A fluorometric assay for peptidyl α-amidation activity using high-performance liquid chromatography
A rapid and sensitive method for the determination of peptidyl α-amidation activity has been developed and is based on reverse-phase high-performance liquid chromatographic separation and fluorometric detection. A dansylated tripeptide, N-dansyl-Tyr-Val-Gly-OH, is used as the substrate in the assay...
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| Veröffentlicht in: | Analytical biochemistry 1988-02, Vol.168 (2), p.272-279 |
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| container_title | Analytical biochemistry |
| container_volume | 168 |
| creator | Jones, Barry N. Tamburini, Paul P. Consalvo, Angelo P. Young, Stanley D. Lovato, Susan J. Gilligan, James P. Jeng, Arco Y. Wennogle, Lawrence P. |
| description | A rapid and sensitive method for the determination of peptidyl α-amidation activity has been developed and is based on reverse-phase high-performance liquid chromatographic separation and fluorometric detection. A dansylated tripeptide,
N-dansyl-Tyr-Val-Gly-OH, is used as the substrate in the assay and the amount of α-amidation activity is determined by quantitating the extent of its conversion to product,
N-dansyl-Tyr-Val-NH
2. Both product and substrate can be detected in a single assay in quantities as low as 5 fmol by isocratic elution using C-18 reversephase columns. The method yields highly reproducible results and requires less than 3 min per sample for separation and quantitation. The assay procedure is applicable to the screening of a large number of samples under different pH conditions and is readily adaptable for use in a variety of studies. For example, the procedure is ideal for detecting α-amidation activity in various tissues, monitoring activity at the different stages during purification of a particular α-amidation enzyme, determining kinetic parameters of the purified enzyme, and identifying both competitive and noncompetitive inhibitors. |
| doi_str_mv | 10.1016/0003-2697(88)90318-1 |
| format | Article |
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N-dansyl-Tyr-Val-Gly-OH, is used as the substrate in the assay and the amount of α-amidation activity is determined by quantitating the extent of its conversion to product,
N-dansyl-Tyr-Val-NH
2. Both product and substrate can be detected in a single assay in quantities as low as 5 fmol by isocratic elution using C-18 reversephase columns. The method yields highly reproducible results and requires less than 3 min per sample for separation and quantitation. The assay procedure is applicable to the screening of a large number of samples under different pH conditions and is readily adaptable for use in a variety of studies. For example, the procedure is ideal for detecting α-amidation activity in various tissues, monitoring activity at the different stages during purification of a particular α-amidation enzyme, determining kinetic parameters of the purified enzyme, and identifying both competitive and noncompetitive inhibitors.</description><identifier>ISSN: 0003-2697</identifier><identifier>EISSN: 1096-0309</identifier><identifier>DOI: 10.1016/0003-2697(88)90318-1</identifier><identifier>PMID: 3364727</identifier><identifier>CODEN: ANBCA2</identifier><language>eng</language><publisher>San Diego, CA: Elsevier Inc</publisher><subject>amidation enzyme ; Amides - analysis ; Biological and medical sciences ; Chromatography, High Pressure Liquid ; Dansyl Compounds - analysis ; Diverse techniques ; fluorescent substrate ; fluorometry ; Fluorometry - methods ; Fundamental and applied biological sciences. Psychology ; high-performance liquid chromatography ; HPLC ; Molecular and cellular biology ; peptide hormones ; peptide processing ; peptide α-amidation ; Peptides - analysis</subject><ispartof>Analytical biochemistry, 1988-02, Vol.168 (2), p.272-279</ispartof><rights>1988</rights><rights>1989 INIST-CNRS</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c417t-f18d77fa7c618414edcd1a7b0cb0766cd66098f7e80ea21e3b6674217204c77a3</citedby><cites>FETCH-LOGICAL-c417t-f18d77fa7c618414edcd1a7b0cb0766cd66098f7e80ea21e3b6674217204c77a3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://dx.doi.org/10.1016/0003-2697(88)90318-1$$EHTML$$P50$$Gelsevier$$H</linktohtml><link.rule.ids>315,781,785,3551,27929,27930,46000</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=7102494$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/3364727$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Jones, Barry N.</creatorcontrib><creatorcontrib>Tamburini, Paul P.</creatorcontrib><creatorcontrib>Consalvo, Angelo P.</creatorcontrib><creatorcontrib>Young, Stanley D.</creatorcontrib><creatorcontrib>Lovato, Susan J.</creatorcontrib><creatorcontrib>Gilligan, James P.</creatorcontrib><creatorcontrib>Jeng, Arco Y.</creatorcontrib><creatorcontrib>Wennogle, Lawrence P.</creatorcontrib><title>A fluorometric assay for peptidyl α-amidation activity using high-performance liquid chromatography</title><title>Analytical biochemistry</title><addtitle>Anal Biochem</addtitle><description>A rapid and sensitive method for the determination of peptidyl α-amidation activity has been developed and is based on reverse-phase high-performance liquid chromatographic separation and fluorometric detection. A dansylated tripeptide,
N-dansyl-Tyr-Val-Gly-OH, is used as the substrate in the assay and the amount of α-amidation activity is determined by quantitating the extent of its conversion to product,
N-dansyl-Tyr-Val-NH
2. Both product and substrate can be detected in a single assay in quantities as low as 5 fmol by isocratic elution using C-18 reversephase columns. The method yields highly reproducible results and requires less than 3 min per sample for separation and quantitation. The assay procedure is applicable to the screening of a large number of samples under different pH conditions and is readily adaptable for use in a variety of studies. For example, the procedure is ideal for detecting α-amidation activity in various tissues, monitoring activity at the different stages during purification of a particular α-amidation enzyme, determining kinetic parameters of the purified enzyme, and identifying both competitive and noncompetitive inhibitors.</description><subject>amidation enzyme</subject><subject>Amides - analysis</subject><subject>Biological and medical sciences</subject><subject>Chromatography, High Pressure Liquid</subject><subject>Dansyl Compounds - analysis</subject><subject>Diverse techniques</subject><subject>fluorescent substrate</subject><subject>fluorometry</subject><subject>Fluorometry - methods</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>high-performance liquid chromatography</subject><subject>HPLC</subject><subject>Molecular and cellular biology</subject><subject>peptide hormones</subject><subject>peptide processing</subject><subject>peptide α-amidation</subject><subject>Peptides - analysis</subject><issn>0003-2697</issn><issn>1096-0309</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1988</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkcFu1DAQhi1EVZbCG4DkA0JwCMw4ru1cKlUV0EqVuMDZ8trOrlGyTm2nUh6LF-kz4WVXe4TTHOb7f42-IeQNwicEFJ8BoG2Y6OQHpT520KJq8BlZIXSigRa652R1Ql6Qlzn_AkDkl-KcnLet4JLJFXHXtB_mmOLoSwqWmpzNQvuY6OSnEtwy0KffjRmDMyXEHTW2hMdQFjrnsNvQbdhsm8mnGhjNzno6hIc5OGq3tdGUuElm2i6vyFlvhuxfH-cF-fn1y4-b2-b--7e7m-v7xnKUpelROSl7I61AxZF7Zx0auQa7BimEdUJAp3rpFXjD0LdrISRnKBlwK6VpL8j7Q--U4sPsc9FjyNYPg9n5OGctFXaStfBfEHl3yTiTFeQH0KaYc_K9nlIYTVo0gt5_Qe8V671irZT--wWNNfb22D-vR-9OoaP2un933JtszdCn6i7kEyYRGO94xa4OmK_SHoNPOtvgq2YXkrdFuxj-fccfP5-k_Q</recordid><startdate>19880201</startdate><enddate>19880201</enddate><creator>Jones, Barry N.</creator><creator>Tamburini, Paul P.</creator><creator>Consalvo, Angelo P.</creator><creator>Young, Stanley D.</creator><creator>Lovato, Susan J.</creator><creator>Gilligan, James P.</creator><creator>Jeng, Arco Y.</creator><creator>Wennogle, Lawrence P.</creator><general>Elsevier Inc</general><general>Elsevier</general><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>C1K</scope><scope>7X8</scope></search><sort><creationdate>19880201</creationdate><title>A fluorometric assay for peptidyl α-amidation activity using high-performance liquid chromatography</title><author>Jones, Barry N. ; Tamburini, Paul P. ; Consalvo, Angelo P. ; Young, Stanley D. ; Lovato, Susan J. ; Gilligan, James P. ; Jeng, Arco Y. ; Wennogle, Lawrence P.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c417t-f18d77fa7c618414edcd1a7b0cb0766cd66098f7e80ea21e3b6674217204c77a3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1988</creationdate><topic>amidation enzyme</topic><topic>Amides - analysis</topic><topic>Biological and medical sciences</topic><topic>Chromatography, High Pressure Liquid</topic><topic>Dansyl Compounds - analysis</topic><topic>Diverse techniques</topic><topic>fluorescent substrate</topic><topic>fluorometry</topic><topic>Fluorometry - methods</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>high-performance liquid chromatography</topic><topic>HPLC</topic><topic>Molecular and cellular biology</topic><topic>peptide hormones</topic><topic>peptide processing</topic><topic>peptide α-amidation</topic><topic>Peptides - analysis</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Jones, Barry N.</creatorcontrib><creatorcontrib>Tamburini, Paul P.</creatorcontrib><creatorcontrib>Consalvo, Angelo P.</creatorcontrib><creatorcontrib>Young, Stanley D.</creatorcontrib><creatorcontrib>Lovato, Susan J.</creatorcontrib><creatorcontrib>Gilligan, James P.</creatorcontrib><creatorcontrib>Jeng, Arco Y.</creatorcontrib><creatorcontrib>Wennogle, Lawrence P.</creatorcontrib><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>Environmental Sciences and Pollution Management</collection><collection>MEDLINE - Academic</collection><jtitle>Analytical biochemistry</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Jones, Barry N.</au><au>Tamburini, Paul P.</au><au>Consalvo, Angelo P.</au><au>Young, Stanley D.</au><au>Lovato, Susan J.</au><au>Gilligan, James P.</au><au>Jeng, Arco Y.</au><au>Wennogle, Lawrence P.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>A fluorometric assay for peptidyl α-amidation activity using high-performance liquid chromatography</atitle><jtitle>Analytical biochemistry</jtitle><addtitle>Anal Biochem</addtitle><date>1988-02-01</date><risdate>1988</risdate><volume>168</volume><issue>2</issue><spage>272</spage><epage>279</epage><pages>272-279</pages><issn>0003-2697</issn><eissn>1096-0309</eissn><coden>ANBCA2</coden><abstract>A rapid and sensitive method for the determination of peptidyl α-amidation activity has been developed and is based on reverse-phase high-performance liquid chromatographic separation and fluorometric detection. A dansylated tripeptide,
N-dansyl-Tyr-Val-Gly-OH, is used as the substrate in the assay and the amount of α-amidation activity is determined by quantitating the extent of its conversion to product,
N-dansyl-Tyr-Val-NH
2. Both product and substrate can be detected in a single assay in quantities as low as 5 fmol by isocratic elution using C-18 reversephase columns. The method yields highly reproducible results and requires less than 3 min per sample for separation and quantitation. The assay procedure is applicable to the screening of a large number of samples under different pH conditions and is readily adaptable for use in a variety of studies. For example, the procedure is ideal for detecting α-amidation activity in various tissues, monitoring activity at the different stages during purification of a particular α-amidation enzyme, determining kinetic parameters of the purified enzyme, and identifying both competitive and noncompetitive inhibitors.</abstract><cop>San Diego, CA</cop><pub>Elsevier Inc</pub><pmid>3364727</pmid><doi>10.1016/0003-2697(88)90318-1</doi><tpages>8</tpages></addata></record> |
| fulltext | fulltext |
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| ispartof | Analytical biochemistry, 1988-02, Vol.168 (2), p.272-279 |
| issn | 0003-2697 1096-0309 |
| language | eng |
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| source | MEDLINE; Access via ScienceDirect (Elsevier) |
| subjects | amidation enzyme Amides - analysis Biological and medical sciences Chromatography, High Pressure Liquid Dansyl Compounds - analysis Diverse techniques fluorescent substrate fluorometry Fluorometry - methods Fundamental and applied biological sciences. Psychology high-performance liquid chromatography HPLC Molecular and cellular biology peptide hormones peptide processing peptide α-amidation Peptides - analysis |
| title | A fluorometric assay for peptidyl α-amidation activity using high-performance liquid chromatography |
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