Covalent Cross-Linking of the Phytophthora megasperma Oligopeptide Elicitor to its Receptor in Parsley Membranes

An oligopeptide elicitor from Phytophthora megasperma f.sp. glycinea (Pep-13) that induces phytoalexin accumulation in cultured parsley cells was radioiodinated and chemically cross-linked to its binding site in microsomal and plasma membrane preparations with each of three homobifunctional reagents...

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Veröffentlicht in:Proceedings of the National Academy of Sciences - PNAS 1995-03, Vol.92 (6), p.2338-2342
Hauptverfasser: Nurnberger, Thorsten, Nennstiel, Dirk, Hahlbrock, Klaus, Scheel, Dierk
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container_title Proceedings of the National Academy of Sciences - PNAS
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Nennstiel, Dirk
Hahlbrock, Klaus
Scheel, Dierk
description An oligopeptide elicitor from Phytophthora megasperma f.sp. glycinea (Pep-13) that induces phytoalexin accumulation in cultured parsley cells was radioiodinated and chemically cross-linked to its binding site in microsomal and plasma membrane preparations with each of three homobifunctional reagents. Analysis by SDS/PAGE and autoradiography of solubilized membrane proteins demonstrated labeling of a 91-kDa protein, regardless of which reagent was used. Cross-linking of this protein was prevented by addition of excess unlabeled Pep-13. The competitor concentration found to half-maximally reduce the intensity of the cross-linked band was 6 nM, which is in good agreement with the IC50value of 4.7 nM, obtained from ligand binding assays. No cross-linking of125I-labeled Pep-13 was observed by using microsomal membranes from three other plant species, indicating species-specific occurrence of the binding site. Coupling of125I-Pep-13 to the parsley 91-kDa protein required the same structural elements within the ligand as was recently reported for binding of125I-Pep-13 to parsley microsomes, elicitor-induced stimulation of ion fluxes across the plasma membrane, the oxidative burst, the expression of defense-related genes, and phytoalexin production. These findings suggest that the 91-kDa protein identified in parsley membranes is the oligopeptide elicitor receptor mediating activation of a multicomponent defense response.
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Analysis by SDS/PAGE and autoradiography of solubilized membrane proteins demonstrated labeling of a 91-kDa protein, regardless of which reagent was used. Cross-linking of this protein was prevented by addition of excess unlabeled Pep-13. The competitor concentration found to half-maximally reduce the intensity of the cross-linked band was 6 nM, which is in good agreement with the IC50value of 4.7 nM, obtained from ligand binding assays. No cross-linking of125I-labeled Pep-13 was observed by using microsomal membranes from three other plant species, indicating species-specific occurrence of the binding site. Coupling of125I-Pep-13 to the parsley 91-kDa protein required the same structural elements within the ligand as was recently reported for binding of125I-Pep-13 to parsley microsomes, elicitor-induced stimulation of ion fluxes across the plasma membrane, the oxidative burst, the expression of defense-related genes, and phytoalexin production. 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Nennstiel, Dirk ; Hahlbrock, Klaus ; Scheel, Dierk</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c554t-1f0c61fc8689c7130d5de71673acbc3023d90cc74ca692395904b7c4cd896edc3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1995</creationdate><topic>Amino Acid Sequence</topic><topic>Binding sites</topic><topic>Binding, Competitive</topic><topic>Cell membranes</topic><topic>Cells, Cultured</topic><topic>Cross-Linking Reagents - pharmacology</topic><topic>Cultured cells</topic><topic>Daucus carota - metabolism</topic><topic>Electrophoresis, Polyacrylamide Gel</topic><topic>Fungal Proteins - isolation &amp; purification</topic><topic>Fungal Proteins - metabolism</topic><topic>Glycine max - metabolism</topic><topic>Glycoproteins</topic><topic>Intracellular Membranes - metabolism</topic><topic>Iodine Radioisotopes</topic><topic>Kinetics</topic><topic>Ligands</topic><topic>Magnoliopsida - metabolism</topic><topic>Membrane Glycoproteins - isolation &amp; purification</topic><topic>Membrane Glycoproteins - metabolism</topic><topic>Membrane Proteins</topic><topic>Microsomes</topic><topic>Microsomes - metabolism</topic><topic>Molecular Sequence Data</topic><topic>P branes</topic><topic>Parsley</topic><topic>Peptide Fragments - chemistry</topic><topic>Peptide Fragments - metabolism</topic><topic>Peptide Fragments - pharmacology</topic><topic>Phytoalexins</topic><topic>Phytophthora - metabolism</topic><topic>Phytophthora megasperma</topic><topic>Plant Extracts - biosynthesis</topic><topic>Plants</topic><topic>Reagents</topic><topic>Receptors</topic><topic>Sesquiterpenes</topic><topic>Substrate Specificity</topic><topic>Succinimides - pharmacology</topic><topic>Terpenes</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Nurnberger, Thorsten</creatorcontrib><creatorcontrib>Nennstiel, Dirk</creatorcontrib><creatorcontrib>Hahlbrock, Klaus</creatorcontrib><creatorcontrib>Scheel, Dierk</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Algology Mycology and Protozoology Abstracts (Microbiology C)</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>Nurnberger, Thorsten</au><au>Nennstiel, Dirk</au><au>Hahlbrock, Klaus</au><au>Scheel, Dierk</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Covalent Cross-Linking of the Phytophthora megasperma Oligopeptide Elicitor to its Receptor in Parsley Membranes</atitle><jtitle>Proceedings of the National Academy of Sciences - PNAS</jtitle><addtitle>Proc Natl Acad Sci U S A</addtitle><date>1995-03-14</date><risdate>1995</risdate><volume>92</volume><issue>6</issue><spage>2338</spage><epage>2342</epage><pages>2338-2342</pages><issn>0027-8424</issn><eissn>1091-6490</eissn><abstract>An oligopeptide elicitor from Phytophthora megasperma f.sp. glycinea (Pep-13) that induces phytoalexin accumulation in cultured parsley cells was radioiodinated and chemically cross-linked to its binding site in microsomal and plasma membrane preparations with each of three homobifunctional reagents. Analysis by SDS/PAGE and autoradiography of solubilized membrane proteins demonstrated labeling of a 91-kDa protein, regardless of which reagent was used. Cross-linking of this protein was prevented by addition of excess unlabeled Pep-13. The competitor concentration found to half-maximally reduce the intensity of the cross-linked band was 6 nM, which is in good agreement with the IC50value of 4.7 nM, obtained from ligand binding assays. No cross-linking of125I-labeled Pep-13 was observed by using microsomal membranes from three other plant species, indicating species-specific occurrence of the binding site. Coupling of125I-Pep-13 to the parsley 91-kDa protein required the same structural elements within the ligand as was recently reported for binding of125I-Pep-13 to parsley microsomes, elicitor-induced stimulation of ion fluxes across the plasma membrane, the oxidative burst, the expression of defense-related genes, and phytoalexin production. These findings suggest that the 91-kDa protein identified in parsley membranes is the oligopeptide elicitor receptor mediating activation of a multicomponent defense response.</abstract><cop>United States</cop><pub>National Academy of Sciences of the United States of America</pub><pmid>7892267</pmid><doi>10.1073/pnas.92.6.2338</doi><tpages>5</tpages><oa>free_for_read</oa></addata></record>
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1091-6490
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subjects Amino Acid Sequence
Binding sites
Binding, Competitive
Cell membranes
Cells, Cultured
Cross-Linking Reagents - pharmacology
Cultured cells
Daucus carota - metabolism
Electrophoresis, Polyacrylamide Gel
Fungal Proteins - isolation & purification
Fungal Proteins - metabolism
Glycine max - metabolism
Glycoproteins
Intracellular Membranes - metabolism
Iodine Radioisotopes
Kinetics
Ligands
Magnoliopsida - metabolism
Membrane Glycoproteins - isolation & purification
Membrane Glycoproteins - metabolism
Membrane Proteins
Microsomes
Microsomes - metabolism
Molecular Sequence Data
P branes
Parsley
Peptide Fragments - chemistry
Peptide Fragments - metabolism
Peptide Fragments - pharmacology
Phytoalexins
Phytophthora - metabolism
Phytophthora megasperma
Plant Extracts - biosynthesis
Plants
Reagents
Receptors
Sesquiterpenes
Substrate Specificity
Succinimides - pharmacology
Terpenes
title Covalent Cross-Linking of the Phytophthora megasperma Oligopeptide Elicitor to its Receptor in Parsley Membranes
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