Determination of Glutamate-Cysteine Ligase (γ-Glutamylcysteine Synthetase) Activity by High-Performance Liquid Chromatography and Electrochemical Detection
The tripeptide glutathione (γ-glutamylcysteinylglycine; GSH) is the predominant low molecular mass thiol in cells. The function of GSH is of considerable interest, with the molecule being implicated in numerous cellular processes in addition to being a major cellular antioxidant. The enzyme glutamat...
Gespeichert in:
| Veröffentlicht in: | Analytical biochemistry 2002-05, Vol.304 (1), p.26-32 |
|---|---|
| Hauptverfasser: | , , |
| Format: | Artikel |
| Sprache: | eng |
| Schlagworte: | |
| Online-Zugang: | Volltext |
| Tags: |
Tag hinzufügen
Keine Tags, Fügen Sie den ersten Tag hinzu!
|
| container_end_page | 32 |
|---|---|
| container_issue | 1 |
| container_start_page | 26 |
| container_title | Analytical biochemistry |
| container_volume | 304 |
| creator | Gegg, Matthew E. Clark, John B. Heales, Simon J.R. |
| description | The tripeptide glutathione (γ-glutamylcysteinylglycine; GSH) is the predominant low molecular mass thiol in cells. The function of GSH is of considerable interest, with the molecule being implicated in numerous cellular processes in addition to being a major cellular antioxidant. The enzyme glutamate-cysteine ligase (GCL) is the rate-limiting step in GSH synthesis. The GCL assay described here is based on high-performance liquid chromatography and exploits the electrochemically active nature of γ-glutamylcysteine (γ-GC), the product of GCL activity. This method allows for the direct detection of γ-GC rather than relying on derivatization of the molecule or linked assays. The sensitivity of the assay is sufficient to allow for the measurement of GCL activity in cultured cells. The specific activity of GCL in rat primary culture astrocytes was 9.7 ± 1.7 nmol γ-GC synthesized/min/mg protein. |
| doi_str_mv | 10.1006/abio.2001.5607 |
| format | Article |
| fullrecord | <record><control><sourceid>proquest_cross</sourceid><recordid>TN_cdi_proquest_miscellaneous_71624751</recordid><sourceformat>XML</sourceformat><sourcesystem>PC</sourcesystem><els_id>S0003269701956076</els_id><sourcerecordid>71624751</sourcerecordid><originalsourceid>FETCH-LOGICAL-c340t-b97e4c3f2640f2b07f465d0ac7a80a8bb37457b4eddab4a7d65ce061538a3db3</originalsourceid><addsrcrecordid>eNp1kc1u1DAUhS0EokNhyxJ5hegiw3V-7GRZTUuLNBJIdG_552ZilMRT26mUd-lb8B48E4lmECtWd3G-e3TvOYS8Z7BlAPyz0s5vcwC2rTiIF2TDoOEZFNC8JBsAKLKcN-KCvInx50KxsuKvyQVjDW9YXW3I8w0mDIMbVXJ-pL6ld_2U1KASZrs5JnQj0r07qIj00-9f2Umde_NX-zGPqcO06Ff02iT35NJM9Uzv3aHLvmNofRjUaFaTx8lZuuuCX9z9IahjN1M1Wnrbo0nBmw4HZ1RP15PMes5b8qpVfcR353lJHr7cPuzus_23u6-7631mihJSphuBpSnanJfQ5hpEW_LKgjJC1aBqrQtRVkKXaK3SpRKWVwaBs6qoVWF1cUk-nmyPwT9OGJMcXDTY92pEP0UpGM9LUbEF3J5AE3yMAVt5DG5QYZYM5FqHXOuQax1yrWNZ-HB2nvSA9h9-zn8B6hOAy3tPDoOMxuESl3VhyUBa7_7n_QdWT55Z</addsrcrecordid><sourcetype>Aggregation Database</sourcetype><iscdi>true</iscdi><recordtype>article</recordtype><pqid>71624751</pqid></control><display><type>article</type><title>Determination of Glutamate-Cysteine Ligase (γ-Glutamylcysteine Synthetase) Activity by High-Performance Liquid Chromatography and Electrochemical Detection</title><source>MEDLINE</source><source>ScienceDirect Journals (5 years ago - present)</source><creator>Gegg, Matthew E. ; Clark, John B. ; Heales, Simon J.R.</creator><creatorcontrib>Gegg, Matthew E. ; Clark, John B. ; Heales, Simon J.R.</creatorcontrib><description>The tripeptide glutathione (γ-glutamylcysteinylglycine; GSH) is the predominant low molecular mass thiol in cells. The function of GSH is of considerable interest, with the molecule being implicated in numerous cellular processes in addition to being a major cellular antioxidant. The enzyme glutamate-cysteine ligase (GCL) is the rate-limiting step in GSH synthesis. The GCL assay described here is based on high-performance liquid chromatography and exploits the electrochemically active nature of γ-glutamylcysteine (γ-GC), the product of GCL activity. This method allows for the direct detection of γ-GC rather than relying on derivatization of the molecule or linked assays. The sensitivity of the assay is sufficient to allow for the measurement of GCL activity in cultured cells. The specific activity of GCL in rat primary culture astrocytes was 9.7 ± 1.7 nmol γ-GC synthesized/min/mg protein.</description><identifier>ISSN: 0003-2697</identifier><identifier>EISSN: 1096-0309</identifier><identifier>DOI: 10.1006/abio.2001.5607</identifier><identifier>PMID: 11969185</identifier><language>eng</language><publisher>United States: Elsevier Inc</publisher><subject>Animals ; astrocytes ; Astrocytes - enzymology ; Buthionine Sulfoximine - pharmacology ; Cells, Cultured ; Chromatography, High Pressure Liquid - methods ; Chromatography, High Pressure Liquid - standards ; Dipeptides - analysis ; electrochemical detection ; Electrochemistry ; Enzyme Inhibitors - pharmacology ; glutamate-cysteine ligase ; Glutamate-Cysteine Ligase - analysis ; Glutamate-Cysteine Ligase - antagonists & inhibitors ; Glutamate-Cysteine Ligase - metabolism ; Glutathione - analysis ; high-performance liquid chromatography ; Kinetics ; Rats ; Reference Standards</subject><ispartof>Analytical biochemistry, 2002-05, Vol.304 (1), p.26-32</ispartof><rights>2002 Elsevier Science (USA)</rights><rights>(c)2002 Elsevier Science (USA).</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c340t-b97e4c3f2640f2b07f465d0ac7a80a8bb37457b4eddab4a7d65ce061538a3db3</citedby><cites>FETCH-LOGICAL-c340t-b97e4c3f2640f2b07f465d0ac7a80a8bb37457b4eddab4a7d65ce061538a3db3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://dx.doi.org/10.1006/abio.2001.5607$$EHTML$$P50$$Gelsevier$$H</linktohtml><link.rule.ids>314,780,784,3550,27924,27925,45995</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/11969185$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Gegg, Matthew E.</creatorcontrib><creatorcontrib>Clark, John B.</creatorcontrib><creatorcontrib>Heales, Simon J.R.</creatorcontrib><title>Determination of Glutamate-Cysteine Ligase (γ-Glutamylcysteine Synthetase) Activity by High-Performance Liquid Chromatography and Electrochemical Detection</title><title>Analytical biochemistry</title><addtitle>Anal Biochem</addtitle><description>The tripeptide glutathione (γ-glutamylcysteinylglycine; GSH) is the predominant low molecular mass thiol in cells. The function of GSH is of considerable interest, with the molecule being implicated in numerous cellular processes in addition to being a major cellular antioxidant. The enzyme glutamate-cysteine ligase (GCL) is the rate-limiting step in GSH synthesis. The GCL assay described here is based on high-performance liquid chromatography and exploits the electrochemically active nature of γ-glutamylcysteine (γ-GC), the product of GCL activity. This method allows for the direct detection of γ-GC rather than relying on derivatization of the molecule or linked assays. The sensitivity of the assay is sufficient to allow for the measurement of GCL activity in cultured cells. The specific activity of GCL in rat primary culture astrocytes was 9.7 ± 1.7 nmol γ-GC synthesized/min/mg protein.</description><subject>Animals</subject><subject>astrocytes</subject><subject>Astrocytes - enzymology</subject><subject>Buthionine Sulfoximine - pharmacology</subject><subject>Cells, Cultured</subject><subject>Chromatography, High Pressure Liquid - methods</subject><subject>Chromatography, High Pressure Liquid - standards</subject><subject>Dipeptides - analysis</subject><subject>electrochemical detection</subject><subject>Electrochemistry</subject><subject>Enzyme Inhibitors - pharmacology</subject><subject>glutamate-cysteine ligase</subject><subject>Glutamate-Cysteine Ligase - analysis</subject><subject>Glutamate-Cysteine Ligase - antagonists & inhibitors</subject><subject>Glutamate-Cysteine Ligase - metabolism</subject><subject>Glutathione - analysis</subject><subject>high-performance liquid chromatography</subject><subject>Kinetics</subject><subject>Rats</subject><subject>Reference Standards</subject><issn>0003-2697</issn><issn>1096-0309</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2002</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNp1kc1u1DAUhS0EokNhyxJ5hegiw3V-7GRZTUuLNBJIdG_552ZilMRT26mUd-lb8B48E4lmECtWd3G-e3TvOYS8Z7BlAPyz0s5vcwC2rTiIF2TDoOEZFNC8JBsAKLKcN-KCvInx50KxsuKvyQVjDW9YXW3I8w0mDIMbVXJ-pL6ld_2U1KASZrs5JnQj0r07qIj00-9f2Umde_NX-zGPqcO06Ff02iT35NJM9Uzv3aHLvmNofRjUaFaTx8lZuuuCX9z9IahjN1M1Wnrbo0nBmw4HZ1RP15PMes5b8qpVfcR353lJHr7cPuzus_23u6-7631mihJSphuBpSnanJfQ5hpEW_LKgjJC1aBqrQtRVkKXaK3SpRKWVwaBs6qoVWF1cUk-nmyPwT9OGJMcXDTY92pEP0UpGM9LUbEF3J5AE3yMAVt5DG5QYZYM5FqHXOuQax1yrWNZ-HB2nvSA9h9-zn8B6hOAy3tPDoOMxuESl3VhyUBa7_7n_QdWT55Z</recordid><startdate>20020501</startdate><enddate>20020501</enddate><creator>Gegg, Matthew E.</creator><creator>Clark, John B.</creator><creator>Heales, Simon J.R.</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>20020501</creationdate><title>Determination of Glutamate-Cysteine Ligase (γ-Glutamylcysteine Synthetase) Activity by High-Performance Liquid Chromatography and Electrochemical Detection</title><author>Gegg, Matthew E. ; Clark, John B. ; Heales, Simon J.R.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c340t-b97e4c3f2640f2b07f465d0ac7a80a8bb37457b4eddab4a7d65ce061538a3db3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2002</creationdate><topic>Animals</topic><topic>astrocytes</topic><topic>Astrocytes - enzymology</topic><topic>Buthionine Sulfoximine - pharmacology</topic><topic>Cells, Cultured</topic><topic>Chromatography, High Pressure Liquid - methods</topic><topic>Chromatography, High Pressure Liquid - standards</topic><topic>Dipeptides - analysis</topic><topic>electrochemical detection</topic><topic>Electrochemistry</topic><topic>Enzyme Inhibitors - pharmacology</topic><topic>glutamate-cysteine ligase</topic><topic>Glutamate-Cysteine Ligase - analysis</topic><topic>Glutamate-Cysteine Ligase - antagonists & inhibitors</topic><topic>Glutamate-Cysteine Ligase - metabolism</topic><topic>Glutathione - analysis</topic><topic>high-performance liquid chromatography</topic><topic>Kinetics</topic><topic>Rats</topic><topic>Reference Standards</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Gegg, Matthew E.</creatorcontrib><creatorcontrib>Clark, John B.</creatorcontrib><creatorcontrib>Heales, Simon J.R.</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>Gegg, Matthew E.</au><au>Clark, John B.</au><au>Heales, Simon J.R.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Determination of Glutamate-Cysteine Ligase (γ-Glutamylcysteine Synthetase) Activity by High-Performance Liquid Chromatography and Electrochemical Detection</atitle><jtitle>Analytical biochemistry</jtitle><addtitle>Anal Biochem</addtitle><date>2002-05-01</date><risdate>2002</risdate><volume>304</volume><issue>1</issue><spage>26</spage><epage>32</epage><pages>26-32</pages><issn>0003-2697</issn><eissn>1096-0309</eissn><abstract>The tripeptide glutathione (γ-glutamylcysteinylglycine; GSH) is the predominant low molecular mass thiol in cells. The function of GSH is of considerable interest, with the molecule being implicated in numerous cellular processes in addition to being a major cellular antioxidant. The enzyme glutamate-cysteine ligase (GCL) is the rate-limiting step in GSH synthesis. The GCL assay described here is based on high-performance liquid chromatography and exploits the electrochemically active nature of γ-glutamylcysteine (γ-GC), the product of GCL activity. This method allows for the direct detection of γ-GC rather than relying on derivatization of the molecule or linked assays. The sensitivity of the assay is sufficient to allow for the measurement of GCL activity in cultured cells. The specific activity of GCL in rat primary culture astrocytes was 9.7 ± 1.7 nmol γ-GC synthesized/min/mg protein.</abstract><cop>United States</cop><pub>Elsevier Inc</pub><pmid>11969185</pmid><doi>10.1006/abio.2001.5607</doi><tpages>7</tpages></addata></record> |
| fulltext | fulltext |
| identifier | ISSN: 0003-2697 |
| ispartof | Analytical biochemistry, 2002-05, Vol.304 (1), p.26-32 |
| issn | 0003-2697 1096-0309 |
| language | eng |
| recordid | cdi_proquest_miscellaneous_71624751 |
| source | MEDLINE; ScienceDirect Journals (5 years ago - present) |
| subjects | Animals astrocytes Astrocytes - enzymology Buthionine Sulfoximine - pharmacology Cells, Cultured Chromatography, High Pressure Liquid - methods Chromatography, High Pressure Liquid - standards Dipeptides - analysis electrochemical detection Electrochemistry Enzyme Inhibitors - pharmacology glutamate-cysteine ligase Glutamate-Cysteine Ligase - analysis Glutamate-Cysteine Ligase - antagonists & inhibitors Glutamate-Cysteine Ligase - metabolism Glutathione - analysis high-performance liquid chromatography Kinetics Rats Reference Standards |
| title | Determination of Glutamate-Cysteine Ligase (γ-Glutamylcysteine Synthetase) Activity by High-Performance Liquid Chromatography and Electrochemical Detection |
| url | https://sfx.bib-bvb.de/sfx_tum?ctx_ver=Z39.88-2004&ctx_enc=info:ofi/enc:UTF-8&ctx_tim=2025-01-04T01%3A45%3A43IST&url_ver=Z39.88-2004&url_ctx_fmt=infofi/fmt:kev:mtx:ctx&rfr_id=info:sid/primo.exlibrisgroup.com:primo3-Article-proquest_cross&rft_val_fmt=info:ofi/fmt:kev:mtx:journal&rft.genre=article&rft.atitle=Determination%20of%20Glutamate-Cysteine%20Ligase%20(%CE%B3-Glutamylcysteine%20Synthetase)%20Activity%20by%20High-Performance%20Liquid%20Chromatography%20and%20Electrochemical%20Detection&rft.jtitle=Analytical%20biochemistry&rft.au=Gegg,%20Matthew%20E.&rft.date=2002-05-01&rft.volume=304&rft.issue=1&rft.spage=26&rft.epage=32&rft.pages=26-32&rft.issn=0003-2697&rft.eissn=1096-0309&rft_id=info:doi/10.1006/abio.2001.5607&rft_dat=%3Cproquest_cross%3E71624751%3C/proquest_cross%3E%3Curl%3E%3C/url%3E&disable_directlink=true&sfx.directlink=off&sfx.report_link=0&rft_id=info:oai/&rft_pqid=71624751&rft_id=info:pmid/11969185&rft_els_id=S0003269701956076&rfr_iscdi=true |