Specific Labeling of Diaminopimelate: A Radioassay for the Determination of the Peptidoglycan Cross-Linking Index

A radioassay for the determination of the peptidoglycan cross-linking index (CLI) was devised. It is based on specific radioactive labeling of diaminopimelic acid (DAP) by diverting [14C]aspartate into the DAP pathway, while inhibiting incorporation of label into other cell wall components. Purified...

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Veröffentlicht in:	Analytical biochemistry 1994-12, Vol.223 (2), p.208-211
Hauptverfasser:	Roten, C.A.H., Pagni, M., Margot, P., Touri, F., Karamata, D.
Format:	Artikel
Sprache:	eng
Schlagworte:	Bacillus subtilis - chemistry Carbon Radioisotopes Cell Wall - chemistry Chromatography, Thin Layer Cross-Linking Reagents Diaminopimelic Acid - analysis Dinitrofluorobenzene Hydrolysis Peptidoglycan - chemistry
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container_title	Analytical biochemistry
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creator	Roten, C.A.H. Pagni, M. Margot, P. Touri, F. Karamata, D.
description	A radioassay for the determination of the peptidoglycan cross-linking index (CLI) was devised. It is based on specific radioactive labeling of diaminopimelic acid (DAP) by diverting [14C]aspartate into the DAP pathway, while inhibiting incorporation of label into other cell wall components. Purified [14C]DAP-labeled cell walls were treated with fluorodinitrobenzene, hydrolyzed, and chromatographed by TLC. The radioactivity in well-separated mono dinitrophenyl-diaminopimelate (DNP-DAP) and DAP spots was counted and the CLI was determined from the ratio of DAP to the total of mono DNP-DAP and DAP counts. The method, suitable for bacteria such as Bacillus subtilis unable to incorporate exogenous DAP, can be applied to other systems. A CLI of 50.8 ± 1.3% and 55.5 ± 0.9% was obtained for B. subtilis 168 cells growing exponentially in rich and minimal medium, respectively. Comparison of these to results previously obtained on B. subtilis suggested the existence of a hitherto unreported peptidoglycan endopeptidase activity.
doi_str_mv	10.1006/abio.1994.1575
format	Article
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Comparison of these to results previously obtained on B. subtilis suggested the existence of a hitherto unreported peptidoglycan endopeptidase activity.</description><identifier>ISSN: 0003-2697</identifier><identifier>EISSN: 1096-0309</identifier><identifier>DOI: 10.1006/abio.1994.1575</identifier><identifier>PMID: 7887465</identifier><language>eng</language><publisher>United States: Elsevier Inc</publisher><subject>Bacillus subtilis - chemistry ; Carbon Radioisotopes ; Cell Wall - chemistry ; Chromatography, Thin Layer ; Cross-Linking Reagents ; Diaminopimelic Acid - analysis ; Dinitrofluorobenzene ; Hydrolysis ; Peptidoglycan - chemistry</subject><ispartof>Analytical biochemistry, 1994-12, Vol.223 (2), p.208-211</ispartof><rights>1994 Academic Press</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c339t-b7910a3a88005028398c586ea48a28350235c2bbe780837814d1555cded86c613</citedby></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://dx.doi.org/10.1006/abio.1994.1575$$EHTML$$P50$$Gelsevier$$H</linktohtml><link.rule.ids>314,780,784,3550,27924,27925,45995</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/7887465$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Roten, C.A.H.</creatorcontrib><creatorcontrib>Pagni, M.</creatorcontrib><creatorcontrib>Margot, P.</creatorcontrib><creatorcontrib>Touri, F.</creatorcontrib><creatorcontrib>Karamata, D.</creatorcontrib><title>Specific Labeling of Diaminopimelate: A Radioassay for the Determination of the Peptidoglycan Cross-Linking Index</title><title>Analytical biochemistry</title><addtitle>Anal Biochem</addtitle><description>A radioassay for the determination of the peptidoglycan cross-linking index (CLI) was devised. 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Comparison of these to results previously obtained on B. subtilis suggested the existence of a hitherto unreported peptidoglycan endopeptidase activity.</description><subject>Bacillus subtilis - chemistry</subject><subject>Carbon Radioisotopes</subject><subject>Cell Wall - chemistry</subject><subject>Chromatography, Thin Layer</subject><subject>Cross-Linking Reagents</subject><subject>Diaminopimelic Acid - analysis</subject><subject>Dinitrofluorobenzene</subject><subject>Hydrolysis</subject><subject>Peptidoglycan - chemistry</subject><issn>0003-2697</issn><issn>1096-0309</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1994</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNp1kM1v1DAQxS0EKkvplRuST9yyjNfxF7dqS2mllaiAni3HnhRDEqd2FnX_-ybaVW-cRjPvzdPMj5APDNYMQH52TUxrZky9ZkKJV2TFwMgKOJjXZAUAvNpIo96Sd6X8AWCsFvKMnCmtVS3Fijz-HNHHNnq6cw12cXigqaVX0fVxSGPssXMTfqGX9IcLMblS3IG2KdPpN9IrnDDPPjfFNCxry_AOxymG9NAdvBvoNqdSql0c_i7Jt0PAp_fkTeu6ghenek7ur7_-2t5Uu-_fbreXu8pzbqaqUYaB405rAAEbzY32Qkt0tXZzN4-48JumQaVBc6VZHZgQwgcMWnrJ-Dn5dMwdc3rcY5lsH4vHrnMDpn2xSimzkULNxvXR6JdjM7Z2zLF3-WAZ2IWxXRjbhbFdGM8LH0_J-6bH8GI_QZ11fdRxfu9fxGyLjzh4DDGjn2xI8X_Rzz5Iis4</recordid><startdate>19941201</startdate><enddate>19941201</enddate><creator>Roten, C.A.H.</creator><creator>Pagni, M.</creator><creator>Margot, P.</creator><creator>Touri, F.</creator><creator>Karamata, D.</creator><general>Elsevier Inc</general><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>7X8</scope></search><sort><creationdate>19941201</creationdate><title>Specific Labeling of Diaminopimelate: A Radioassay for the Determination of the Peptidoglycan Cross-Linking Index</title><author>Roten, C.A.H. ; Pagni, M. ; Margot, P. ; Touri, F. ; Karamata, D.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c339t-b7910a3a88005028398c586ea48a28350235c2bbe780837814d1555cded86c613</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1994</creationdate><topic>Bacillus subtilis - chemistry</topic><topic>Carbon Radioisotopes</topic><topic>Cell Wall - chemistry</topic><topic>Chromatography, Thin Layer</topic><topic>Cross-Linking Reagents</topic><topic>Diaminopimelic Acid - analysis</topic><topic>Dinitrofluorobenzene</topic><topic>Hydrolysis</topic><topic>Peptidoglycan - chemistry</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Roten, C.A.H.</creatorcontrib><creatorcontrib>Pagni, M.</creatorcontrib><creatorcontrib>Margot, P.</creatorcontrib><creatorcontrib>Touri, F.</creatorcontrib><creatorcontrib>Karamata, D.</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><jtitle>Analytical biochemistry</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Roten, C.A.H.</au><au>Pagni, M.</au><au>Margot, P.</au><au>Touri, F.</au><au>Karamata, D.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Specific Labeling of Diaminopimelate: A Radioassay for the Determination of the Peptidoglycan Cross-Linking Index</atitle><jtitle>Analytical biochemistry</jtitle><addtitle>Anal Biochem</addtitle><date>1994-12-01</date><risdate>1994</risdate><volume>223</volume><issue>2</issue><spage>208</spage><epage>211</epage><pages>208-211</pages><issn>0003-2697</issn><eissn>1096-0309</eissn><abstract>A radioassay for the determination of the peptidoglycan cross-linking index (CLI) was devised. It is based on specific radioactive labeling of diaminopimelic acid (DAP) by diverting [14C]aspartate into the DAP pathway, while inhibiting incorporation of label into other cell wall components. Purified [14C]DAP-labeled cell walls were treated with fluorodinitrobenzene, hydrolyzed, and chromatographed by TLC. The radioactivity in well-separated mono dinitrophenyl-diaminopimelate (DNP-DAP) and DAP spots was counted and the CLI was determined from the ratio of DAP to the total of mono DNP-DAP and DAP counts. The method, suitable for bacteria such as Bacillus subtilis unable to incorporate exogenous DAP, can be applied to other systems. A CLI of 50.8 ± 1.3% and 55.5 ± 0.9% was obtained for B. subtilis 168 cells growing exponentially in rich and minimal medium, respectively. Comparison of these to results previously obtained on B. subtilis suggested the existence of a hitherto unreported peptidoglycan endopeptidase activity.</abstract><cop>United States</cop><pub>Elsevier Inc</pub><pmid>7887465</pmid><doi>10.1006/abio.1994.1575</doi><tpages>4</tpages></addata></record>
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subjects	Bacillus subtilis - chemistry Carbon Radioisotopes Cell Wall - chemistry Chromatography, Thin Layer Cross-Linking Reagents Diaminopimelic Acid - analysis Dinitrofluorobenzene Hydrolysis Peptidoglycan - chemistry
title	Specific Labeling of Diaminopimelate: A Radioassay for the Determination of the Peptidoglycan Cross-Linking Index
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