Human Cytochrome P450 3A4: Enzymatic Properties of a Purified Recombinant Fusion Protein Containing NADPH-P450 Reductase
Human cytochrome P450 3A4 is recognized as the catalyst for the oxygen-dependent metabolism of a diverse group of medically important chemicals, including the immunosuppressive agent cyclosporin; macrolide antibiotics, such as erythromycin; drugs such as benzphetamine, nifedipine, and cocaine; and s...
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| Veröffentlicht in: | Proceedings of the National Academy of Sciences - PNAS 1993-12, Vol.90 (24), p.11748-11752 |
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| creator | Shet, Manjunath S. Fisher, Charles W. Holmans, Priscilla L. Estabrook, Ronald W. |
| description | Human cytochrome P450 3A4 is recognized as the catalyst for the oxygen-dependent metabolism of a diverse group of medically important chemicals, including the immunosuppressive agent cyclosporin; macrolide antibiotics, such as erythromycin; drugs such as benzphetamine, nifedipine, and cocaine; and steroids, such as cortisol and testosterone to name but a few. We have engineered the cDNA for human cytochrome P450 3A4 by linkage to the cDNA for the rat or human flavoprotein, NADPH-P450 reductase (NADPH:ferrihemoprotein oxidoreductase, EC 1.6.2.4). An enzymatically active fusion protein (rF450[mHum3A4/mRatOR]L1) has been expressed at high levels in Escherichia coli and purified to homogeneity. Enzymatic studies show a requirement for lipid, detergent, and cytochrome b5for the 6β-hydroxylation of steroids and the N-oxidation of nifedipine. In contrast, these additions are not required for the N-demethylation of erythromycin or benzphetamine. A spectrophotometrically detectable metabolite complex of P450 3A4 is formed during the metabolism of triacetyloleandomycin, and this has a pronounced inhibitory effect on the metabolism of both testosterone and erythromycin. These results relate to the interpretation of current methods used to assess the in vivo activity of P450 3A4. |
| doi_str_mv | 10.1073/pnas.90.24.11748 |
| format | Article |
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We have engineered the cDNA for human cytochrome P450 3A4 by linkage to the cDNA for the rat or human flavoprotein, NADPH-P450 reductase (NADPH:ferrihemoprotein oxidoreductase, EC 1.6.2.4). An enzymatically active fusion protein (rF450[mHum3A4/mRatOR]L1) has been expressed at high levels in Escherichia coli and purified to homogeneity. Enzymatic studies show a requirement for lipid, detergent, and cytochrome b5for the 6β-hydroxylation of steroids and the N-oxidation of nifedipine. In contrast, these additions are not required for the N-demethylation of erythromycin or benzphetamine. A spectrophotometrically detectable metabolite complex of P450 3A4 is formed during the metabolism of triacetyloleandomycin, and this has a pronounced inhibitory effect on the metabolism of both testosterone and erythromycin. These results relate to the interpretation of current methods used to assess the in vivo activity of P450 3A4.</description><identifier>ISSN: 0027-8424</identifier><identifier>EISSN: 1091-6490</identifier><identifier>DOI: 10.1073/pnas.90.24.11748</identifier><identifier>PMID: 8265621</identifier><identifier>CODEN: PNASA6</identifier><language>eng</language><publisher>Washington, DC: National Academy of Sciences of the United States of America</publisher><subject>Analytical, structural and metabolic biochemistry ; Base Sequence ; Biochemistry ; Biological and medical sciences ; Catalysts ; Cloning, Molecular ; Complementary DNA ; Cytochrome P-450 CYP3A ; Cytochrome P-450 Enzyme System ; Cytochromes ; Cytochromes b5 - metabolism ; Detergents ; DNA Primers ; Enzymes ; Enzymes and enzyme inhibitors ; Erythromycin - metabolism ; Escherichia coli ; Fundamental and applied biological sciences. Psychology ; Humans ; Hydroxylation ; Kinetics ; Lipid metabolism ; Lipids ; Liver - enzymology ; Mixed Function Oxygenases ; Molecular Sequence Data ; Mutagenesis ; NADPH-Ferrihemoprotein Reductase - isolation & purification ; NADPH-Ferrihemoprotein Reductase - metabolism ; Nifedipine - metabolism ; Oxidoreductases ; Pharmacology ; Plasmids ; Polymerase Chain Reaction ; Protein metabolism ; Recombinant Fusion Proteins - isolation & purification ; Recombinant Fusion Proteins - metabolism ; Rodents ; Steroid metabolism ; Steroids ; Substrate Specificity ; Testosterone ; Testosterone - metabolism</subject><ispartof>Proceedings of the National Academy of Sciences - PNAS, 1993-12, Vol.90 (24), p.11748-11752</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 Dec 15, 1993</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c620t-5fa9d0d0f1938d77f2608d3668364a34835b4f5f59593d14ae37e901e8b3f1913</citedby></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Uhttp://www.pnas.org/content/90/24.cover.gif</thumbnail><linktopdf>$$Uhttps://www.jstor.org/stable/pdf/2363540$$EPDF$$P50$$Gjstor$$H</linktopdf><linktohtml>$$Uhttps://www.jstor.org/stable/2363540$$EHTML$$P50$$Gjstor$$H</linktohtml><link.rule.ids>230,314,727,780,784,803,885,27922,27923,53789,53791,58015,58248</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=3880405$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/8265621$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Shet, Manjunath S.</creatorcontrib><creatorcontrib>Fisher, Charles W.</creatorcontrib><creatorcontrib>Holmans, Priscilla L.</creatorcontrib><creatorcontrib>Estabrook, Ronald W.</creatorcontrib><title>Human Cytochrome P450 3A4: Enzymatic Properties of a Purified Recombinant Fusion Protein Containing NADPH-P450 Reductase</title><title>Proceedings of the National Academy of Sciences - PNAS</title><addtitle>Proc Natl Acad Sci U S A</addtitle><description>Human cytochrome P450 3A4 is recognized as the catalyst for the oxygen-dependent metabolism of a diverse group of medically important chemicals, including the immunosuppressive agent cyclosporin; macrolide antibiotics, such as erythromycin; drugs such as benzphetamine, nifedipine, and cocaine; and steroids, such as cortisol and testosterone to name but a few. We have engineered the cDNA for human cytochrome P450 3A4 by linkage to the cDNA for the rat or human flavoprotein, NADPH-P450 reductase (NADPH:ferrihemoprotein oxidoreductase, EC 1.6.2.4). An enzymatically active fusion protein (rF450[mHum3A4/mRatOR]L1) has been expressed at high levels in Escherichia coli and purified to homogeneity. Enzymatic studies show a requirement for lipid, detergent, and cytochrome b5for the 6β-hydroxylation of steroids and the N-oxidation of nifedipine. In contrast, these additions are not required for the N-demethylation of erythromycin or benzphetamine. A spectrophotometrically detectable metabolite complex of P450 3A4 is formed during the metabolism of triacetyloleandomycin, and this has a pronounced inhibitory effect on the metabolism of both testosterone and erythromycin. These results relate to the interpretation of current methods used to assess the in vivo activity of P450 3A4.</description><subject>Analytical, structural and metabolic biochemistry</subject><subject>Base Sequence</subject><subject>Biochemistry</subject><subject>Biological and medical sciences</subject><subject>Catalysts</subject><subject>Cloning, Molecular</subject><subject>Complementary DNA</subject><subject>Cytochrome P-450 CYP3A</subject><subject>Cytochrome P-450 Enzyme System</subject><subject>Cytochromes</subject><subject>Cytochromes b5 - metabolism</subject><subject>Detergents</subject><subject>DNA Primers</subject><subject>Enzymes</subject><subject>Enzymes and enzyme inhibitors</subject><subject>Erythromycin - metabolism</subject><subject>Escherichia coli</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Humans</subject><subject>Hydroxylation</subject><subject>Kinetics</subject><subject>Lipid metabolism</subject><subject>Lipids</subject><subject>Liver - enzymology</subject><subject>Mixed Function Oxygenases</subject><subject>Molecular Sequence Data</subject><subject>Mutagenesis</subject><subject>NADPH-Ferrihemoprotein Reductase - isolation & purification</subject><subject>NADPH-Ferrihemoprotein Reductase - metabolism</subject><subject>Nifedipine - metabolism</subject><subject>Oxidoreductases</subject><subject>Pharmacology</subject><subject>Plasmids</subject><subject>Polymerase Chain Reaction</subject><subject>Protein metabolism</subject><subject>Recombinant Fusion Proteins - isolation & purification</subject><subject>Recombinant Fusion Proteins - metabolism</subject><subject>Rodents</subject><subject>Steroid metabolism</subject><subject>Steroids</subject><subject>Substrate Specificity</subject><subject>Testosterone</subject><subject>Testosterone - metabolism</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>eNp9kU1vEzEYhFcIVELhzgHECiHEZcPrj_XaiEsUWoJUQVTB2XJ27dbRrp3aXtTw63GaEBUOnHyYZ-Yda4riOYIpgoa83zgVpwKmmE4Raih_UEwQCFQxKuBhMQHATcUppo-LJzGuAUDUHE6KE45ZzTCaFLeLcVCunG-Tb6-DH3S5pDWUZEY_lGfu13ZQybblMviNDsnqWHpTqnI5Bmus7spL3fphZZ1yqTwfo_VuxyZtc6R3SVln3VX5dfZpuajugi91N7ZJRf20eGRUH_Wzw3ta_Dg_-z5fVBffPn-Zzy6qlmFIVW2U6KADgwThXdMYzIB3hDFOGFWEclKvqKlNLWpBOkSVJo0WgDRfkexB5LT4uM_djKtBd612KaheboIdVNhKr6z8W3H2Wl75n5JyYDv724M9-JtRxyQHG1vd98ppP0aJWAOEYJLB1_-Aaz8Gl78mMSCci2KRIdhDbfAxBm2OPRDI3aByN6gUIDGVd4Nmy8v7_Y-Gw4JZf3PQVWxVb4JyrY1HjHAOFOqMvTtguwN_1HuHpBn7PunblNFX_0cz8WJPrGPy4YhgwkhNgfwGkp3Jdw</recordid><startdate>19931215</startdate><enddate>19931215</enddate><creator>Shet, Manjunath S.</creator><creator>Fisher, Charles W.</creator><creator>Holmans, Priscilla L.</creator><creator>Estabrook, Ronald W.</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>7QO</scope><scope>5PM</scope></search><sort><creationdate>19931215</creationdate><title>Human Cytochrome P450 3A4: Enzymatic Properties of a Purified Recombinant Fusion Protein Containing NADPH-P450 Reductase</title><author>Shet, Manjunath S. ; Fisher, Charles W. ; Holmans, Priscilla L. ; Estabrook, Ronald W.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c620t-5fa9d0d0f1938d77f2608d3668364a34835b4f5f59593d14ae37e901e8b3f1913</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1993</creationdate><topic>Analytical, structural and metabolic biochemistry</topic><topic>Base Sequence</topic><topic>Biochemistry</topic><topic>Biological and medical sciences</topic><topic>Catalysts</topic><topic>Cloning, Molecular</topic><topic>Complementary DNA</topic><topic>Cytochrome P-450 CYP3A</topic><topic>Cytochrome P-450 Enzyme System</topic><topic>Cytochromes</topic><topic>Cytochromes b5 - metabolism</topic><topic>Detergents</topic><topic>DNA Primers</topic><topic>Enzymes</topic><topic>Enzymes and enzyme inhibitors</topic><topic>Erythromycin - metabolism</topic><topic>Escherichia coli</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>Humans</topic><topic>Hydroxylation</topic><topic>Kinetics</topic><topic>Lipid metabolism</topic><topic>Lipids</topic><topic>Liver - enzymology</topic><topic>Mixed Function Oxygenases</topic><topic>Molecular Sequence Data</topic><topic>Mutagenesis</topic><topic>NADPH-Ferrihemoprotein Reductase - isolation & purification</topic><topic>NADPH-Ferrihemoprotein Reductase - metabolism</topic><topic>Nifedipine - metabolism</topic><topic>Oxidoreductases</topic><topic>Pharmacology</topic><topic>Plasmids</topic><topic>Polymerase Chain Reaction</topic><topic>Protein metabolism</topic><topic>Recombinant Fusion Proteins - isolation & purification</topic><topic>Recombinant Fusion Proteins - metabolism</topic><topic>Rodents</topic><topic>Steroid metabolism</topic><topic>Steroids</topic><topic>Substrate Specificity</topic><topic>Testosterone</topic><topic>Testosterone - metabolism</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Shet, Manjunath S.</creatorcontrib><creatorcontrib>Fisher, Charles W.</creatorcontrib><creatorcontrib>Holmans, Priscilla L.</creatorcontrib><creatorcontrib>Estabrook, Ronald W.</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>Biotechnology Research Abstracts</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>Shet, Manjunath S.</au><au>Fisher, Charles W.</au><au>Holmans, Priscilla L.</au><au>Estabrook, Ronald W.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Human Cytochrome P450 3A4: Enzymatic Properties of a Purified Recombinant Fusion Protein Containing NADPH-P450 Reductase</atitle><jtitle>Proceedings of the National Academy of Sciences - PNAS</jtitle><addtitle>Proc Natl Acad Sci U S A</addtitle><date>1993-12-15</date><risdate>1993</risdate><volume>90</volume><issue>24</issue><spage>11748</spage><epage>11752</epage><pages>11748-11752</pages><issn>0027-8424</issn><eissn>1091-6490</eissn><coden>PNASA6</coden><abstract>Human cytochrome P450 3A4 is recognized as the catalyst for the oxygen-dependent metabolism of a diverse group of medically important chemicals, including the immunosuppressive agent cyclosporin; macrolide antibiotics, such as erythromycin; drugs such as benzphetamine, nifedipine, and cocaine; and steroids, such as cortisol and testosterone to name but a few. We have engineered the cDNA for human cytochrome P450 3A4 by linkage to the cDNA for the rat or human flavoprotein, NADPH-P450 reductase (NADPH:ferrihemoprotein oxidoreductase, EC 1.6.2.4). An enzymatically active fusion protein (rF450[mHum3A4/mRatOR]L1) has been expressed at high levels in Escherichia coli and purified to homogeneity. Enzymatic studies show a requirement for lipid, detergent, and cytochrome b5for the 6β-hydroxylation of steroids and the N-oxidation of nifedipine. In contrast, these additions are not required for the N-demethylation of erythromycin or benzphetamine. A spectrophotometrically detectable metabolite complex of P450 3A4 is formed during the metabolism of triacetyloleandomycin, and this has a pronounced inhibitory effect on the metabolism of both testosterone and erythromycin. These results relate to the interpretation of current methods used to assess the in vivo activity of P450 3A4.</abstract><cop>Washington, DC</cop><pub>National Academy of Sciences of the United States of America</pub><pmid>8265621</pmid><doi>10.1073/pnas.90.24.11748</doi><tpages>5</tpages><oa>free_for_read</oa></addata></record> |
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| subjects | Analytical, structural and metabolic biochemistry Base Sequence Biochemistry Biological and medical sciences Catalysts Cloning, Molecular Complementary DNA Cytochrome P-450 CYP3A Cytochrome P-450 Enzyme System Cytochromes Cytochromes b5 - metabolism Detergents DNA Primers Enzymes Enzymes and enzyme inhibitors Erythromycin - metabolism Escherichia coli Fundamental and applied biological sciences. Psychology Humans Hydroxylation Kinetics Lipid metabolism Lipids Liver - enzymology Mixed Function Oxygenases Molecular Sequence Data Mutagenesis NADPH-Ferrihemoprotein Reductase - isolation & purification NADPH-Ferrihemoprotein Reductase - metabolism Nifedipine - metabolism Oxidoreductases Pharmacology Plasmids Polymerase Chain Reaction Protein metabolism Recombinant Fusion Proteins - isolation & purification Recombinant Fusion Proteins - metabolism Rodents Steroid metabolism Steroids Substrate Specificity Testosterone Testosterone - metabolism |
| title | Human Cytochrome P450 3A4: Enzymatic Properties of a Purified Recombinant Fusion Protein Containing NADPH-P450 Reductase |
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