanding of the complexity, identity, and topography of these antigens, many of which are proteinaceous in nature, has been facilitated by the us...

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Combination of line immunoelectrophoresis and sodium dodecyl sulfate‐polyacrylamide gel electrophoresis for molecular weight evaluation of isozymes: Application to iso‐α‐amylases identified in barley and rice seeds

Kerhardy¹,

Hara‐Nishimura²,

Nishimura³

et al. 1987

Electrophoresis

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Combination of line immunoelectrophoresis and sodium dodec yl sulfate-polyacrylamide gel electrophoresis for molecular weight evaluation of isozymes: Application to iso-a-amylases ideniified in barley and rice seeds A procedure for enzyme purification by means of line immunoelectrophoresis compatible with subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme and Coomassie Blue staining is described. Electrophoresis was carried out in an agarose gel backed to GelBond film. The enzyme activity was detected after immunoelectrophoresis on the wet gel and the immunoprecipitate by protein staining on the dried gel. The strip bearing the maximum amount of precipitate was excised and the dried agarose gel scratched offthe GelBond support. The dried gel was extracted with the Laemmli sample buffer, heated at 100 "C for 10 min and submitted to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins were stained with Coomassie Blue. Difficulties due to the presence of the immunoglobulin G subunits were encountered. The validity of the procedure was checked with an a-amylase from barley purified by immunoafinity chromatography. The procedure was used to compare the molecular weight of a-amylase isozymes identified in barley and in rice seeds. In barley, a-amylase I, the minor enzyme group appearing during germination, had a slightly higher molecular weight than c1-amylase 11, the main enzyme group. In germinating rice seeds, the minor enzyme group, antigen D, was found to have a molecular weight slightly lower than the one of the other rice a-amylases. A new a-amylase antigen identified in rice as the constituent with the lowest p1 was called pre-A. Its molecular weight was found to be the same as the one of the main enzyme group, antigen (A + B).

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An improved procedure for polypeptide analysis of radiolabeled antigens resolved by crossed immunoelectrophoresis and its application to the study of inner and outer membranes of Escherichia coli

Cited by 9 publications

References 40 publications

Identification and partial characterization of a novel bipartite protein antigen associated with the outer membrane of Escherichia coli

Identification and partial characterization of a novel bipartite protein antigen associated with the outer membrane of Escherichia coli

Identification, immunochemical characterization, and purification of a major lipoprotein antigen associated with the inner (cytoplasmic) membrane of Escherichia coli

Combination of line immunoelectrophoresis and sodium dodecyl sulfate‐polyacrylamide gel electrophoresis for molecular weight evaluation of isozymes: Application to iso‐α‐amylases identified in barley and rice seeds

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