Immunochemical analysis of inner and outer membranes of Escherichia coli by crossed immunoelectrophoresis

Smyth, Cyril J.; Siegel, Johanna; Saltón, M. R. J.; Owen, Philip

doi:10.1128/jb.133.1.306-319.1978

Cited by 66 publications

(34 citation statements)

References 50 publications

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“…Additional chromatophore polypeptide components have been observed in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (6,15, 19), but the function of these components has remained largely unknown because this procedure results in the loss of any biological activity. Recently, crossed immunoelectrophoresis (CIE) has been applied to the study of bacterial membranes (7,14,17,18). Since nondenaturing detergents are used for membrane disruption before CIE, both enzymatic and antigenic activities are usually retained, thus permitting a functional analysis of membrane components.…”

mentioning

confidence: 99%

Crossed immunoelectrophoretic analysis of chromatophore membranes from Rhodopseudomonas sphaeroides

Collins¹,

Mallon²,

Niederman³

1979

J Bacteriol

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Triton extracts of intracytoplasmic photosynthetic membranes (chromatophores) purified from Rhodopseudomonas sphaeroides were subjected to crossed immunoelectrophoresis with antiserum raised in rabbits to purified chromatophores. A total of 31 immunoprecipitates was visualized; 2 of the immunoprecipitates were identified as reduced nicotinamide adenine dinucleotide (EC 1.6.99.3) and L-lactate dehydrogenases by enzyme staining techniques. Reaction with a monospecific antiserum identified the photochemical reaction center. Photopigments were associated with a major precipitate in the pattern which was identified on the basis of immunological identity as light-harvesting bacteriochlorophyll aprotein complex. These results provide the basis for a detailed structural and functional analysis of the chromatophore membrane by crossed immunoelectrophoresis.Growth of the facultative photoheterotrophic bacterium Rhodopseudomonas sphaeroides under low oxygen tension results in the development of intracytoplasmic (chromatophore) membranes in which the photosynthetic apparatus is localized. Formation of the chromatophore membrane is repressed under high aeration and derepressed upon reduction of oxygen tension. Fractionation and analysis of functional components of the R. sphaeroides chromatophore membrane has been restricted mainly to the photochemical reaction center (RC) (2,13), the light-harvesting (LH) bacteriochlorophyll a (BCHL) complex (1,5, 16), and the L-lactate dehydrogenase (LDH) (10). Additional chromatophore polypeptide components have been observed in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (6,15, 19), but the function of these components has remained largely unknown because this procedure results in the loss of any biological activity. Recently, crossed immunoelectrophoresis (CIE) has been applied to the study of bacterial membranes (7,14,17,18). Since nondenaturing detergents are used for membrane disruption before CIE, both enzymatic and antigenic activities are usually retained, thus permitting a functional analysis of membrane components. In addition, a high order of resolution is achieved through CIE by virtue of the two-dimensional distribution of antigenic components. This communication describes a CIE analysis of chromatophores isolated from R. sphaeroides.(This work was presented in part at the 79th Annual Meeting of the American Society for Microbiology, Los Angeles, Calif., 4-8 May 1979.) Triton X-100 was selected as the detergent for the solubilization of chromatophores for CIE because a recent study demonstrated that it was superior to other detergents for the maximal recovery of Micrococcus lysodeikticus membrane components (4). Chromatophores were purified from phototrophically grown (15) R. sphaeroides NCIB8253 by differential and sucrose density gradient centrifugation (12). Chromatophore preparations for immunization were purified further by a second cycle of differential and rate-zone sedimentation. Previous reconstitution studies have shown that such chromato...

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confidence: 99%

Crossed immunoelectrophoretic analysis of chromatophore membranes from Rhodopseudomonas sphaeroides

Collins¹,

Mallon²,

Niederman³

1979

J Bacteriol

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“…In addition to staining the CIE plates for heme, zymograms were also made for the most common dehydrogenases, as previously reported (27,36). Precipitin arcs were identified which contained residual D-lactate, succinate, and NADH dehydrogenase activities.…”

Section: Discussionmentioning

confidence: 98%

“…One purpose of this paper is to demonstrate the utility of both isoelectric focusing (IEF) and crossed immunoelectrophoresis (CIE) for the resolution and analysis of the E. coli cytochromes. Both techniques have been previously applied successfully to the study of membrane proteins (14,26,27,36). In our study, four heme-containing protein complexes were resolved under nondenaturing conditions in the presence of Triton X-100.…”

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confidence: 99%

Isoelectric focusing and crossed immunoelectrophoresis of heme proteins in the Escherichia coli cytoplasmic membrane

Kranz¹,

Gennis²

1982

J Bacteriol

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Isoelectric focusing (IEF), agarose electrophoresis, and crossed immunoelectrophoresis (CIE) were used to resolve the heme-containing proteins of the Escherichia coli cytoplasmic membrane after solubilization by Triton X-100. Two bands in IEF stained for heme with pI values of 4.7 and 5.3. One of the bands, with an isoelectric point of pH 5.3, was present only when the cells were grown to late log or stationary phase and possessed N,N,N,'N'-tetramethyl-p-phenylene-diamine (TMPD) oxidase activity. The pI 4.7 band was present in cells harvested in both mid-log and stationary phases. Agarose electrophoresis, using larger samples, revealed the same two components apparent by IEF, and, in addition, a third component. The heme-containing fractions were extracted after agarose electrophoresis and subjected to further study. The component which was present in cells grown to stationary phase contained hemes b, a1, and d. The other two fractions contained only b heme. One of these corresponded to the component with pI 4.7 in IEF and had catalase activity. Antisera were raised against Triton X-100-solubilized cytoplasmic membranes and against the focused TMPD oxidase complex. With these anti-sera, CIE in the presence of Triton X-100 revealed four precipitin complexes containing heme. Three of these corresponded to the components identified by IEF and agarose electrophoresis. We demonstrate that the combined use of IEF and CIE is valuable for analysis of membrane proteins. In particular, this work represents a substantial initial step toward a structural elucidation of the E. coli aerobic respiratory chain.

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“…Recently, attention is focussed on the possible use of outer membrane protein, particularly porins, as subunit vaccines against S. typhi, S. typhimurium, Escherichia coli, Pseudomonas aeruginosa, etc. (17,19,27,34). Results showed that they are good immunogens.…”

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confidence: 99%

Protective Efficacy and Immunogenicity of Vi‐Porin Conjugate against Salmonella typhi

Singh

Ganguly

Kumar

et al. 1999

Microbiology and Immunology

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A conjugate vaccine against Salmonella typhi was prepared by covalently binding capsular polysaccharide (Vi) with porin, both isolated from S. typhi. First, Vi and porins were extracted. The Vi was purified from S. typhi Ty2. The purified Vi conformed to the requirements of the World Health Organization. Porins were purified from S. typhi 0901. The Vi was bound to the porins by a heterobifunctional cross‐linking reagent, N‐succinimidyl‐3‐(2‐pyridyl dithio)‐propionate (SPDP). After preparing the Vi‐porin conjugate, its protective ability and immunogenicity were studied in mice following systemic immunization. The results showed that the conjugate is 6.5‐fold more protective than Vi alone against S. typhi. The mice immunized with conjugate elicited higher anti‐Vi antibody (IgG) levels (P<0.01) than the mice immunized with Vi alone. Anti‐porin antibodies were also induced by the conjugate. To study the mucosal immune responses, secretory IgA (sIgA) in the intestinal fluid was measured. Conjugate‐immunized mice showed the induction of sIgA as compared to Vi alone. The results showed that when Vi is bound to porins, both isolated from same organism, the resultant conjugate induced both systemic and mucosal immune responses and provided better protection against S. typhi than Vi alone.

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Immunochemical analysis of inner and outer membranes of Escherichia coli by crossed immunoelectrophoresis

Cited by 66 publications

References 50 publications

Crossed immunoelectrophoretic analysis of chromatophore membranes from Rhodopseudomonas sphaeroides

Crossed immunoelectrophoretic analysis of chromatophore membranes from Rhodopseudomonas sphaeroides

Isoelectric focusing and crossed immunoelectrophoresis of heme proteins in the Escherichia coli cytoplasmic membrane

Protective Efficacy and Immunogenicity of Vi‐Porin Conjugate against Salmonella typhi

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