Purification and properties of the major nuclease from mitochondria of Saccharomyces cerevisiae
The vast majority of nuclease activity in yeast mitochondria is due to a single polypeptide with an apparent molecular weight of 38,000. The enzyme is located in the mitochondrial inner membrane and requires non-ionic detergents for solubilization and activity. A combination of heparin-agarose and C...
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| Veröffentlicht in: | The Journal of biological chemistry 1988-06, Vol.263 (16), p.7691-7702 |
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| creator | Dake, E Hofmann, T J McIntire, S Hudson, A Zassenhaus, H P |
| description | The vast majority of nuclease activity in yeast mitochondria is due to a single polypeptide with an apparent molecular weight of 38,000. The enzyme is located in the mitochondrial inner membrane and requires non-ionic detergents for solubilization and activity. A combination of heparin-agarose and Cibacron blue-agarose chromatography was employed to purify the nuclease to approximately 90% homogeneity. The purified enzyme shows multiple activities: 1) RNase activity on single-stranded, but not double-stranded RNA, 2) endonuclease activity on single- and double-stranded DNA, and 3) a 5‘-exonuclease activity on double-stranded DNA. Digestion products with DNA contain 5‘-phosphorylated termini. Antibody raised against an analogous enzyme purified from Neurospora crassa (Chow, T. Y. K., and Fraser, M. (1983) J. Biol. Chem. 258, 12010-12018) inhibits and immunoprecipitates the yeast enzyme. This antibody inhibits 90-95% of all nuclease activity present in solubilized mitochondria, indicating that the purified nuclease accounts for the bulk of mitochondrial nucleolytic activity. Analysis of a mutant strain in which the gene for the nuclease has been disrupted supports this conclusion and shows that all detectable DNase activity and most nonspecific RNase activity in the mitochondria is due to this single enzyme. |
| doi_str_mv | 10.1016/S0021-9258(18)68554-0 |
| format | Article |
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The enzyme is located in the mitochondrial inner membrane and requires non-ionic detergents for solubilization and activity. A combination of heparin-agarose and Cibacron blue-agarose chromatography was employed to purify the nuclease to approximately 90% homogeneity. The purified enzyme shows multiple activities: 1) RNase activity on single-stranded, but not double-stranded RNA, 2) endonuclease activity on single- and double-stranded DNA, and 3) a 5‘-exonuclease activity on double-stranded DNA. Digestion products with DNA contain 5‘-phosphorylated termini. Antibody raised against an analogous enzyme purified from Neurospora crassa (Chow, T. Y. K., and Fraser, M. (1983) J. Biol. Chem. 258, 12010-12018) inhibits and immunoprecipitates the yeast enzyme. This antibody inhibits 90-95% of all nuclease activity present in solubilized mitochondria, indicating that the purified nuclease accounts for the bulk of mitochondrial nucleolytic activity. Analysis of a mutant strain in which the gene for the nuclease has been disrupted supports this conclusion and shows that all detectable DNase activity and most nonspecific RNase activity in the mitochondria is due to this single enzyme.</description><identifier>ISSN: 0021-9258</identifier><identifier>EISSN: 1083-351X</identifier><identifier>DOI: 10.1016/S0021-9258(18)68554-0</identifier><identifier>PMID: 3286639</identifier><identifier>CODEN: JBCHA3</identifier><language>eng</language><publisher>Bethesda, MD: Elsevier Inc</publisher><subject>Analytical, structural and metabolic biochemistry ; Biological and medical sciences ; Cations, Divalent - analysis ; Deoxyribonucleases - isolation & purification ; Enzymes and enzyme inhibitors ; EPURATION ; Ethidium - pharmacology ; Fundamental and applied biological sciences. Psychology ; Hydrogen-Ion Concentration ; MITOCHONDRIA ; Mitochondria - enzymology ; MITOCHONDRIE ; MITOCONDRIA ; Molecular Weight ; NUCLEOSIDASAS ; NUCLEOSIDASE ; NUCLEOSIDASES ; PURIFICACION ; PURIFICATION ; Ribonucleases - isolation & purification ; SACCHAROMYCES CEREVISIAE ; Saccharomyces cerevisiae - enzymology ; Saccharomyces cerevisiae - ultrastructure ; Spermidine - pharmacology ; Temperature</subject><ispartof>The Journal of biological chemistry, 1988-06, Vol.263 (16), p.7691-7702</ispartof><rights>1988 © 1988 ASBMB. Currently published by Elsevier Inc; originally published by American Society for Biochemistry and Molecular Biology.</rights><rights>1991 INIST-CNRS</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c581t-385b0bcdcb8dd8f24124c2775282a835c6af6079b38b8d56e13155ac7d6a8e223</citedby><cites>FETCH-LOGICAL-c581t-385b0bcdcb8dd8f24124c2775282a835c6af6079b38b8d56e13155ac7d6a8e223</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,780,784,27923,27924</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=19563037$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/3286639$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Dake, E</creatorcontrib><creatorcontrib>Hofmann, T J</creatorcontrib><creatorcontrib>McIntire, S</creatorcontrib><creatorcontrib>Hudson, A</creatorcontrib><creatorcontrib>Zassenhaus, H P</creatorcontrib><title>Purification and properties of the major nuclease from mitochondria of Saccharomyces cerevisiae</title><title>The Journal of biological chemistry</title><addtitle>J Biol Chem</addtitle><description>The vast majority of nuclease activity in yeast mitochondria is due to a single polypeptide with an apparent molecular weight of 38,000. The enzyme is located in the mitochondrial inner membrane and requires non-ionic detergents for solubilization and activity. A combination of heparin-agarose and Cibacron blue-agarose chromatography was employed to purify the nuclease to approximately 90% homogeneity. The purified enzyme shows multiple activities: 1) RNase activity on single-stranded, but not double-stranded RNA, 2) endonuclease activity on single- and double-stranded DNA, and 3) a 5‘-exonuclease activity on double-stranded DNA. Digestion products with DNA contain 5‘-phosphorylated termini. Antibody raised against an analogous enzyme purified from Neurospora crassa (Chow, T. Y. K., and Fraser, M. (1983) J. Biol. Chem. 258, 12010-12018) inhibits and immunoprecipitates the yeast enzyme. This antibody inhibits 90-95% of all nuclease activity present in solubilized mitochondria, indicating that the purified nuclease accounts for the bulk of mitochondrial nucleolytic activity. Analysis of a mutant strain in which the gene for the nuclease has been disrupted supports this conclusion and shows that all detectable DNase activity and most nonspecific RNase activity in the mitochondria is due to this single enzyme.</description><subject>Analytical, structural and metabolic biochemistry</subject><subject>Biological and medical sciences</subject><subject>Cations, Divalent - analysis</subject><subject>Deoxyribonucleases - isolation & purification</subject><subject>Enzymes and enzyme inhibitors</subject><subject>EPURATION</subject><subject>Ethidium - pharmacology</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Hydrogen-Ion Concentration</subject><subject>MITOCHONDRIA</subject><subject>Mitochondria - enzymology</subject><subject>MITOCHONDRIE</subject><subject>MITOCONDRIA</subject><subject>Molecular Weight</subject><subject>NUCLEOSIDASAS</subject><subject>NUCLEOSIDASE</subject><subject>NUCLEOSIDASES</subject><subject>PURIFICACION</subject><subject>PURIFICATION</subject><subject>Ribonucleases - isolation & purification</subject><subject>SACCHAROMYCES CEREVISIAE</subject><subject>Saccharomyces cerevisiae - enzymology</subject><subject>Saccharomyces cerevisiae - ultrastructure</subject><subject>Spermidine - pharmacology</subject><subject>Temperature</subject><issn>0021-9258</issn><issn>1083-351X</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1988</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkc2L1TAUxYMo43P0HxAGCqLoopqPJk1XIoNfMKDwHHAXbm9vpxlem2fSjsx_b970MS4nmyzO756bnMPYmeDvBRfmw5ZzKcpGavtW2HfGal2V_BHbCG5VqbT4_Zht7pGn7FlK1zyfqhEn7ERJa4xqNsz9XKLvPcLsw1TA1BX7GPYUZ0-pCH0xD1SMcB1iMS24I0hU9DGMxejngEOYuujhwG0BcYCs3GIeRIp045MHes6e9LBL9OJ4n7LLL59_nX8rL358_X7-6aJEbcVcKqtb3mKHre0628tKyAplXWtpJVil0UBveN20ymZCGxJKaA1YdwYsSalO2ZvVNz__z0JpdqNPSLsdTBSW5GorFdd19SAoqkY23Bwc9QpiDClF6t0--hHirRPcHRpwdw24Q7xOWHfXgON57uy4YGlH6u6njpFn_fVRh4Sw6yNM6NN_80YbxVWduVcrN_ir4a-P5FqfI6fRSaNcXl-bRmTq5Ur1EBxcxex0ubW2qo1QWfy4ipSTv_EUXUJPE1KX7XB2XfAPfOYfswe26g</recordid><startdate>19880605</startdate><enddate>19880605</enddate><creator>Dake, E</creator><creator>Hofmann, T J</creator><creator>McIntire, S</creator><creator>Hudson, A</creator><creator>Zassenhaus, H P</creator><general>Elsevier Inc</general><general>American Society for Biochemistry and Molecular Biology</general><scope>6I.</scope><scope>AAFTH</scope><scope>FBQ</scope><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>7TM</scope><scope>M7N</scope><scope>7X8</scope></search><sort><creationdate>19880605</creationdate><title>Purification and properties of the major nuclease from mitochondria of Saccharomyces cerevisiae</title><author>Dake, E ; Hofmann, T J ; McIntire, S ; Hudson, A ; Zassenhaus, H P</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c581t-385b0bcdcb8dd8f24124c2775282a835c6af6079b38b8d56e13155ac7d6a8e223</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1988</creationdate><topic>Analytical, structural and metabolic biochemistry</topic><topic>Biological and medical sciences</topic><topic>Cations, Divalent - analysis</topic><topic>Deoxyribonucleases - isolation & purification</topic><topic>Enzymes and enzyme inhibitors</topic><topic>EPURATION</topic><topic>Ethidium - pharmacology</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>Hydrogen-Ion Concentration</topic><topic>MITOCHONDRIA</topic><topic>Mitochondria - enzymology</topic><topic>MITOCHONDRIE</topic><topic>MITOCONDRIA</topic><topic>Molecular Weight</topic><topic>NUCLEOSIDASAS</topic><topic>NUCLEOSIDASE</topic><topic>NUCLEOSIDASES</topic><topic>PURIFICACION</topic><topic>PURIFICATION</topic><topic>Ribonucleases - isolation & purification</topic><topic>SACCHAROMYCES CEREVISIAE</topic><topic>Saccharomyces cerevisiae - enzymology</topic><topic>Saccharomyces cerevisiae - ultrastructure</topic><topic>Spermidine - pharmacology</topic><topic>Temperature</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Dake, E</creatorcontrib><creatorcontrib>Hofmann, T J</creatorcontrib><creatorcontrib>McIntire, S</creatorcontrib><creatorcontrib>Hudson, A</creatorcontrib><creatorcontrib>Zassenhaus, H P</creatorcontrib><collection>ScienceDirect Open Access Titles</collection><collection>Elsevier:ScienceDirect:Open Access</collection><collection>AGRIS</collection><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>Nucleic Acids Abstracts</collection><collection>Algology Mycology and Protozoology Abstracts (Microbiology C)</collection><collection>MEDLINE - Academic</collection><jtitle>The Journal of biological chemistry</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Dake, E</au><au>Hofmann, T J</au><au>McIntire, S</au><au>Hudson, A</au><au>Zassenhaus, H P</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Purification and properties of the major nuclease from mitochondria of Saccharomyces cerevisiae</atitle><jtitle>The Journal of biological chemistry</jtitle><addtitle>J Biol Chem</addtitle><date>1988-06-05</date><risdate>1988</risdate><volume>263</volume><issue>16</issue><spage>7691</spage><epage>7702</epage><pages>7691-7702</pages><issn>0021-9258</issn><eissn>1083-351X</eissn><coden>JBCHA3</coden><abstract>The vast majority of nuclease activity in yeast mitochondria is due to a single polypeptide with an apparent molecular weight of 38,000. The enzyme is located in the mitochondrial inner membrane and requires non-ionic detergents for solubilization and activity. A combination of heparin-agarose and Cibacron blue-agarose chromatography was employed to purify the nuclease to approximately 90% homogeneity. The purified enzyme shows multiple activities: 1) RNase activity on single-stranded, but not double-stranded RNA, 2) endonuclease activity on single- and double-stranded DNA, and 3) a 5‘-exonuclease activity on double-stranded DNA. Digestion products with DNA contain 5‘-phosphorylated termini. Antibody raised against an analogous enzyme purified from Neurospora crassa (Chow, T. Y. K., and Fraser, M. (1983) J. Biol. Chem. 258, 12010-12018) inhibits and immunoprecipitates the yeast enzyme. This antibody inhibits 90-95% of all nuclease activity present in solubilized mitochondria, indicating that the purified nuclease accounts for the bulk of mitochondrial nucleolytic activity. Analysis of a mutant strain in which the gene for the nuclease has been disrupted supports this conclusion and shows that all detectable DNase activity and most nonspecific RNase activity in the mitochondria is due to this single enzyme.</abstract><cop>Bethesda, MD</cop><pub>Elsevier Inc</pub><pmid>3286639</pmid><doi>10.1016/S0021-9258(18)68554-0</doi><tpages>12</tpages><oa>free_for_read</oa></addata></record> |
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| subjects | Analytical, structural and metabolic biochemistry Biological and medical sciences Cations, Divalent - analysis Deoxyribonucleases - isolation & purification Enzymes and enzyme inhibitors EPURATION Ethidium - pharmacology Fundamental and applied biological sciences. Psychology Hydrogen-Ion Concentration MITOCHONDRIA Mitochondria - enzymology MITOCHONDRIE MITOCONDRIA Molecular Weight NUCLEOSIDASAS NUCLEOSIDASE NUCLEOSIDASES PURIFICACION PURIFICATION Ribonucleases - isolation & purification SACCHAROMYCES CEREVISIAE Saccharomyces cerevisiae - enzymology Saccharomyces cerevisiae - ultrastructure Spermidine - pharmacology Temperature |
| title | Purification and properties of the major nuclease from mitochondria of Saccharomyces cerevisiae |
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