Selective extraction of gonococcal envelope antigens with thiocyanates

Saltón, M. R. J.; Urban, Carl

doi:10.1111/j.1574-6968.1978.tb02885.x

Cited by 8 publications

(5 citation statements)

References 13 publications

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“…The extraction of M-2 from cells with GuHCI as described above suggested that GuHCI might also be used to dissociate M-2 from the peptidoglycan-enzyme complex. GuHCI (6 M) extracts of peptidoglycan-enzyme complex yielded about twice the M-2 activity yielded by alkaline extracts (pH 12) and 4 (4,10,25,26,33). We found that the chaotropic salts GuHCI, guanidine-thiocyanate, sodium thiocyanate, and lithium thiocyanate could be used to extract both muramidases from the intact bacterial cells and from the insoluble residue (crude cell wall fraction) of mechanically disrupted E. hirae.…”

Section: Resultsmentioning

confidence: 88%

Extracellular and cellular distribution of muramidase-2 and muramidase-1 of Enterococcus hirae ATCC 9790

Kariyama

Shockman

1992

J Bacteriol

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A substantial portion of the second peptidoglycan hydrolase (muramidase-2) activity of Enterococcus hirae ATCC 9790 (formerly Streptococcusfaecium) is present in the supernatant culture medium. In contrast, nearly all muramidase-1 activity is associated with cells in the latent, proteinase-activatable form. Muramidase-2 activity is produced and secreted throughout growth, with maximal levels attained at or near the end of exponential growth in a rich organic medium. Muramidase-2 activity in the culture medium remained high even during overnight incubations in the absence of proteinase inhibitors. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of supernatant culture medium concentrated by 60% saturated ammonium sulfate precipitation showed the presence of several Coomassie blue-staining bands. One intensely staining protein band, at about 71 kDa, selectively adsorbed to the insoluble peptidoglycan fraction of cell walls of E. hirae, retained muramidase-2 activity, and reacted in Western immunoblots with monoclonal antibodies to muramidase-2. The mobility of extracellular muramidase-2 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis was indistinguishable from that of muramidase-2 extracted with 6 M guanidine hydrochloride from intact bacteria. Muramidase-2 appears to have only a limited number of binding sites on the peptidoglycan of E. hirae cell walls but binds with high affinity. Although high levels of muramidase-2 activity were present in supernatants of stationary-phase cultures, the bacteria were resistant to autolysis. Thus it appears that the peptidoglycan in walls of intact cells of E. hirae is somehow protected from the hydrolytic action of extracellular muramidase-2.Enterococcus hirae ATCC 9790 (formerly Streptococcus faecium) was shown to possess two separate and distinct peptidoglycan hydrolase activities; both enzymes are N-acetylmuramoylhydrolases (muramidases) (18,29). Both enzymes are high-molecular-weight, complex proteins that possess a number of rather unusual properties, especially in contrast to avian and bacteriophage lysozymes that hydrolyze the same bond in the cell wall peptidoglycan. Muramidase-1 (M-1) was isolated and purified to homogeneity and was shown to occur in a latent, 130-kDa form that can be proteolytically hydrolyzed to an active, 87-kDa form (17,24,30). M-1 was also shown to be a glycoenzyme containing covalently attached monomeric and oligomeric glucose (17) and to possess approximately 12 phosphodiester-linked monomeric 5-mercaptouridine monophosphate residues (8). In addition, M-1 was shown to processively hydrolyze linear, soluble, un-cross-linked peptidoglycan chains (1).Muramidase-2 (M-2) was partially purified from the supernatant culture medium and was shown to be a 70-to 75-kDa protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (18). M-2 was also purified to apparent homogeneity from the insoluble pellet of disrupted E. hirae (7). Two polypeptide bands, one of about 125 kDa and the other of about 71 kDa, were show...

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Section: Resultsmentioning

confidence: 88%

Extracellular and cellular distribution of muramidase-2 and muramidase-1 of Enterococcus hirae ATCC 9790

Kariyama

Shockman

1992

J Bacteriol

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“…It facilitated the characterization of the major outer membrane proteins of several Enterobacteriaceae species as common envelope antigens. Previous studies using crossed immunoelectrophoresis resulted in a detailed immunochemical analysis of membrane material derived from Micrococcus lysodeikticus (23), Neisseria gonorrhoeae (28,31), and N. meningitidis (11). Recently, crossed immunoelectrophoresis was used for the resolution of cytoplasmic and outer membranes of Escherichia coli (32).…”

Section: Resultsmentioning

confidence: 99%

“…Crossed immunoelectrophoresis has been widely used for the immunochemical analysis of bacterial envelope structures (23,28,31). However, attempts to identify the major outer membrane proteins of E. coli by this technique have been unsuccessful (32).…”

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confidence: 99%

Cross-reactivity of major outer membrane proteins of Enterobacteriaceae, studied by crossed immunoelectrophoresis

Hofstra¹,

Tol²,

Dankert³

1980

J Bacteriol

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Outer membrane fractions were prepared from 11 bacteria in the family Enterobacteriaceae: Escherichia coli serotypes 01K-, 04K2, 026K60, 075K-, and O111K58, Shigella flexneri, Salmonella typhimurium, Klebsiella pneumoniae, Serratia marcescens, Proteus vulgaris, Proteus mirabilis, and Providencia stuartii. All strains studied were found to contain one non-peptidoglycan-bound, heat-modifiable outer membrane protein, and one or two peptidoglycan-associated major outer membrane proteins in the 27,000to 40,000-dalton range. Crossed immunoelectrophoresis using sodium dodecyl sulfate-polyacrylamide gel electrophoresis for separation of the antigens in the first dimension of the procedure was shown to provide a useful model system for studying the antigenic relationships of the major outer membrane proteins in Enterobacteriaceae species. Peptidoglycan-bound major outer membrane proteins of all bacteria studied reacted with antiserum against the purified peptidoglycan-bound matrix protein I of E. coli 026K60 in this system. Non-peptidoglycan-associated proteins of all strains crossreacted with protein II* of E. coli 026K60 in both their unmodified and in their heat-modified forms. These results indicate that the genes coding for the major outer membrane proteins in the family Enterobacteriaceae have been well enough conserved during the course of evolution to allow significant antigenic crossreactivity between the corresponding proteins in different enterobacterial species. ter (4), we identified the peptidoglycan-bound 328 on August 6, 2020 by guest

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“…The antigens of Neisseria gonorrhoeae are of interest for their potential usefulness in serodiagnosis, immunoprophylaxis, and in the investigation of the interaction of the gonococcus with the host and the role of individual antigenic components in the mechanism of pathogenesis. In order to resolve these antigens for analytical purposes, this laboratory has applied crossed immunoelectrophoresis (CIE) to gonococcal antigens (25,29,30) and explored the possibility of using these techniques to observe interaction between sera of patients and these bacterial antigens (23).…”

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confidence: 99%

Preparation and crossed immunoelectrophoretic analysis of cytoplasmic and outer membrane fractions from Neisseria gonorrhoeae

Collins¹,

Saltón²

1980

Infect Immun

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Cell envelopes were obtained from lysates of Neisseria gonorrhoeae, colony type T1, prepared with lysozyme, ethylenediaminetetraacetate, and Brij 58. This preparation was separated into cytoplasmic (inner) and outer membrane fractions by equilibrium sucrose density gradient centrifugation. The former fraction was 10-fold enriched in L-lactate dehydrogenase activity with respect to the latter. On the basis of buoyant density in sucrose, polypeptide patterns in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and enzymatic activity, these preparations appear similar to cytoplasmic and outer membrane preparations from other gram-negative bacteria. The membrane preparations were analyzed by high-resolution crossed immunoelectrophoretic procedures. This technique permitted the identification of antigens originating from the structural components of the gonococcal cell. Among those found to be cytoplasmic membrane components was the fast-moving antigen which occurs widely in gram-negative bacteria.

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Selective extraction of gonococcal envelope antigens with thiocyanates

Cited by 8 publications

References 13 publications

Extracellular and cellular distribution of muramidase-2 and muramidase-1 of Enterococcus hirae ATCC 9790

Extracellular and cellular distribution of muramidase-2 and muramidase-1 of Enterococcus hirae ATCC 9790

Cross-reactivity of major outer membrane proteins of Enterobacteriaceae, studied by crossed immunoelectrophoresis

Preparation and crossed immunoelectrophoretic analysis of cytoplasmic and outer membrane fractions from Neisseria gonorrhoeae

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