Coupling of a Purified Goldfish Brain Kainate Receptor with a Pertussis Toxin-Sensitive G Protein
Goldfish brain has a high density of [3H]kainate-binding sites, a subpopulation of which appears to be coupled to a pertussis toxin-sensitive G protein. We show here that a purified kainate receptor preparation reconstituted into phospholipid vesicles exhibits guanine nucleotide-sensitive high-affin...
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| Veröffentlicht in: | Proceedings of the National Academy of Sciences - PNAS 1992-05, Vol.89 (9), p.4134-4138 |
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| container_title | Proceedings of the National Academy of Sciences - PNAS |
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| creator | Ziegra, Cynthia J. Willard, James M. Oswald, Robert E. |
| description | Goldfish brain has a high density of [3H]kainate-binding sites, a subpopulation of which appears to be coupled to a pertussis toxin-sensitive G protein. We show here that a purified kainate receptor preparation reconstituted into phospholipid vesicles exhibits guanine nucleotide-sensitive high-affinity [3H]kainate binding. Pertussis toxin treatment abolishes the guanine nucleotide-sensitive portion of the [3H]kainate binding, and kainate promotes [3H]guanosine 5'-[β,γ-imido]triphosphate binding and [γ-32P]GTP hydrolysis. Guanosine 5'-[γ-thio]triphosphate (GTP[γ S]) decreases the apparent Stokes radius of the soluble purified receptor preparation, consistent with dissociation of the kainate receptor-G protein complexes. The affinity-purified preparations contain proteins of 45, 41, and 35 kDa. The 45- and 41-kDa proteins crossreact with antibodies against the kainate receptor cloned from frog brain. The 35-kDa protein is recognized by an antiserum (SW) directed against the β subunit of G proteins. When kainate receptors are purified in the presence of GTP[γS], the 35-kDa protein is no longer present. Also, [3H]kainate affinity is decreased and is no longer guanine nucleotide sensitive. Upon reconstitution with purified G proteins, high-affinity guanine nucleotide-sensitive binding and kainate-stimulated GTPase activity can be restored. These observations indicate that a kainate receptor from goldfish brain functionally interacts with a pertussis toxin-sensitive G protein. |
| doi_str_mv | 10.1073/pnas.89.9.4134 |
| format | Article |
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We show here that a purified kainate receptor preparation reconstituted into phospholipid vesicles exhibits guanine nucleotide-sensitive high-affinity [3H]kainate binding. Pertussis toxin treatment abolishes the guanine nucleotide-sensitive portion of the [3H]kainate binding, and kainate promotes [3H]guanosine 5'-[β,γ-imido]triphosphate binding and [γ-32P]GTP hydrolysis. Guanosine 5'-[γ-thio]triphosphate (GTP[γ S]) decreases the apparent Stokes radius of the soluble purified receptor preparation, consistent with dissociation of the kainate receptor-G protein complexes. The affinity-purified preparations contain proteins of 45, 41, and 35 kDa. The 45- and 41-kDa proteins crossreact with antibodies against the kainate receptor cloned from frog brain. The 35-kDa protein is recognized by an antiserum (SW) directed against the β subunit of G proteins. When kainate receptors are purified in the presence of GTP[γS], the 35-kDa protein is no longer present. Also, [3H]kainate affinity is decreased and is no longer guanine nucleotide sensitive. Upon reconstitution with purified G proteins, high-affinity guanine nucleotide-sensitive binding and kainate-stimulated GTPase activity can be restored. These observations indicate that a kainate receptor from goldfish brain functionally interacts with a pertussis toxin-sensitive G protein.</description><identifier>ISSN: 0027-8424</identifier><identifier>EISSN: 1091-6490</identifier><identifier>DOI: 10.1073/pnas.89.9.4134</identifier><identifier>PMID: 1315052</identifier><identifier>CODEN: PNASA6</identifier><language>eng</language><publisher>Washington, DC: National Academy of Sciences of the United States of America</publisher><subject>Aminoacid receptors (glycine, glutamate, gaba) ; Animals ; Biological and medical sciences ; Brain ; Carassius auratus ; Cattle ; Cell receptors ; Cell structures and functions ; Elution ; Fish ; Freshwater ; Frogs ; Fundamental and applied biological sciences. Psychology ; Gels ; Goldfish ; GTP-Binding Proteins - isolation & purification ; GTP-Binding Proteins - metabolism ; guanine nucleotide-binding protein ; Guanine nucleotides ; In Vitro Techniques ; kainic acid ; Kainic acid receptors ; Macromolecular Substances ; Molecular and cellular biology ; neurotransmitters ; pertussis ; Pertussis Toxin ; Protein Binding ; Proteins ; Receptors ; Receptors, Kainic Acid ; Receptors, Neurotransmitter - isolation & purification ; Receptors, Neurotransmitter - metabolism ; Toxins ; Virulence Factors, Bordetella - pharmacology ; Whooping cough</subject><ispartof>Proceedings of the National Academy of Sciences - PNAS, 1992-05, Vol.89 (9), p.4134-4138</ispartof><rights>Copyright 1992 The National Academy of Sciences of the United States of America</rights><rights>1992 INIST-CNRS</rights><rights>Copyright National Academy of Sciences May 1, 1992</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c545t-4f63bbaf953c734926ebd084c7ba160e8005fd5382e6da860bcd85a3bbda303c3</citedby></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Uhttp://www.pnas.org/content/89/9.cover.gif</thumbnail><linktopdf>$$Uhttps://www.jstor.org/stable/pdf/2359822$$EPDF$$P50$$Gjstor$$H</linktopdf><linktohtml>$$Uhttps://www.jstor.org/stable/2359822$$EHTML$$P50$$Gjstor$$H</linktohtml><link.rule.ids>230,314,725,778,782,801,883,27907,27908,53774,53776,58000,58233</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=5284279$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/1315052$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Ziegra, Cynthia J.</creatorcontrib><creatorcontrib>Willard, James M.</creatorcontrib><creatorcontrib>Oswald, Robert E.</creatorcontrib><title>Coupling of a Purified Goldfish Brain Kainate Receptor with a Pertussis Toxin-Sensitive G Protein</title><title>Proceedings of the National Academy of Sciences - PNAS</title><addtitle>Proc Natl Acad Sci U S A</addtitle><description>Goldfish brain has a high density of [3H]kainate-binding sites, a subpopulation of which appears to be coupled to a pertussis toxin-sensitive G protein. We show here that a purified kainate receptor preparation reconstituted into phospholipid vesicles exhibits guanine nucleotide-sensitive high-affinity [3H]kainate binding. Pertussis toxin treatment abolishes the guanine nucleotide-sensitive portion of the [3H]kainate binding, and kainate promotes [3H]guanosine 5'-[β,γ-imido]triphosphate binding and [γ-32P]GTP hydrolysis. Guanosine 5'-[γ-thio]triphosphate (GTP[γ S]) decreases the apparent Stokes radius of the soluble purified receptor preparation, consistent with dissociation of the kainate receptor-G protein complexes. The affinity-purified preparations contain proteins of 45, 41, and 35 kDa. The 45- and 41-kDa proteins crossreact with antibodies against the kainate receptor cloned from frog brain. The 35-kDa protein is recognized by an antiserum (SW) directed against the β subunit of G proteins. When kainate receptors are purified in the presence of GTP[γS], the 35-kDa protein is no longer present. Also, [3H]kainate affinity is decreased and is no longer guanine nucleotide sensitive. Upon reconstitution with purified G proteins, high-affinity guanine nucleotide-sensitive binding and kainate-stimulated GTPase activity can be restored. These observations indicate that a kainate receptor from goldfish brain functionally interacts with a pertussis toxin-sensitive G protein.</description><subject>Aminoacid receptors (glycine, glutamate, gaba)</subject><subject>Animals</subject><subject>Biological and medical sciences</subject><subject>Brain</subject><subject>Carassius auratus</subject><subject>Cattle</subject><subject>Cell receptors</subject><subject>Cell structures and functions</subject><subject>Elution</subject><subject>Fish</subject><subject>Freshwater</subject><subject>Frogs</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Gels</subject><subject>Goldfish</subject><subject>GTP-Binding Proteins - isolation & purification</subject><subject>GTP-Binding Proteins - metabolism</subject><subject>guanine nucleotide-binding protein</subject><subject>Guanine nucleotides</subject><subject>In Vitro Techniques</subject><subject>kainic acid</subject><subject>Kainic acid receptors</subject><subject>Macromolecular Substances</subject><subject>Molecular and cellular biology</subject><subject>neurotransmitters</subject><subject>pertussis</subject><subject>Pertussis Toxin</subject><subject>Protein Binding</subject><subject>Proteins</subject><subject>Receptors</subject><subject>Receptors, Kainic Acid</subject><subject>Receptors, Neurotransmitter - isolation & purification</subject><subject>Receptors, Neurotransmitter - metabolism</subject><subject>Toxins</subject><subject>Virulence Factors, Bordetella - pharmacology</subject><subject>Whooping cough</subject><issn>0027-8424</issn><issn>1091-6490</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1992</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFks9v0zAcxSMEGmVw5QSShabdEvwzsQ8coIKCmMQE42w5jrO6Su3Mdrbx3-OopRQOcLEP7_Oef7xvUTxHsEKwIa9Hp2LFRSUqigh9UCwQFKisqYAPiwWEuCk5xfRx8STGDYRQMA5PihNEEIMMLwq19NM4WHcNfA8UuJyC7a3pwMoPXW_jGrwLyjrwOS8qGfDVaDMmH8CdTeuZNyFNMdoIrvy9deU346JN9taAFbgMPhnrnhaPejVE82y_nxbfP7y_Wn4sL76sPi3fXpSaUZZK2tekbVUvGNENoQLXpu0gp7ppFaqh4RCyvmOEY1N3itew1R1nKns6RSDR5LR4s8sdp3ZrOm1cCmqQY7BbFX5Ir6z8U3F2La_9rWSY1bTJ_vO9P_ibycQktzZqMwzKGT9F2WAuGGv4f0FUI47rBmfw1V_gxk_B5U-QGCJMEWtghqodpIOPMZj-cGME5dywnBuWXEgh54az4eXxO3_ju0qzfrbXVdRq6INy2sYDxnCeh0Ycxczxv9TjY87_pct-GoZk7lMGX-zATcyDcSAxYYJjTH4CQu3QZw</recordid><startdate>19920501</startdate><enddate>19920501</enddate><creator>Ziegra, Cynthia J.</creator><creator>Willard, James M.</creator><creator>Oswald, Robert E.</creator><general>National Academy of Sciences of the United States of America</general><general>National Acad Sciences</general><general>National Academy of Sciences</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>7QG</scope><scope>7QL</scope><scope>7QP</scope><scope>7QR</scope><scope>7SN</scope><scope>7SS</scope><scope>7T5</scope><scope>7TK</scope><scope>7TM</scope><scope>7TO</scope><scope>7U9</scope><scope>8FD</scope><scope>C1K</scope><scope>FR3</scope><scope>H94</scope><scope>M7N</scope><scope>P64</scope><scope>RC3</scope><scope>7TN</scope><scope>F1W</scope><scope>H95</scope><scope>L.G</scope><scope>M7Z</scope><scope>7X8</scope><scope>5PM</scope></search><sort><creationdate>19920501</creationdate><title>Coupling of a Purified Goldfish Brain Kainate Receptor with a Pertussis Toxin-Sensitive G Protein</title><author>Ziegra, Cynthia J. ; Willard, James M. ; Oswald, Robert E.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c545t-4f63bbaf953c734926ebd084c7ba160e8005fd5382e6da860bcd85a3bbda303c3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1992</creationdate><topic>Aminoacid receptors (glycine, glutamate, gaba)</topic><topic>Animals</topic><topic>Biological and medical sciences</topic><topic>Brain</topic><topic>Carassius auratus</topic><topic>Cattle</topic><topic>Cell receptors</topic><topic>Cell structures and functions</topic><topic>Elution</topic><topic>Fish</topic><topic>Freshwater</topic><topic>Frogs</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>Gels</topic><topic>Goldfish</topic><topic>GTP-Binding Proteins - isolation & purification</topic><topic>GTP-Binding Proteins - metabolism</topic><topic>guanine nucleotide-binding protein</topic><topic>Guanine nucleotides</topic><topic>In Vitro Techniques</topic><topic>kainic acid</topic><topic>Kainic acid receptors</topic><topic>Macromolecular Substances</topic><topic>Molecular and cellular biology</topic><topic>neurotransmitters</topic><topic>pertussis</topic><topic>Pertussis Toxin</topic><topic>Protein Binding</topic><topic>Proteins</topic><topic>Receptors</topic><topic>Receptors, Kainic Acid</topic><topic>Receptors, Neurotransmitter - isolation & purification</topic><topic>Receptors, Neurotransmitter - metabolism</topic><topic>Toxins</topic><topic>Virulence Factors, Bordetella - pharmacology</topic><topic>Whooping cough</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Ziegra, Cynthia J.</creatorcontrib><creatorcontrib>Willard, James M.</creatorcontrib><creatorcontrib>Oswald, Robert E.</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>Animal Behavior Abstracts</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Calcium & Calcified Tissue Abstracts</collection><collection>Chemoreception Abstracts</collection><collection>Ecology Abstracts</collection><collection>Entomology Abstracts (Full archive)</collection><collection>Immunology Abstracts</collection><collection>Neurosciences Abstracts</collection><collection>Nucleic Acids Abstracts</collection><collection>Oncogenes and Growth Factors Abstracts</collection><collection>Virology and AIDS Abstracts</collection><collection>Technology Research Database</collection><collection>Environmental Sciences and Pollution Management</collection><collection>Engineering Research Database</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>Algology Mycology and Protozoology Abstracts (Microbiology C)</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>Genetics Abstracts</collection><collection>Oceanic Abstracts</collection><collection>ASFA: Aquatic Sciences and Fisheries Abstracts</collection><collection>Aquatic Science & Fisheries Abstracts (ASFA) 1: Biological Sciences & Living Resources</collection><collection>Aquatic Science & Fisheries Abstracts (ASFA) Professional</collection><collection>Biochemistry Abstracts 1</collection><collection>MEDLINE - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Proceedings of the National Academy of Sciences - PNAS</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Ziegra, Cynthia J.</au><au>Willard, James M.</au><au>Oswald, Robert E.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Coupling of a Purified Goldfish Brain Kainate Receptor with a Pertussis Toxin-Sensitive G Protein</atitle><jtitle>Proceedings of the National Academy of Sciences - PNAS</jtitle><addtitle>Proc Natl Acad Sci U S A</addtitle><date>1992-05-01</date><risdate>1992</risdate><volume>89</volume><issue>9</issue><spage>4134</spage><epage>4138</epage><pages>4134-4138</pages><issn>0027-8424</issn><eissn>1091-6490</eissn><coden>PNASA6</coden><abstract>Goldfish brain has a high density of [3H]kainate-binding sites, a subpopulation of which appears to be coupled to a pertussis toxin-sensitive G protein. We show here that a purified kainate receptor preparation reconstituted into phospholipid vesicles exhibits guanine nucleotide-sensitive high-affinity [3H]kainate binding. Pertussis toxin treatment abolishes the guanine nucleotide-sensitive portion of the [3H]kainate binding, and kainate promotes [3H]guanosine 5'-[β,γ-imido]triphosphate binding and [γ-32P]GTP hydrolysis. Guanosine 5'-[γ-thio]triphosphate (GTP[γ S]) decreases the apparent Stokes radius of the soluble purified receptor preparation, consistent with dissociation of the kainate receptor-G protein complexes. The affinity-purified preparations contain proteins of 45, 41, and 35 kDa. The 45- and 41-kDa proteins crossreact with antibodies against the kainate receptor cloned from frog brain. The 35-kDa protein is recognized by an antiserum (SW) directed against the β subunit of G proteins. When kainate receptors are purified in the presence of GTP[γS], the 35-kDa protein is no longer present. Also, [3H]kainate affinity is decreased and is no longer guanine nucleotide sensitive. Upon reconstitution with purified G proteins, high-affinity guanine nucleotide-sensitive binding and kainate-stimulated GTPase activity can be restored. These observations indicate that a kainate receptor from goldfish brain functionally interacts with a pertussis toxin-sensitive G protein.</abstract><cop>Washington, DC</cop><pub>National Academy of Sciences of the United States of America</pub><pmid>1315052</pmid><doi>10.1073/pnas.89.9.4134</doi><tpages>5</tpages><oa>free_for_read</oa></addata></record> |
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| ispartof | Proceedings of the National Academy of Sciences - PNAS, 1992-05, Vol.89 (9), p.4134-4138 |
| issn | 0027-8424 1091-6490 |
| language | eng |
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| source | MEDLINE; Jstor Complete Legacy; PubMed Central; Alma/SFX Local Collection; Free Full-Text Journals in Chemistry |
| subjects | Aminoacid receptors (glycine, glutamate, gaba) Animals Biological and medical sciences Brain Carassius auratus Cattle Cell receptors Cell structures and functions Elution Fish Freshwater Frogs Fundamental and applied biological sciences. Psychology Gels Goldfish GTP-Binding Proteins - isolation & purification GTP-Binding Proteins - metabolism guanine nucleotide-binding protein Guanine nucleotides In Vitro Techniques kainic acid Kainic acid receptors Macromolecular Substances Molecular and cellular biology neurotransmitters pertussis Pertussis Toxin Protein Binding Proteins Receptors Receptors, Kainic Acid Receptors, Neurotransmitter - isolation & purification Receptors, Neurotransmitter - metabolism Toxins Virulence Factors, Bordetella - pharmacology Whooping cough |
| title | Coupling of a Purified Goldfish Brain Kainate Receptor with a Pertussis Toxin-Sensitive G Protein |
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