Purification and Characterization of Heat-Labile Toxin from Bordetella bronchiseptica

The heat-labile toxin (HLT) of Bordetella bronchiseptica was purified successively from sonic extracts of phase I organisms grown in Stainer-Scholte medium, by partition in hydrophobic interaction, sucrose density gradient centrifugation, gel filtration through Sepharose 4B and 6B, isoelectric preci...

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Veröffentlicht in:MICROBIOLOGY and IMMUNOLOGY 1986, Vol.30(7), pp.659-673
Hauptverfasser: Endoh, Masahiko, Amitani, Makoto, Nakase, Yasukiyo
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Amitani, Makoto
Nakase, Yasukiyo
description The heat-labile toxin (HLT) of Bordetella bronchiseptica was purified successively from sonic extracts of phase I organisms grown in Stainer-Scholte medium, by partition in hydrophobic interaction, sucrose density gradient centrifugation, gel filtration through Sepharose 4B and 6B, isoelectric precipitation and isoelectric focusing. The purified HLT was homogeneous by disc polyacrylamide gel electrophoresis and the gel diffusion-test, and free of detectable hemagglutinin and endotoxin activity. A 386-fold purification over the crude extract was obtained at a yield of about 28%, and a minimum dose of 0.9ng was dermonecrotizing with a lesion 5mm in diameter in guinea pigs and induced splenoatrophy. The mouse LD50 was 200ng (intraperitoneal) or 70ng (intravenous). The HLT was found to be a simple protein with an isoelectric point of pI 6.9. It has a molecular weight of 102, 000 estimated by Sepharose 6B gel filtration and was found to consist of two different types of polypeptide by SDS-polyacrylamide gel electrophoresis, their molecular weights being 30, 000 and 20, 000. Amino acid analysis showed 15 common amino acid residues, and methionine, cysteine and tryptophan were undetectable. The HLT crystallized by methylpentanediol showed a block form. The HLT was inactivated at 56C when heated for 10min, and at above pH 9 and below pH 4.
doi_str_mv 10.1111/j.1348-0421.1986.tb02992.x
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The purified HLT was homogeneous by disc polyacrylamide gel electrophoresis and the gel diffusion-test, and free of detectable hemagglutinin and endotoxin activity. A 386-fold purification over the crude extract was obtained at a yield of about 28%, and a minimum dose of 0.9ng was dermonecrotizing with a lesion 5mm in diameter in guinea pigs and induced splenoatrophy. The mouse LD50 was 200ng (intraperitoneal) or 70ng (intravenous). The HLT was found to be a simple protein with an isoelectric point of pI 6.9. It has a molecular weight of 102, 000 estimated by Sepharose 6B gel filtration and was found to consist of two different types of polypeptide by SDS-polyacrylamide gel electrophoresis, their molecular weights being 30, 000 and 20, 000. Amino acid analysis showed 15 common amino acid residues, and methionine, cysteine and tryptophan were undetectable. The HLT crystallized by methylpentanediol showed a block form. The HLT was inactivated at 56C when heated for 10min, and at above pH 9 and below pH 4.</description><identifier>ISSN: 0385-5600</identifier><identifier>EISSN: 1348-0421</identifier><identifier>DOI: 10.1111/j.1348-0421.1986.tb02992.x</identifier><identifier>PMID: 3773790</identifier><identifier>CODEN: MIIMDV</identifier><language>eng</language><publisher>Tokyo: Blackwell Publishing Ltd</publisher><subject>Amino Acids - analysis ; Animals ; Bacterial Toxins - isolation &amp; purification ; Bacterial Toxins - toxicity ; Bacteriology ; Biological and medical sciences ; Bordetella - isolation &amp; purification ; Bordetella - pathogenicity ; Bordetella bronchiseptica ; Bordetella Infections - veterinary ; Crystallization ; Female ; Fundamental and applied biological sciences. 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The purified HLT was homogeneous by disc polyacrylamide gel electrophoresis and the gel diffusion-test, and free of detectable hemagglutinin and endotoxin activity. A 386-fold purification over the crude extract was obtained at a yield of about 28%, and a minimum dose of 0.9ng was dermonecrotizing with a lesion 5mm in diameter in guinea pigs and induced splenoatrophy. The mouse LD50 was 200ng (intraperitoneal) or 70ng (intravenous). The HLT was found to be a simple protein with an isoelectric point of pI 6.9. It has a molecular weight of 102, 000 estimated by Sepharose 6B gel filtration and was found to consist of two different types of polypeptide by SDS-polyacrylamide gel electrophoresis, their molecular weights being 30, 000 and 20, 000. Amino acid analysis showed 15 common amino acid residues, and methionine, cysteine and tryptophan were undetectable. The HLT crystallized by methylpentanediol showed a block form. The HLT was inactivated at 56C when heated for 10min, and at above pH 9 and below pH 4.</description><subject>Amino Acids - analysis</subject><subject>Animals</subject><subject>Bacterial Toxins - isolation &amp; purification</subject><subject>Bacterial Toxins - toxicity</subject><subject>Bacteriology</subject><subject>Biological and medical sciences</subject><subject>Bordetella - isolation &amp; purification</subject><subject>Bordetella - pathogenicity</subject><subject>Bordetella bronchiseptica</subject><subject>Bordetella Infections - veterinary</subject><subject>Crystallization</subject><subject>Female</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Guinea Pigs</subject><subject>Mice</subject><subject>Mice, Inbred ICR</subject><subject>Microbiology</subject><subject>Molecular Weight</subject><subject>Necrosis</subject><subject>Pathogenicity, virulence, toxins, bacteriocins, pyrogens, host-bacteria relations, miscellaneous strains</subject><subject>Skin - drug effects</subject><subject>Skin - pathology</subject><subject>Swine</subject><subject>Swine Diseases - microbiology</subject><subject>Transglutaminases</subject><subject>Virulence Factors, Bordetella</subject><issn>0385-5600</issn><issn>1348-0421</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1986</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqVkk1v1DAQhiMEKtvCT0CKEEJcEvwVf3ACVt226i4gUeA4chyHeskmi51Vt_x6HBJF3BA-2NbMO4_Hr50kzzHKcRyvtzmmTGaIEZxjJXnel4goRfLjg2Qxpx4mC0RlkRUcocfJaQhbhIggkp0kJ1QIKhRaJF8-HbyrndG969pUt1W6vNVem95692sMdnV6aXWfrXXpGpvedEfXprXvdun7zle2t02j09J3rbl1we77CHuSPKp1E-zTaT1LPq_Ob5aX2frjxdXy3TozAlGSCcE4EVzRmpdIFDXmqtRSKmQqjQpTyRjkkhlLqaotRbgSihaSqMowVtKz5NVI3fvu58GGHnYumKGd1naHAJgpwlmhmIjSl_-QMo65YFH4ZhQa34XgbQ1773ba3wNGMJgPWxgchsFhGMyHyXw4xuJn0ymHcmeruXRyO-ZfTHkdjG5qr1vjwiyTOHIkj7K3o-wu-n3_Hw3A5mrzZxsR5yNiG3r93c4M7ePzNBZ28aUdVkIARTBNvFBz3sRPALaNnGzkuNDb41-YH8DjnQr49uECvq5WxbW4JrChvwGaNchd</recordid><startdate>19860101</startdate><enddate>19860101</enddate><creator>Endoh, Masahiko</creator><creator>Amitani, Makoto</creator><creator>Nakase, Yasukiyo</creator><general>Blackwell Publishing Ltd</general><general>Center For Academic Publications Japan</general><general>Center for Academic Publications Japan</general><scope>BSCLL</scope><scope>IQODW</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7QL</scope><scope>7T7</scope><scope>8FD</scope><scope>C1K</scope><scope>FR3</scope><scope>P64</scope></search><sort><creationdate>19860101</creationdate><title>Purification and Characterization of Heat-Labile Toxin from Bordetella bronchiseptica</title><author>Endoh, Masahiko ; Amitani, Makoto ; Nakase, Yasukiyo</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c7032-774627693f6b075f169ba8890cda05cd8075684ce339fe301d7935829dc44b3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1986</creationdate><topic>Amino Acids - analysis</topic><topic>Animals</topic><topic>Bacterial Toxins - isolation &amp; purification</topic><topic>Bacterial Toxins - toxicity</topic><topic>Bacteriology</topic><topic>Biological and medical sciences</topic><topic>Bordetella - isolation &amp; purification</topic><topic>Bordetella - pathogenicity</topic><topic>Bordetella bronchiseptica</topic><topic>Bordetella Infections - veterinary</topic><topic>Crystallization</topic><topic>Female</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>Guinea Pigs</topic><topic>Mice</topic><topic>Mice, Inbred ICR</topic><topic>Microbiology</topic><topic>Molecular Weight</topic><topic>Necrosis</topic><topic>Pathogenicity, virulence, toxins, bacteriocins, pyrogens, host-bacteria relations, miscellaneous strains</topic><topic>Skin - drug effects</topic><topic>Skin - pathology</topic><topic>Swine</topic><topic>Swine Diseases - microbiology</topic><topic>Transglutaminases</topic><topic>Virulence Factors, Bordetella</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Endoh, Masahiko</creatorcontrib><creatorcontrib>Amitani, Makoto</creatorcontrib><creatorcontrib>Nakase, Yasukiyo</creatorcontrib><collection>Istex</collection><collection>Pascal-Francis</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Industrial and Applied Microbiology Abstracts (Microbiology A)</collection><collection>Technology Research Database</collection><collection>Environmental Sciences and Pollution Management</collection><collection>Engineering Research Database</collection><collection>Biotechnology and BioEngineering Abstracts</collection><jtitle>MICROBIOLOGY and IMMUNOLOGY</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Endoh, Masahiko</au><au>Amitani, Makoto</au><au>Nakase, Yasukiyo</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Purification and Characterization of Heat-Labile Toxin from Bordetella bronchiseptica</atitle><jtitle>MICROBIOLOGY and IMMUNOLOGY</jtitle><addtitle>Microbiology and Immunology</addtitle><date>1986-01-01</date><risdate>1986</risdate><volume>30</volume><issue>7</issue><spage>659</spage><epage>673</epage><pages>659-673</pages><issn>0385-5600</issn><eissn>1348-0421</eissn><coden>MIIMDV</coden><abstract>The heat-labile toxin (HLT) of Bordetella bronchiseptica was purified successively from sonic extracts of phase I organisms grown in Stainer-Scholte medium, by partition in hydrophobic interaction, sucrose density gradient centrifugation, gel filtration through Sepharose 4B and 6B, isoelectric precipitation and isoelectric focusing. The purified HLT was homogeneous by disc polyacrylamide gel electrophoresis and the gel diffusion-test, and free of detectable hemagglutinin and endotoxin activity. A 386-fold purification over the crude extract was obtained at a yield of about 28%, and a minimum dose of 0.9ng was dermonecrotizing with a lesion 5mm in diameter in guinea pigs and induced splenoatrophy. The mouse LD50 was 200ng (intraperitoneal) or 70ng (intravenous). The HLT was found to be a simple protein with an isoelectric point of pI 6.9. It has a molecular weight of 102, 000 estimated by Sepharose 6B gel filtration and was found to consist of two different types of polypeptide by SDS-polyacrylamide gel electrophoresis, their molecular weights being 30, 000 and 20, 000. Amino acid analysis showed 15 common amino acid residues, and methionine, cysteine and tryptophan were undetectable. The HLT crystallized by methylpentanediol showed a block form. 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subjects Amino Acids - analysis
Animals
Bacterial Toxins - isolation & purification
Bacterial Toxins - toxicity
Bacteriology
Biological and medical sciences
Bordetella - isolation & purification
Bordetella - pathogenicity
Bordetella bronchiseptica
Bordetella Infections - veterinary
Crystallization
Female
Fundamental and applied biological sciences. Psychology
Guinea Pigs
Mice
Mice, Inbred ICR
Microbiology
Molecular Weight
Necrosis
Pathogenicity, virulence, toxins, bacteriocins, pyrogens, host-bacteria relations, miscellaneous strains
Skin - drug effects
Skin - pathology
Swine
Swine Diseases - microbiology
Transglutaminases
Virulence Factors, Bordetella
title Purification and Characterization of Heat-Labile Toxin from Bordetella bronchiseptica
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