Regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase and lipid metabolism in a concanavalin A-resistant chinese hamster ovary cell line
Lipid metabolism in a concanavalin A-resistant, glycosylation-defective mutant cell line was investigated by comparing growth properties, lipid composition, and lipid biosynthesis in wild-type (WT), mutant (C R-7), and revertant (RC R-7) cells. In contrast to WT and RC R-7, the mutant was auxotrophi...
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| Veröffentlicht in: | Archives of biochemistry and biophysics 1986-02, Vol.244 (2), p.502-516 |
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| creator | Borgford, Thor J. Hurta, Robert A. Tough, David F. Burton, David N. |
| description | Lipid metabolism in a concanavalin A-resistant, glycosylation-defective mutant cell line was investigated by comparing growth properties, lipid composition, and lipid biosynthesis in wild-type (WT), mutant (C
R-7), and revertant (RC
R-7) cells. In contrast to WT and RC
R-7, the mutant was auxotrophic for cholesterol, but mevalonolactone did not restore growth on lipoprotein-deficient medium. The use of
R-[2-
14C]mevalonolactone revealed that C
R-7 was deficient in the conversion of lanosterol to cholesterol. Total lipid and phospholipid content and composition were similar in all three cell lines, but C
R-7 displayed subnormal content and biosynthesis of cholesterol and unsaturated fatty acids. The mutant was hypersensitive to compactin and was unable to upregulate either 3-hydroxy-3methylglutaryl coenzyme A (HMG-CoA) reductase activity or the binding and internalization of
125I-labeled low-density lipoprotein (LDL) in response to lipoprotein deprivation. HMG-CoA reductase activity in all three cell lines showed similar kinetics and phosphorylation status, and the binding kinetics and degradation of
125I-LDL were also similar, suggesting that C
R-7 possesses kinetically normal reductase and LDL binding sites, but is deficient in their coordinate regulation. Tunicamycin (1–2 μg/ml) strongly and reversibly suppressed reductase activity in WT and RC
R-7. C
R-7 was resistant to this inhibitor. In WT cells this suppressive effect was accompanied by inhibition of
3H-labeled mannose incorporation into cellular protein, but
3H-labeled leucine incorporation was unaffected. Immunotitration of HMG-CoA reductase activity in extracts of WT cells, cultured in the presence and absence of tunicamycin, showed that suppression of reductase activity reflected the presence of reduced amounts of reductase protein, implying that glycosylation plays an important role in the coordinate regulation of HMG-CoA reductase activity and LDL binding. |
| doi_str_mv | 10.1016/0003-9861(86)90619-3 |
| format | Article |
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R-7), and revertant (RC
R-7) cells. In contrast to WT and RC
R-7, the mutant was auxotrophic for cholesterol, but mevalonolactone did not restore growth on lipoprotein-deficient medium. The use of
R-[2-
14C]mevalonolactone revealed that C
R-7 was deficient in the conversion of lanosterol to cholesterol. Total lipid and phospholipid content and composition were similar in all three cell lines, but C
R-7 displayed subnormal content and biosynthesis of cholesterol and unsaturated fatty acids. The mutant was hypersensitive to compactin and was unable to upregulate either 3-hydroxy-3methylglutaryl coenzyme A (HMG-CoA) reductase activity or the binding and internalization of
125I-labeled low-density lipoprotein (LDL) in response to lipoprotein deprivation. HMG-CoA reductase activity in all three cell lines showed similar kinetics and phosphorylation status, and the binding kinetics and degradation of
125I-LDL were also similar, suggesting that C
R-7 possesses kinetically normal reductase and LDL binding sites, but is deficient in their coordinate regulation. Tunicamycin (1–2 μg/ml) strongly and reversibly suppressed reductase activity in WT and RC
R-7. C
R-7 was resistant to this inhibitor. In WT cells this suppressive effect was accompanied by inhibition of
3H-labeled mannose incorporation into cellular protein, but
3H-labeled leucine incorporation was unaffected. Immunotitration of HMG-CoA reductase activity in extracts of WT cells, cultured in the presence and absence of tunicamycin, showed that suppression of reductase activity reflected the presence of reduced amounts of reductase protein, implying that glycosylation plays an important role in the coordinate regulation of HMG-CoA reductase activity and LDL binding.</description><identifier>ISSN: 0003-9861</identifier><identifier>EISSN: 1096-0384</identifier><identifier>DOI: 10.1016/0003-9861(86)90619-3</identifier><identifier>PMID: 3947077</identifier><identifier>CODEN: ABBIA4</identifier><language>eng</language><publisher>San Diego, CA: Elsevier Inc</publisher><subject>Acetates - metabolism ; Acetic Acid ; Animals ; Applied sciences ; Carbon Radioisotopes ; Cell Line ; Cholesterol - metabolism ; Concanavalin A - pharmacology ; Cricetinae ; Cricetulus ; Culture Media ; Dolichol - metabolism ; Drug Resistance ; Exact sciences and technology ; Fatty Acids - analysis ; Female ; Hydroxymethylglutaryl CoA Reductases - analysis ; Iodine Radioisotopes ; Lipid Metabolism ; Lipids - analysis ; Lipoproteins, LDL - metabolism ; Mannose - metabolism ; Mevalonic Acid - analogs & derivatives ; Mevalonic Acid - metabolism ; Mutation ; Other techniques and industries ; Ovary - metabolism ; Tunicamycin - pharmacology</subject><ispartof>Archives of biochemistry and biophysics, 1986-02, Vol.244 (2), p.502-516</ispartof><rights>1986</rights><rights>1987 INIST-CNRS</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c386t-3b5cd2f9b19ad340990e6228521c7942eb11adadbc46cdb0ab625331eb8793b3</citedby><cites>FETCH-LOGICAL-c386t-3b5cd2f9b19ad340990e6228521c7942eb11adadbc46cdb0ab625331eb8793b3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://dx.doi.org/10.1016/0003-9861(86)90619-3$$EHTML$$P50$$Gelsevier$$H</linktohtml><link.rule.ids>314,780,784,3550,27924,27925,45995</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=8030429$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/3947077$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Borgford, Thor J.</creatorcontrib><creatorcontrib>Hurta, Robert A.</creatorcontrib><creatorcontrib>Tough, David F.</creatorcontrib><creatorcontrib>Burton, David N.</creatorcontrib><title>Regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase and lipid metabolism in a concanavalin A-resistant chinese hamster ovary cell line</title><title>Archives of biochemistry and biophysics</title><addtitle>Arch Biochem Biophys</addtitle><description>Lipid metabolism in a concanavalin A-resistant, glycosylation-defective mutant cell line was investigated by comparing growth properties, lipid composition, and lipid biosynthesis in wild-type (WT), mutant (C
R-7), and revertant (RC
R-7) cells. In contrast to WT and RC
R-7, the mutant was auxotrophic for cholesterol, but mevalonolactone did not restore growth on lipoprotein-deficient medium. The use of
R-[2-
14C]mevalonolactone revealed that C
R-7 was deficient in the conversion of lanosterol to cholesterol. Total lipid and phospholipid content and composition were similar in all three cell lines, but C
R-7 displayed subnormal content and biosynthesis of cholesterol and unsaturated fatty acids. The mutant was hypersensitive to compactin and was unable to upregulate either 3-hydroxy-3methylglutaryl coenzyme A (HMG-CoA) reductase activity or the binding and internalization of
125I-labeled low-density lipoprotein (LDL) in response to lipoprotein deprivation. HMG-CoA reductase activity in all three cell lines showed similar kinetics and phosphorylation status, and the binding kinetics and degradation of
125I-LDL were also similar, suggesting that C
R-7 possesses kinetically normal reductase and LDL binding sites, but is deficient in their coordinate regulation. Tunicamycin (1–2 μg/ml) strongly and reversibly suppressed reductase activity in WT and RC
R-7. C
R-7 was resistant to this inhibitor. In WT cells this suppressive effect was accompanied by inhibition of
3H-labeled mannose incorporation into cellular protein, but
3H-labeled leucine incorporation was unaffected. Immunotitration of HMG-CoA reductase activity in extracts of WT cells, cultured in the presence and absence of tunicamycin, showed that suppression of reductase activity reflected the presence of reduced amounts of reductase protein, implying that glycosylation plays an important role in the coordinate regulation of HMG-CoA reductase activity and LDL binding.</description><subject>Acetates - metabolism</subject><subject>Acetic Acid</subject><subject>Animals</subject><subject>Applied sciences</subject><subject>Carbon Radioisotopes</subject><subject>Cell Line</subject><subject>Cholesterol - metabolism</subject><subject>Concanavalin A - pharmacology</subject><subject>Cricetinae</subject><subject>Cricetulus</subject><subject>Culture Media</subject><subject>Dolichol - metabolism</subject><subject>Drug Resistance</subject><subject>Exact sciences and technology</subject><subject>Fatty Acids - analysis</subject><subject>Female</subject><subject>Hydroxymethylglutaryl CoA Reductases - analysis</subject><subject>Iodine Radioisotopes</subject><subject>Lipid Metabolism</subject><subject>Lipids - analysis</subject><subject>Lipoproteins, LDL - metabolism</subject><subject>Mannose - metabolism</subject><subject>Mevalonic Acid - analogs & derivatives</subject><subject>Mevalonic Acid - metabolism</subject><subject>Mutation</subject><subject>Other techniques and industries</subject><subject>Ovary - metabolism</subject><subject>Tunicamycin - pharmacology</subject><issn>0003-9861</issn><issn>1096-0384</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1986</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNp9kc2KFDEUhYMoYzv6BgpZiOgimlSqU5XNQDP4BwOCzD7k5_Z0JJW0SaqxfAof2bTd9NLV5XK_czici9BLRt8zysQHSiknchTs7SjeSSqYJPwRWjEqBaF87B-j1QV5ip6V8oNSxnrRXaErLvuBDsMK_fkOD3PQ1aeI0xZzsltcTr8WwskEdbeEhzBXnZeAbYL4e5kAb3AGN9uqC2AdHQ5-7x1utDYp-DJhH7FueLQ66oMObd2QDMWXqmPFducjNOlOT6VCxunQ7LGFEJpThOfoyVaHAi_O8xrdf_p4f_uF3H37_PV2c0csH0Ul3Kyt67bSMKkd76mUFETXjeuO2UH2HRjGtNPO2F5YZ6g2oltzzsCMg-SGX6M3J9t9Tj9nKFVNvhxD6AhpLmoQA-NyHBrYn0CbUykZtmqf_dQiK0bV8Q_qWLI6lqxGof79QfEme3X2n80E7iI6F9_ur893XawO26yj9eWCjZTTvpMNuzlh0Ko4eMiqWA_RgvMZbFUu-f_n-As9NaaO</recordid><startdate>19860201</startdate><enddate>19860201</enddate><creator>Borgford, Thor J.</creator><creator>Hurta, Robert A.</creator><creator>Tough, David F.</creator><creator>Burton, David N.</creator><general>Elsevier Inc</general><general>Elsevier</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>7X8</scope></search><sort><creationdate>19860201</creationdate><title>Regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase and lipid metabolism in a concanavalin A-resistant chinese hamster ovary cell line</title><author>Borgford, Thor J. ; Hurta, Robert A. ; Tough, David F. ; Burton, David N.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c386t-3b5cd2f9b19ad340990e6228521c7942eb11adadbc46cdb0ab625331eb8793b3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1986</creationdate><topic>Acetates - metabolism</topic><topic>Acetic Acid</topic><topic>Animals</topic><topic>Applied sciences</topic><topic>Carbon Radioisotopes</topic><topic>Cell Line</topic><topic>Cholesterol - metabolism</topic><topic>Concanavalin A - pharmacology</topic><topic>Cricetinae</topic><topic>Cricetulus</topic><topic>Culture Media</topic><topic>Dolichol - metabolism</topic><topic>Drug Resistance</topic><topic>Exact sciences and technology</topic><topic>Fatty Acids - analysis</topic><topic>Female</topic><topic>Hydroxymethylglutaryl CoA Reductases - analysis</topic><topic>Iodine Radioisotopes</topic><topic>Lipid Metabolism</topic><topic>Lipids - analysis</topic><topic>Lipoproteins, LDL - metabolism</topic><topic>Mannose - metabolism</topic><topic>Mevalonic Acid - analogs & derivatives</topic><topic>Mevalonic Acid - metabolism</topic><topic>Mutation</topic><topic>Other techniques and industries</topic><topic>Ovary - metabolism</topic><topic>Tunicamycin - pharmacology</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Borgford, Thor J.</creatorcontrib><creatorcontrib>Hurta, Robert A.</creatorcontrib><creatorcontrib>Tough, David F.</creatorcontrib><creatorcontrib>Burton, David N.</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>MEDLINE - Academic</collection><jtitle>Archives of biochemistry and biophysics</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Borgford, Thor J.</au><au>Hurta, Robert A.</au><au>Tough, David F.</au><au>Burton, David N.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase and lipid metabolism in a concanavalin A-resistant chinese hamster ovary cell line</atitle><jtitle>Archives of biochemistry and biophysics</jtitle><addtitle>Arch Biochem Biophys</addtitle><date>1986-02-01</date><risdate>1986</risdate><volume>244</volume><issue>2</issue><spage>502</spage><epage>516</epage><pages>502-516</pages><issn>0003-9861</issn><eissn>1096-0384</eissn><coden>ABBIA4</coden><abstract>Lipid metabolism in a concanavalin A-resistant, glycosylation-defective mutant cell line was investigated by comparing growth properties, lipid composition, and lipid biosynthesis in wild-type (WT), mutant (C
R-7), and revertant (RC
R-7) cells. In contrast to WT and RC
R-7, the mutant was auxotrophic for cholesterol, but mevalonolactone did not restore growth on lipoprotein-deficient medium. The use of
R-[2-
14C]mevalonolactone revealed that C
R-7 was deficient in the conversion of lanosterol to cholesterol. Total lipid and phospholipid content and composition were similar in all three cell lines, but C
R-7 displayed subnormal content and biosynthesis of cholesterol and unsaturated fatty acids. The mutant was hypersensitive to compactin and was unable to upregulate either 3-hydroxy-3methylglutaryl coenzyme A (HMG-CoA) reductase activity or the binding and internalization of
125I-labeled low-density lipoprotein (LDL) in response to lipoprotein deprivation. HMG-CoA reductase activity in all three cell lines showed similar kinetics and phosphorylation status, and the binding kinetics and degradation of
125I-LDL were also similar, suggesting that C
R-7 possesses kinetically normal reductase and LDL binding sites, but is deficient in their coordinate regulation. Tunicamycin (1–2 μg/ml) strongly and reversibly suppressed reductase activity in WT and RC
R-7. C
R-7 was resistant to this inhibitor. In WT cells this suppressive effect was accompanied by inhibition of
3H-labeled mannose incorporation into cellular protein, but
3H-labeled leucine incorporation was unaffected. Immunotitration of HMG-CoA reductase activity in extracts of WT cells, cultured in the presence and absence of tunicamycin, showed that suppression of reductase activity reflected the presence of reduced amounts of reductase protein, implying that glycosylation plays an important role in the coordinate regulation of HMG-CoA reductase activity and LDL binding.</abstract><cop>San Diego, CA</cop><pub>Elsevier Inc</pub><pmid>3947077</pmid><doi>10.1016/0003-9861(86)90619-3</doi><tpages>15</tpages></addata></record> |
| fulltext | fulltext |
| identifier | ISSN: 0003-9861 |
| ispartof | Archives of biochemistry and biophysics, 1986-02, Vol.244 (2), p.502-516 |
| issn | 0003-9861 1096-0384 |
| language | eng |
| recordid | cdi_proquest_miscellaneous_76713987 |
| source | MEDLINE; Access via ScienceDirect (Elsevier) |
| subjects | Acetates - metabolism Acetic Acid Animals Applied sciences Carbon Radioisotopes Cell Line Cholesterol - metabolism Concanavalin A - pharmacology Cricetinae Cricetulus Culture Media Dolichol - metabolism Drug Resistance Exact sciences and technology Fatty Acids - analysis Female Hydroxymethylglutaryl CoA Reductases - analysis Iodine Radioisotopes Lipid Metabolism Lipids - analysis Lipoproteins, LDL - metabolism Mannose - metabolism Mevalonic Acid - analogs & derivatives Mevalonic Acid - metabolism Mutation Other techniques and industries Ovary - metabolism Tunicamycin - pharmacology |
| title | Regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase and lipid metabolism in a concanavalin A-resistant chinese hamster ovary cell line |
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