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 |
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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 & 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</subject><ispartof>MICROBIOLOGY and IMMUNOLOGY, 1986, Vol.30(7), pp.659-673</ispartof><rights>Center for Academic Publications Japan</rights><rights>owned by Center for Academic Publications Japan (Publisher)</rights><rights>1987 INIST-CNRS</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c7032-774627693f6b075f169ba8890cda05cd8075684ce339fe301d7935829dc44b3</citedby><cites>FETCH-LOGICAL-c7032-774627693f6b075f169ba8890cda05cd8075684ce339fe301d7935829dc44b3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,776,780,1877,27901,27902</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=8129986$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/3773790$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Endoh, Masahiko</creatorcontrib><creatorcontrib>Amitani, Makoto</creatorcontrib><creatorcontrib>Nakase, Yasukiyo</creatorcontrib><title>Purification and Characterization of Heat-Labile Toxin from Bordetella bronchiseptica</title><title>MICROBIOLOGY and IMMUNOLOGY</title><addtitle>Microbiology and Immunology</addtitle><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.</description><subject>Amino Acids - analysis</subject><subject>Animals</subject><subject>Bacterial Toxins - isolation & purification</subject><subject>Bacterial Toxins - toxicity</subject><subject>Bacteriology</subject><subject>Biological and medical sciences</subject><subject>Bordetella - isolation & 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 & purification</topic><topic>Bacterial Toxins - toxicity</topic><topic>Bacteriology</topic><topic>Biological and medical sciences</topic><topic>Bordetella - isolation & 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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