Immunochemical Detection of 4-Hydroxynonenal Protein Adducts in Oxidized Hepatocytes
We report here the development of an immunochemical procedure that uses an antibody specific to the 4-hydroxynonenal (HNE) moiety for the detection of HNE-protein adducts. The HNE-specific antibody was prepared by immunizing rabbits with a HNE-keyhole limpet hemocyanin conjugate and purifying the ra...
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| Veröffentlicht in: | Proceedings of the National Academy of Sciences - PNAS 1993-09, Vol.90 (18), p.8742-8746 |
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| creator | Uchida, Koji Szweda, Luke I. Chae, Ho-Zoon Stadtman, Earl R. |
| description | We report here the development of an immunochemical procedure that uses an antibody specific to the 4-hydroxynonenal (HNE) moiety for the detection of HNE-protein adducts. The HNE-specific antibody was prepared by immunizing rabbits with a HNE-keyhole limpet hemocyanin conjugate and purifying the rabbit serum on an affinity gel prepared by covalent attachment of a HNE-conjugated heptapeptide. When various preparations of glyceraldehyde-3-phosphate dehydrogenase containing 0-7.0 equivalent of HNE-histidine residues per subunit were obtained by incubating samples of glyceraldehyde-3-phosphate dehydrogenase with increased amounts of HNE and subjected to immunoblotting with the HNE-specific antibody, the intensities of the blots were directly proportional to the number of HNE-histidine adducts as measured directly by amino acid analysis. Binding of the HNE-conjugated glyceraldehyde-3-phosphate dehydrogenase to the HNE-specific antibody could be completely inhibited by HNE-N-acetylhistidine, HNE-N-acetyllysine, or HNE-glutathione, suggesting that the antigenic determinant recognized by the antibody is the HNE moiety, not the HNE-amino acid conjugates, such as HNE-histidine, HNE-lysine, and HNE-cysteine. The utility of the HNE-specific antibody was demonstrated by its ability to react selectively with a number of HNE-protein adducts in immunoblot analyses of crude homogenates of rat liver hepatocytes that had been exposed to HNE or oxidative stresses with tert-butylhydroperoxide or metal-ion-catalyzed oxidation systems. |
| doi_str_mv | 10.1073/pnas.90.18.8742 |
| format | Article |
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The HNE-specific antibody was prepared by immunizing rabbits with a HNE-keyhole limpet hemocyanin conjugate and purifying the rabbit serum on an affinity gel prepared by covalent attachment of a HNE-conjugated heptapeptide. When various preparations of glyceraldehyde-3-phosphate dehydrogenase containing 0-7.0 equivalent of HNE-histidine residues per subunit were obtained by incubating samples of glyceraldehyde-3-phosphate dehydrogenase with increased amounts of HNE and subjected to immunoblotting with the HNE-specific antibody, the intensities of the blots were directly proportional to the number of HNE-histidine adducts as measured directly by amino acid analysis. Binding of the HNE-conjugated glyceraldehyde-3-phosphate dehydrogenase to the HNE-specific antibody could be completely inhibited by HNE-N-acetylhistidine, HNE-N-acetyllysine, or HNE-glutathione, suggesting that the antigenic determinant recognized by the antibody is the HNE moiety, not the HNE-amino acid conjugates, such as HNE-histidine, HNE-lysine, and HNE-cysteine. The utility of the HNE-specific antibody was demonstrated by its ability to react selectively with a number of HNE-protein adducts in immunoblot analyses of crude homogenates of rat liver hepatocytes that had been exposed to HNE or oxidative stresses with tert-butylhydroperoxide or metal-ion-catalyzed oxidation systems.</description><identifier>ISSN: 0027-8424</identifier><identifier>EISSN: 1091-6490</identifier><identifier>DOI: 10.1073/pnas.90.18.8742</identifier><identifier>PMID: 8378358</identifier><identifier>CODEN: PNASA6</identifier><language>eng</language><publisher>Washington, DC: National Academy of Sciences of the United States of America</publisher><subject>Adducts ; Aldehydes - analysis ; Aldehydes - metabolism ; Amino Acid Sequence ; Amino acids ; Animals ; Antibodies ; Biochemistry ; Biological and medical sciences ; Cell metabolism, cell oxidation ; Cell physiology ; Cells, Cultured ; Electrophoresis, Polyacrylamide Gel ; Enzyme-Linked Immunosorbent Assay ; Epitopes ; Fundamental and applied biological sciences. Psychology ; Hepatocytes ; Histidine - metabolism ; Immunity (Disease) ; Immunoblotting ; Immunohistochemistry - methods ; Kinetics ; Lipid Peroxidation ; Lipids ; Liver ; Liver - metabolism ; Medical research ; Molecular and cellular biology ; Molecular Sequence Data ; Oligopeptides - chemical synthesis ; Oligopeptides - metabolism ; Oxidation ; Phosphates ; Protein Binding ; Proteins ; Proteins - analysis ; Proteins - metabolism ; Rats ; Rats, Inbred BN ; Rats, Inbred F344</subject><ispartof>Proceedings of the National Academy of Sciences - PNAS, 1993-09, Vol.90 (18), p.8742-8746</ispartof><rights>Copyright 1993 The National Academy of Sciences of the United States of America</rights><rights>1994 INIST-CNRS</rights><rights>Copyright National Academy of Sciences Sep 15, 1993</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c615t-3fc00d90e2383b64354fff596357367af8027fa0506ad54e121c81c762cd07773</citedby></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Uhttp://www.pnas.org/content/90/18.cover.gif</thumbnail><linktopdf>$$Uhttps://www.jstor.org/stable/pdf/2362927$$EPDF$$P50$$Gjstor$$H</linktopdf><linktohtml>$$Uhttps://www.jstor.org/stable/2362927$$EHTML$$P50$$Gjstor$$H</linktohtml><link.rule.ids>230,314,723,776,780,799,881,27901,27902,53766,53768,57992,58225</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=3773142$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/8378358$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Uchida, Koji</creatorcontrib><creatorcontrib>Szweda, Luke I.</creatorcontrib><creatorcontrib>Chae, Ho-Zoon</creatorcontrib><creatorcontrib>Stadtman, Earl R.</creatorcontrib><title>Immunochemical Detection of 4-Hydroxynonenal Protein Adducts in Oxidized Hepatocytes</title><title>Proceedings of the National Academy of Sciences - PNAS</title><addtitle>Proc Natl Acad Sci U S A</addtitle><description>We report here the development of an immunochemical procedure that uses an antibody specific to the 4-hydroxynonenal (HNE) moiety for the detection of HNE-protein adducts. The HNE-specific antibody was prepared by immunizing rabbits with a HNE-keyhole limpet hemocyanin conjugate and purifying the rabbit serum on an affinity gel prepared by covalent attachment of a HNE-conjugated heptapeptide. When various preparations of glyceraldehyde-3-phosphate dehydrogenase containing 0-7.0 equivalent of HNE-histidine residues per subunit were obtained by incubating samples of glyceraldehyde-3-phosphate dehydrogenase with increased amounts of HNE and subjected to immunoblotting with the HNE-specific antibody, the intensities of the blots were directly proportional to the number of HNE-histidine adducts as measured directly by amino acid analysis. Binding of the HNE-conjugated glyceraldehyde-3-phosphate dehydrogenase to the HNE-specific antibody could be completely inhibited by HNE-N-acetylhistidine, HNE-N-acetyllysine, or HNE-glutathione, suggesting that the antigenic determinant recognized by the antibody is the HNE moiety, not the HNE-amino acid conjugates, such as HNE-histidine, HNE-lysine, and HNE-cysteine. The utility of the HNE-specific antibody was demonstrated by its ability to react selectively with a number of HNE-protein adducts in immunoblot analyses of crude homogenates of rat liver hepatocytes that had been exposed to HNE or oxidative stresses with tert-butylhydroperoxide or metal-ion-catalyzed oxidation systems.</description><subject>Adducts</subject><subject>Aldehydes - analysis</subject><subject>Aldehydes - metabolism</subject><subject>Amino Acid Sequence</subject><subject>Amino acids</subject><subject>Animals</subject><subject>Antibodies</subject><subject>Biochemistry</subject><subject>Biological and medical sciences</subject><subject>Cell metabolism, cell oxidation</subject><subject>Cell physiology</subject><subject>Cells, Cultured</subject><subject>Electrophoresis, Polyacrylamide Gel</subject><subject>Enzyme-Linked Immunosorbent Assay</subject><subject>Epitopes</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Hepatocytes</subject><subject>Histidine - metabolism</subject><subject>Immunity (Disease)</subject><subject>Immunoblotting</subject><subject>Immunohistochemistry - methods</subject><subject>Kinetics</subject><subject>Lipid Peroxidation</subject><subject>Lipids</subject><subject>Liver</subject><subject>Liver - metabolism</subject><subject>Medical research</subject><subject>Molecular and cellular biology</subject><subject>Molecular Sequence Data</subject><subject>Oligopeptides - chemical synthesis</subject><subject>Oligopeptides - metabolism</subject><subject>Oxidation</subject><subject>Phosphates</subject><subject>Protein Binding</subject><subject>Proteins</subject><subject>Proteins - analysis</subject><subject>Proteins - metabolism</subject><subject>Rats</subject><subject>Rats, Inbred BN</subject><subject>Rats, Inbred F344</subject><issn>0027-8424</issn><issn>1091-6490</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1993</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkc1v1DAQxS0EKkvhzAVQhCo4ZetvOxKXqgW2UqVyKGfLdWyaVWJvbQft8tfjsGFFOcDJHr3fjJ_nAfASwSWCgpxuvE7LphRyKQXFj8ACwQbVnDbwMVhAiEUtKaZPwbOU1hDChkl4BI4kEZIwuQA3l8Mw-mDu7NAZ3VcXNluTu-Cr4Cpar3ZtDNudD976on6JIdvOV2dtO5qcqnK93nZt98O21cpudA5ml216Dp443Sf7Yj6PwddPH2_OV_XV9efL87Or2nDEck2cgbBtoMVEkltOCaPOOdZwwgThQjtZ7DsNGeS6ZdQijIxERnBsWiiEIMfgw37uZrwdbGusz1H3ahO7QcedCrpTDxXf3alv4buighJa2t_N7THcjzZlNXTJ2L7X3oYxKVGsQMjYf0HEeYOQwAV8-xe4DmMsm0sKQ4QZbdDk-nQPmRhSitYdDCOoplDVFKpqSiHVFGrpeP3nPw_8nGLRT2ZdpxKii9qbLh0wUlaFfo15M2PT_N_qg3fe_xNQbuz7bLe5kK_25DrlEA8oJhw3WJCfO5jLzA</recordid><startdate>19930915</startdate><enddate>19930915</enddate><creator>Uchida, Koji</creator><creator>Szweda, Luke I.</creator><creator>Chae, Ho-Zoon</creator><creator>Stadtman, Earl R.</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>7X8</scope><scope>5PM</scope></search><sort><creationdate>19930915</creationdate><title>Immunochemical Detection of 4-Hydroxynonenal Protein Adducts in Oxidized Hepatocytes</title><author>Uchida, Koji ; Szweda, Luke I. ; Chae, Ho-Zoon ; Stadtman, Earl R.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c615t-3fc00d90e2383b64354fff596357367af8027fa0506ad54e121c81c762cd07773</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1993</creationdate><topic>Adducts</topic><topic>Aldehydes - analysis</topic><topic>Aldehydes - metabolism</topic><topic>Amino Acid Sequence</topic><topic>Amino acids</topic><topic>Animals</topic><topic>Antibodies</topic><topic>Biochemistry</topic><topic>Biological and medical sciences</topic><topic>Cell metabolism, cell oxidation</topic><topic>Cell physiology</topic><topic>Cells, Cultured</topic><topic>Electrophoresis, Polyacrylamide Gel</topic><topic>Enzyme-Linked Immunosorbent Assay</topic><topic>Epitopes</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>Hepatocytes</topic><topic>Histidine - metabolism</topic><topic>Immunity (Disease)</topic><topic>Immunoblotting</topic><topic>Immunohistochemistry - methods</topic><topic>Kinetics</topic><topic>Lipid Peroxidation</topic><topic>Lipids</topic><topic>Liver</topic><topic>Liver - metabolism</topic><topic>Medical research</topic><topic>Molecular and cellular biology</topic><topic>Molecular Sequence Data</topic><topic>Oligopeptides - chemical synthesis</topic><topic>Oligopeptides - metabolism</topic><topic>Oxidation</topic><topic>Phosphates</topic><topic>Protein Binding</topic><topic>Proteins</topic><topic>Proteins - analysis</topic><topic>Proteins - metabolism</topic><topic>Rats</topic><topic>Rats, Inbred BN</topic><topic>Rats, Inbred F344</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Uchida, Koji</creatorcontrib><creatorcontrib>Szweda, Luke I.</creatorcontrib><creatorcontrib>Chae, Ho-Zoon</creatorcontrib><creatorcontrib>Stadtman, Earl R.</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>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>Uchida, Koji</au><au>Szweda, Luke I.</au><au>Chae, Ho-Zoon</au><au>Stadtman, Earl R.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Immunochemical Detection of 4-Hydroxynonenal Protein Adducts in Oxidized Hepatocytes</atitle><jtitle>Proceedings of the National Academy of Sciences - PNAS</jtitle><addtitle>Proc Natl Acad Sci U S A</addtitle><date>1993-09-15</date><risdate>1993</risdate><volume>90</volume><issue>18</issue><spage>8742</spage><epage>8746</epage><pages>8742-8746</pages><issn>0027-8424</issn><eissn>1091-6490</eissn><coden>PNASA6</coden><abstract>We report here the development of an immunochemical procedure that uses an antibody specific to the 4-hydroxynonenal (HNE) moiety for the detection of HNE-protein adducts. The HNE-specific antibody was prepared by immunizing rabbits with a HNE-keyhole limpet hemocyanin conjugate and purifying the rabbit serum on an affinity gel prepared by covalent attachment of a HNE-conjugated heptapeptide. When various preparations of glyceraldehyde-3-phosphate dehydrogenase containing 0-7.0 equivalent of HNE-histidine residues per subunit were obtained by incubating samples of glyceraldehyde-3-phosphate dehydrogenase with increased amounts of HNE and subjected to immunoblotting with the HNE-specific antibody, the intensities of the blots were directly proportional to the number of HNE-histidine adducts as measured directly by amino acid analysis. Binding of the HNE-conjugated glyceraldehyde-3-phosphate dehydrogenase to the HNE-specific antibody could be completely inhibited by HNE-N-acetylhistidine, HNE-N-acetyllysine, or HNE-glutathione, suggesting that the antigenic determinant recognized by the antibody is the HNE moiety, not the HNE-amino acid conjugates, such as HNE-histidine, HNE-lysine, and HNE-cysteine. The utility of the HNE-specific antibody was demonstrated by its ability to react selectively with a number of HNE-protein adducts in immunoblot analyses of crude homogenates of rat liver hepatocytes that had been exposed to HNE or oxidative stresses with tert-butylhydroperoxide or metal-ion-catalyzed oxidation systems.</abstract><cop>Washington, DC</cop><pub>National Academy of Sciences of the United States of America</pub><pmid>8378358</pmid><doi>10.1073/pnas.90.18.8742</doi><tpages>5</tpages><oa>free_for_read</oa></addata></record> |
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| ispartof | Proceedings of the National Academy of Sciences - PNAS, 1993-09, Vol.90 (18), p.8742-8746 |
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| source | Jstor Complete Legacy; MEDLINE; PubMed Central; Alma/SFX Local Collection; Free Full-Text Journals in Chemistry |
| subjects | Adducts Aldehydes - analysis Aldehydes - metabolism Amino Acid Sequence Amino acids Animals Antibodies Biochemistry Biological and medical sciences Cell metabolism, cell oxidation Cell physiology Cells, Cultured Electrophoresis, Polyacrylamide Gel Enzyme-Linked Immunosorbent Assay Epitopes Fundamental and applied biological sciences. Psychology Hepatocytes Histidine - metabolism Immunity (Disease) Immunoblotting Immunohistochemistry - methods Kinetics Lipid Peroxidation Lipids Liver Liver - metabolism Medical research Molecular and cellular biology Molecular Sequence Data Oligopeptides - chemical synthesis Oligopeptides - metabolism Oxidation Phosphates Protein Binding Proteins Proteins - analysis Proteins - metabolism Rats Rats, Inbred BN Rats, Inbred F344 |
| title | Immunochemical Detection of 4-Hydroxynonenal Protein Adducts in Oxidized Hepatocytes |
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