Loading…

Heteromeric assembly of the cytosolic glutamine synthetase polypeptides of Medicago truncatula: complementation of a glnA Escherichia coli mutant with a plant domain-swapped enzyme

We have cloned and sequenced the cDNAs corresponding to the two cytosolic glutamine synthetase (GS) polypeptides (a and b) of Medicago truncatula. Using these two cDNAs we have prepared a construct encoding the N-terminal domain of b and the C-terminal domain of a in order to produce a domain-swappe...

Full description

Saved in:
Bibliographic Details
Published in:Plant molecular biology 1997-11, Vol.35 (5), p.623-632
Main Authors: Carvalho, H, Sunkel, C, Salema, R, Cullimore, J.V
Format: Article
Language:English
Subjects:
Citations: Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
cited_by cdi_FETCH-LOGICAL-c334t-fcf8ca1dc2e4dae14eff142a31a4f94faad1595886bbedae74eca753fffc65223
cites
container_end_page 632
container_issue 5
container_start_page 623
container_title Plant molecular biology
container_volume 35
creator Carvalho, H
Sunkel, C
Salema, R
Cullimore, J.V
description We have cloned and sequenced the cDNAs corresponding to the two cytosolic glutamine synthetase (GS) polypeptides (a and b) of Medicago truncatula. Using these two cDNAs we have prepared a construct encoding the N-terminal domain of b and the C-terminal domain of a in order to produce a domain-swapped polypeptide which should assemble to give an enzyme containing chimeric active sites. Both the native and the domain-swapped enzymes were expressed in Escherichia coli where they were catalytically and physiologically active as they were able to rescue a glnA deletion mutant. The expressed polypeptides were of the correct size and the isoenzymes behaved similarly to their native homologues on ion-exchange chromatography. We have found slight differences in the kinetic properties of the purified enzymes and in the modulation of their activities by several putative cellular effectors. In vitro dissociation of the purified a and b homo-octamers, followed by reassociation, showed that the subunits are able to self-assemble, perhaps randomly, to form heteromeric isoenzymes. Moreover, heteromeric isoenzymes occur in the plant as revealed by studies on the GS isoenzymes of nodules, roots, stems and stipules.
doi_str_mv 10.1023/A:1005884304303
format article
fullrecord <record><control><sourceid>proquest_pubme</sourceid><recordid>TN_cdi_proquest_miscellaneous_79362424</recordid><sourceformat>XML</sourceformat><sourcesystem>PC</sourcesystem><sourcerecordid>16255646</sourcerecordid><originalsourceid>FETCH-LOGICAL-c334t-fcf8ca1dc2e4dae14eff142a31a4f94faad1595886bbedae74eca753fffc65223</originalsourceid><addsrcrecordid>eNqFkj1vFDEQhi0ECkegpkJYKegW_LVf6U5RSJCCKCD1as47vnPkj2XtVbT8Ln4gPuWqNEgjjaz38bz2zBDynrPPnAn5ZXvJGau7TklWQr4gG163sqqZ6F6SDeNNWynFxWvyJqUHxgosmzNy1kvVi05uyN9bzDhHj7PVFFJCv3MrjYbmA1K95piiK8reLRm8DUjTGoqUISGdolsnnLIdMR2vfMfRathHmuclaMiLg0uqo58cegwZso3hyEEpF7b0OunD0fZgoVDOUl88QqaPNh8KM7njYYwebKjSI0wTjhTDn9XjW_LKgEv47pTPyf3X619Xt9Xdj5tvV9u7SkupcmW06TTwUQtUIyBXaAxXAiQHZXplAEZe96V5zW6HBWgVamhraYzRTS2EPCefnupOc_y9YMqDt0mjKy_DuKSh7WUjlFD_BXkj6rpRTQEvnoEPcZlD-cTQtlzVopeyQB9O0LLzOA7TbD3M63AaWtE_PukG4gD72abh_qdgXJaht2UVmPwHOien7A</addsrcrecordid><sourcetype>Aggregation Database</sourcetype><iscdi>true</iscdi><recordtype>article</recordtype><pqid>771452933</pqid></control><display><type>article</type><title>Heteromeric assembly of the cytosolic glutamine synthetase polypeptides of Medicago truncatula: complementation of a glnA Escherichia coli mutant with a plant domain-swapped enzyme</title><source>Springer Link</source><creator>Carvalho, H ; Sunkel, C ; Salema, R ; Cullimore, J.V</creator><creatorcontrib>Carvalho, H ; Sunkel, C ; Salema, R ; Cullimore, J.V</creatorcontrib><description>We have cloned and sequenced the cDNAs corresponding to the two cytosolic glutamine synthetase (GS) polypeptides (a and b) of Medicago truncatula. Using these two cDNAs we have prepared a construct encoding the N-terminal domain of b and the C-terminal domain of a in order to produce a domain-swapped polypeptide which should assemble to give an enzyme containing chimeric active sites. Both the native and the domain-swapped enzymes were expressed in Escherichia coli where they were catalytically and physiologically active as they were able to rescue a glnA deletion mutant. The expressed polypeptides were of the correct size and the isoenzymes behaved similarly to their native homologues on ion-exchange chromatography. We have found slight differences in the kinetic properties of the purified enzymes and in the modulation of their activities by several putative cellular effectors. In vitro dissociation of the purified a and b homo-octamers, followed by reassociation, showed that the subunits are able to self-assemble, perhaps randomly, to form heteromeric isoenzymes. Moreover, heteromeric isoenzymes occur in the plant as revealed by studies on the GS isoenzymes of nodules, roots, stems and stipules.</description><identifier>ISSN: 0167-4412</identifier><identifier>EISSN: 1573-5028</identifier><identifier>DOI: 10.1023/A:1005884304303</identifier><identifier>PMID: 9349283</identifier><language>eng</language><publisher>Netherlands: Springer Nature B.V</publisher><subject>Alfalfa ; Amino Acid Sequence ; amino acid sequences ; Binding Sites ; Cloning, Molecular ; complementary DNA ; cytosol ; Cytosol - enzymology ; E coli ; enzyme activity ; Enzymes ; Escherichia coli ; Escherichia coli - genetics ; genbank/y10267 ; genbank/y10268 ; genetic complementation ; Genetic Complementation Test ; glutamate-ammonia ligase ; Glutamate-Ammonia Ligase - biosynthesis ; Glutamate-Ammonia Ligase - chemistry ; Glutamate-Ammonia Ligase - genetics ; Glutamate-Ammonia Ligase - metabolism ; interactions ; Isoenzymes - biosynthesis ; Isoenzymes - chemistry ; Isoenzymes - genetics ; Isoenzymes - metabolism ; isozymes ; Kinetics ; Medicago sativa - enzymology ; Medicago sativa - genetics ; Medicago truncatula ; Molecular Sequence Data ; mutants ; nucleotide sequences ; Peptides - chemistry ; Polypeptides ; Recombinant Fusion Proteins - biosynthesis ; Recombinant Fusion Proteins - chemistry ; Recombinant Fusion Proteins - metabolism ; Sequence Analysis, DNA ; Sequence Deletion</subject><ispartof>Plant molecular biology, 1997-11, Vol.35 (5), p.623-632</ispartof><rights>Kluwer Academic Publishers 1997</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c334t-fcf8ca1dc2e4dae14eff142a31a4f94faad1595886bbedae74eca753fffc65223</citedby></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,780,784,27924,27925</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/9349283$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Carvalho, H</creatorcontrib><creatorcontrib>Sunkel, C</creatorcontrib><creatorcontrib>Salema, R</creatorcontrib><creatorcontrib>Cullimore, J.V</creatorcontrib><title>Heteromeric assembly of the cytosolic glutamine synthetase polypeptides of Medicago truncatula: complementation of a glnA Escherichia coli mutant with a plant domain-swapped enzyme</title><title>Plant molecular biology</title><addtitle>Plant Mol Biol</addtitle><description>We have cloned and sequenced the cDNAs corresponding to the two cytosolic glutamine synthetase (GS) polypeptides (a and b) of Medicago truncatula. Using these two cDNAs we have prepared a construct encoding the N-terminal domain of b and the C-terminal domain of a in order to produce a domain-swapped polypeptide which should assemble to give an enzyme containing chimeric active sites. Both the native and the domain-swapped enzymes were expressed in Escherichia coli where they were catalytically and physiologically active as they were able to rescue a glnA deletion mutant. The expressed polypeptides were of the correct size and the isoenzymes behaved similarly to their native homologues on ion-exchange chromatography. We have found slight differences in the kinetic properties of the purified enzymes and in the modulation of their activities by several putative cellular effectors. In vitro dissociation of the purified a and b homo-octamers, followed by reassociation, showed that the subunits are able to self-assemble, perhaps randomly, to form heteromeric isoenzymes. Moreover, heteromeric isoenzymes occur in the plant as revealed by studies on the GS isoenzymes of nodules, roots, stems and stipules.</description><subject>Alfalfa</subject><subject>Amino Acid Sequence</subject><subject>amino acid sequences</subject><subject>Binding Sites</subject><subject>Cloning, Molecular</subject><subject>complementary DNA</subject><subject>cytosol</subject><subject>Cytosol - enzymology</subject><subject>E coli</subject><subject>enzyme activity</subject><subject>Enzymes</subject><subject>Escherichia coli</subject><subject>Escherichia coli - genetics</subject><subject>genbank/y10267</subject><subject>genbank/y10268</subject><subject>genetic complementation</subject><subject>Genetic Complementation Test</subject><subject>glutamate-ammonia ligase</subject><subject>Glutamate-Ammonia Ligase - biosynthesis</subject><subject>Glutamate-Ammonia Ligase - chemistry</subject><subject>Glutamate-Ammonia Ligase - genetics</subject><subject>Glutamate-Ammonia Ligase - metabolism</subject><subject>interactions</subject><subject>Isoenzymes - biosynthesis</subject><subject>Isoenzymes - chemistry</subject><subject>Isoenzymes - genetics</subject><subject>Isoenzymes - metabolism</subject><subject>isozymes</subject><subject>Kinetics</subject><subject>Medicago sativa - enzymology</subject><subject>Medicago sativa - genetics</subject><subject>Medicago truncatula</subject><subject>Molecular Sequence Data</subject><subject>mutants</subject><subject>nucleotide sequences</subject><subject>Peptides - chemistry</subject><subject>Polypeptides</subject><subject>Recombinant Fusion Proteins - biosynthesis</subject><subject>Recombinant Fusion Proteins - chemistry</subject><subject>Recombinant Fusion Proteins - metabolism</subject><subject>Sequence Analysis, DNA</subject><subject>Sequence Deletion</subject><issn>0167-4412</issn><issn>1573-5028</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1997</creationdate><recordtype>article</recordtype><recordid>eNqFkj1vFDEQhi0ECkegpkJYKegW_LVf6U5RSJCCKCD1as47vnPkj2XtVbT8Ln4gPuWqNEgjjaz38bz2zBDynrPPnAn5ZXvJGau7TklWQr4gG163sqqZ6F6SDeNNWynFxWvyJqUHxgosmzNy1kvVi05uyN9bzDhHj7PVFFJCv3MrjYbmA1K95piiK8reLRm8DUjTGoqUISGdolsnnLIdMR2vfMfRathHmuclaMiLg0uqo58cegwZso3hyEEpF7b0OunD0fZgoVDOUl88QqaPNh8KM7njYYwebKjSI0wTjhTDn9XjW_LKgEv47pTPyf3X619Xt9Xdj5tvV9u7SkupcmW06TTwUQtUIyBXaAxXAiQHZXplAEZe96V5zW6HBWgVamhraYzRTS2EPCefnupOc_y9YMqDt0mjKy_DuKSh7WUjlFD_BXkj6rpRTQEvnoEPcZlD-cTQtlzVopeyQB9O0LLzOA7TbD3M63AaWtE_PukG4gD72abh_qdgXJaht2UVmPwHOien7A</recordid><startdate>19971101</startdate><enddate>19971101</enddate><creator>Carvalho, H</creator><creator>Sunkel, C</creator><creator>Salema, R</creator><creator>Cullimore, J.V</creator><general>Springer Nature B.V</general><scope>FBQ</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>3V.</scope><scope>7TM</scope><scope>7X7</scope><scope>7XB</scope><scope>88A</scope><scope>88E</scope><scope>8AO</scope><scope>8FD</scope><scope>8FE</scope><scope>8FH</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>8G5</scope><scope>ABUWG</scope><scope>AFKRA</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BHPHI</scope><scope>CCPQU</scope><scope>DWQXO</scope><scope>FR3</scope><scope>FYUFA</scope><scope>GHDGH</scope><scope>GNUQQ</scope><scope>GUQSH</scope><scope>HCIFZ</scope><scope>K9.</scope><scope>LK8</scope><scope>M0S</scope><scope>M1P</scope><scope>M2O</scope><scope>M7P</scope><scope>MBDVC</scope><scope>P64</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>Q9U</scope><scope>RC3</scope><scope>7X8</scope></search><sort><creationdate>19971101</creationdate><title>Heteromeric assembly of the cytosolic glutamine synthetase polypeptides of Medicago truncatula: complementation of a glnA Escherichia coli mutant with a plant domain-swapped enzyme</title><author>Carvalho, H ; Sunkel, C ; Salema, R ; Cullimore, J.V</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c334t-fcf8ca1dc2e4dae14eff142a31a4f94faad1595886bbedae74eca753fffc65223</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1997</creationdate><topic>Alfalfa</topic><topic>Amino Acid Sequence</topic><topic>amino acid sequences</topic><topic>Binding Sites</topic><topic>Cloning, Molecular</topic><topic>complementary DNA</topic><topic>cytosol</topic><topic>Cytosol - enzymology</topic><topic>E coli</topic><topic>enzyme activity</topic><topic>Enzymes</topic><topic>Escherichia coli</topic><topic>Escherichia coli - genetics</topic><topic>genbank/y10267</topic><topic>genbank/y10268</topic><topic>genetic complementation</topic><topic>Genetic Complementation Test</topic><topic>glutamate-ammonia ligase</topic><topic>Glutamate-Ammonia Ligase - biosynthesis</topic><topic>Glutamate-Ammonia Ligase - chemistry</topic><topic>Glutamate-Ammonia Ligase - genetics</topic><topic>Glutamate-Ammonia Ligase - metabolism</topic><topic>interactions</topic><topic>Isoenzymes - biosynthesis</topic><topic>Isoenzymes - chemistry</topic><topic>Isoenzymes - genetics</topic><topic>Isoenzymes - metabolism</topic><topic>isozymes</topic><topic>Kinetics</topic><topic>Medicago sativa - enzymology</topic><topic>Medicago sativa - genetics</topic><topic>Medicago truncatula</topic><topic>Molecular Sequence Data</topic><topic>mutants</topic><topic>nucleotide sequences</topic><topic>Peptides - chemistry</topic><topic>Polypeptides</topic><topic>Recombinant Fusion Proteins - biosynthesis</topic><topic>Recombinant Fusion Proteins - chemistry</topic><topic>Recombinant Fusion Proteins - metabolism</topic><topic>Sequence Analysis, DNA</topic><topic>Sequence Deletion</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Carvalho, H</creatorcontrib><creatorcontrib>Sunkel, C</creatorcontrib><creatorcontrib>Salema, R</creatorcontrib><creatorcontrib>Cullimore, J.V</creatorcontrib><collection>AGRIS</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>ProQuest Central (Corporate)</collection><collection>Nucleic Acids Abstracts</collection><collection>Health &amp; Medical Collection (Proquest)</collection><collection>ProQuest Central (purchase pre-March 2016)</collection><collection>Biology Database (Alumni Edition)</collection><collection>Medical Database (Alumni Edition)</collection><collection>ProQuest Pharma Collection</collection><collection>Technology Research Database</collection><collection>ProQuest SciTech Collection</collection><collection>ProQuest Natural Science Collection</collection><collection>Hospital Premium Collection</collection><collection>Hospital Premium Collection (Alumni Edition)</collection><collection>ProQuest Central (Alumni) (purchase pre-March 2016)</collection><collection>Research Library (Alumni Edition)</collection><collection>ProQuest Central (Alumni)</collection><collection>ProQuest Central</collection><collection>ProQuest Central Essentials</collection><collection>Biological Science Collection</collection><collection>AUTh Library subscriptions: ProQuest Central</collection><collection>ProQuest Natural Science Collection</collection><collection>ProQuest One Community College</collection><collection>ProQuest Central</collection><collection>Engineering Research Database</collection><collection>Health Research Premium Collection</collection><collection>Health Research Premium Collection (Alumni)</collection><collection>ProQuest Central Student</collection><collection>Research Library Prep</collection><collection>SciTech Premium Collection</collection><collection>ProQuest Health &amp; Medical Complete (Alumni)</collection><collection>ProQuest Biological Science Collection</collection><collection>Health &amp; Medical Collection (Alumni Edition)</collection><collection>PML(ProQuest Medical Library)</collection><collection>ProQuest research library</collection><collection>ProQuest Biological Science Journals</collection><collection>Research Library (Corporate)</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>ProQuest One Academic Eastern Edition (DO NOT USE)</collection><collection>ProQuest One Academic</collection><collection>ProQuest One Academic UKI Edition</collection><collection>ProQuest Central Basic</collection><collection>Genetics Abstracts</collection><collection>MEDLINE - Academic</collection><jtitle>Plant molecular biology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Carvalho, H</au><au>Sunkel, C</au><au>Salema, R</au><au>Cullimore, J.V</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Heteromeric assembly of the cytosolic glutamine synthetase polypeptides of Medicago truncatula: complementation of a glnA Escherichia coli mutant with a plant domain-swapped enzyme</atitle><jtitle>Plant molecular biology</jtitle><addtitle>Plant Mol Biol</addtitle><date>1997-11-01</date><risdate>1997</risdate><volume>35</volume><issue>5</issue><spage>623</spage><epage>632</epage><pages>623-632</pages><issn>0167-4412</issn><eissn>1573-5028</eissn><abstract>We have cloned and sequenced the cDNAs corresponding to the two cytosolic glutamine synthetase (GS) polypeptides (a and b) of Medicago truncatula. Using these two cDNAs we have prepared a construct encoding the N-terminal domain of b and the C-terminal domain of a in order to produce a domain-swapped polypeptide which should assemble to give an enzyme containing chimeric active sites. Both the native and the domain-swapped enzymes were expressed in Escherichia coli where they were catalytically and physiologically active as they were able to rescue a glnA deletion mutant. The expressed polypeptides were of the correct size and the isoenzymes behaved similarly to their native homologues on ion-exchange chromatography. We have found slight differences in the kinetic properties of the purified enzymes and in the modulation of their activities by several putative cellular effectors. In vitro dissociation of the purified a and b homo-octamers, followed by reassociation, showed that the subunits are able to self-assemble, perhaps randomly, to form heteromeric isoenzymes. Moreover, heteromeric isoenzymes occur in the plant as revealed by studies on the GS isoenzymes of nodules, roots, stems and stipules.</abstract><cop>Netherlands</cop><pub>Springer Nature B.V</pub><pmid>9349283</pmid><doi>10.1023/A:1005884304303</doi><tpages>10</tpages></addata></record>
fulltext fulltext
identifier ISSN: 0167-4412
ispartof Plant molecular biology, 1997-11, Vol.35 (5), p.623-632
issn 0167-4412
1573-5028
language eng
recordid cdi_proquest_miscellaneous_79362424
source Springer Link
subjects Alfalfa
Amino Acid Sequence
amino acid sequences
Binding Sites
Cloning, Molecular
complementary DNA
cytosol
Cytosol - enzymology
E coli
enzyme activity
Enzymes
Escherichia coli
Escherichia coli - genetics
genbank/y10267
genbank/y10268
genetic complementation
Genetic Complementation Test
glutamate-ammonia ligase
Glutamate-Ammonia Ligase - biosynthesis
Glutamate-Ammonia Ligase - chemistry
Glutamate-Ammonia Ligase - genetics
Glutamate-Ammonia Ligase - metabolism
interactions
Isoenzymes - biosynthesis
Isoenzymes - chemistry
Isoenzymes - genetics
Isoenzymes - metabolism
isozymes
Kinetics
Medicago sativa - enzymology
Medicago sativa - genetics
Medicago truncatula
Molecular Sequence Data
mutants
nucleotide sequences
Peptides - chemistry
Polypeptides
Recombinant Fusion Proteins - biosynthesis
Recombinant Fusion Proteins - chemistry
Recombinant Fusion Proteins - metabolism
Sequence Analysis, DNA
Sequence Deletion
title Heteromeric assembly of the cytosolic glutamine synthetase polypeptides of Medicago truncatula: complementation of a glnA Escherichia coli mutant with a plant domain-swapped enzyme
url http://sfxeu10.hosted.exlibrisgroup.com/loughborough?ctx_ver=Z39.88-2004&ctx_enc=info:ofi/enc:UTF-8&ctx_tim=2024-12-25T04%3A55%3A31IST&url_ver=Z39.88-2004&url_ctx_fmt=infofi/fmt:kev:mtx:ctx&rfr_id=info:sid/primo.exlibrisgroup.com:primo3-Article-proquest_pubme&rft_val_fmt=info:ofi/fmt:kev:mtx:journal&rft.genre=article&rft.atitle=Heteromeric%20assembly%20of%20the%20cytosolic%20glutamine%20synthetase%20polypeptides%20of%20Medicago%20truncatula:%20complementation%20of%20a%20glnA%20Escherichia%20coli%20mutant%20with%20a%20plant%20domain-swapped%20enzyme&rft.jtitle=Plant%20molecular%20biology&rft.au=Carvalho,%20H&rft.date=1997-11-01&rft.volume=35&rft.issue=5&rft.spage=623&rft.epage=632&rft.pages=623-632&rft.issn=0167-4412&rft.eissn=1573-5028&rft_id=info:doi/10.1023/A:1005884304303&rft_dat=%3Cproquest_pubme%3E16255646%3C/proquest_pubme%3E%3Cgrp_id%3Ecdi_FETCH-LOGICAL-c334t-fcf8ca1dc2e4dae14eff142a31a4f94faad1595886bbedae74eca753fffc65223%3C/grp_id%3E%3Coa%3E%3C/oa%3E%3Curl%3E%3C/url%3E&rft_id=info:oai/&rft_pqid=771452933&rft_id=info:pmid/9349283&rfr_iscdi=true