In Vitro Hybridization and Separation of Hybrids of Human Adenylosuccinate Lyase from Wild-Type and Disease-Associated Mutant Enzymes
Human adenylosuccinate lyase (ASL) deficiency is an inherited metabolic disease in which the majority of the patients are compound heterozygotes for the mutations that occur in the ASL gene. Starting with purified wild-type (WT) and single-mutant human ASL, we generated in vitro hybrids that mimic c...
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| Veröffentlicht in: | Biochemistry (Easton) 2011-03, Vol.50 (8), p.1336-1346 |
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| creator | De Zoysa Ariyananda, Lushanti Antonopoulos, Christina Currier, Jenna Colman, Roberta F |
| description | Human adenylosuccinate lyase (ASL) deficiency is an inherited metabolic disease in which the majority of the patients are compound heterozygotes for the mutations that occur in the ASL gene. Starting with purified wild-type (WT) and single-mutant human ASL, we generated in vitro hybrids that mimic compound heterozygote ASL. For this study, we used His-tagged WT/non-His-tagged WT, His-tagged WT/non-His-tagged R396C, His-tagged WT/non-His-tagged R396H, His-tagged R194C/non-His-tagged R396C, and His-tagged L311V/non-His-tagged R396H enzyme pairs. We generated various hybrids by denaturing pairs of enzymes in 1 M guanidinium chloride and renaturing them by removing the denaturant. The hybrids were separated on a nickel−nitrilotriacetic acid−agarose column based on the number of His tags present in the enzyme tetramer. Analytical ultracentrifuge data indicate that the hybrids have predominant amounts of heterotetramers. Analysis of the V max values of the hybrids indicates that most of the subunits behave independently; however, the hybrid tetramers retain weak positive cooperativity, indicating that there is some interaction between the different subunit types. The interactions between WT and mutant subunits may be advantageous to the parents of ASL deficient patients, while the interactions between some mutant subunits may assist heterozygote ASL deficient patients. |
| doi_str_mv | 10.1021/bi101734q |
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Starting with purified wild-type (WT) and single-mutant human ASL, we generated in vitro hybrids that mimic compound heterozygote ASL. For this study, we used His-tagged WT/non-His-tagged WT, His-tagged WT/non-His-tagged R396C, His-tagged WT/non-His-tagged R396H, His-tagged R194C/non-His-tagged R396C, and His-tagged L311V/non-His-tagged R396H enzyme pairs. We generated various hybrids by denaturing pairs of enzymes in 1 M guanidinium chloride and renaturing them by removing the denaturant. The hybrids were separated on a nickel−nitrilotriacetic acid−agarose column based on the number of His tags present in the enzyme tetramer. Analytical ultracentrifuge data indicate that the hybrids have predominant amounts of heterotetramers. Analysis of the V max values of the hybrids indicates that most of the subunits behave independently; however, the hybrid tetramers retain weak positive cooperativity, indicating that there is some interaction between the different subunit types. The interactions between WT and mutant subunits may be advantageous to the parents of ASL deficient patients, while the interactions between some mutant subunits may assist heterozygote ASL deficient patients.</description><identifier>ISSN: 0006-2960</identifier><identifier>EISSN: 1520-4995</identifier><identifier>DOI: 10.1021/bi101734q</identifier><identifier>PMID: 21210713</identifier><language>eng</language><publisher>United States: American Chemical Society</publisher><subject>Adenylosuccinate Lyase - chemistry ; Adenylosuccinate Lyase - genetics ; Adenylosuccinate Lyase - isolation & purification ; Adenylosuccinate Lyase - metabolism ; Amino Acid Sequence ; Aminoimidazole Carboxamide - analogs & derivatives ; Aminoimidazole Carboxamide - metabolism ; Area Under Curve ; Electrophoresis, Polyacrylamide Gel ; Enzyme Stability ; Histidine ; Humans ; Kinetics ; Metabolism, Inborn Errors - enzymology ; Metabolism, Inborn Errors - genetics ; Models, Molecular ; Molecular Sequence Data ; Mutagenesis, Site-Directed ; Mutant Proteins - chemistry ; Mutant Proteins - genetics ; Mutant Proteins - isolation & purification ; Mutant Proteins - metabolism ; Mutation ; Protein Conformation ; Protein Denaturation ; Protein Engineering - methods ; Protein Renaturation ; Recombinant Fusion Proteins - chemistry ; Recombinant Fusion Proteins - genetics ; Recombinant Fusion Proteins - isolation & purification ; Recombinant Fusion Proteins - metabolism ; Ribonucleotides - metabolism ; Succinic Acid - chemistry ; Succinic Acid - metabolism ; Temperature</subject><ispartof>Biochemistry (Easton), 2011-03, Vol.50 (8), p.1336-1346</ispartof><rights>Copyright © 2011 American Chemical Society</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-a404t-66f56f4024c2a0475a83a56cdf0546273035c87ac8023131bbf78378524c28d33</citedby><cites>FETCH-LOGICAL-a404t-66f56f4024c2a0475a83a56cdf0546273035c87ac8023131bbf78378524c28d33</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://pubs.acs.org/doi/pdf/10.1021/bi101734q$$EPDF$$P50$$Gacs$$H</linktopdf><linktohtml>$$Uhttps://pubs.acs.org/doi/10.1021/bi101734q$$EHTML$$P50$$Gacs$$H</linktohtml><link.rule.ids>230,314,776,780,881,2751,27055,27903,27904,56716,56766</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/21210713$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>De Zoysa Ariyananda, Lushanti</creatorcontrib><creatorcontrib>Antonopoulos, Christina</creatorcontrib><creatorcontrib>Currier, Jenna</creatorcontrib><creatorcontrib>Colman, Roberta F</creatorcontrib><title>In Vitro Hybridization and Separation of Hybrids of Human Adenylosuccinate Lyase from Wild-Type and Disease-Associated Mutant Enzymes</title><title>Biochemistry (Easton)</title><addtitle>Biochemistry</addtitle><description>Human adenylosuccinate lyase (ASL) deficiency is an inherited metabolic disease in which the majority of the patients are compound heterozygotes for the mutations that occur in the ASL gene. Starting with purified wild-type (WT) and single-mutant human ASL, we generated in vitro hybrids that mimic compound heterozygote ASL. For this study, we used His-tagged WT/non-His-tagged WT, His-tagged WT/non-His-tagged R396C, His-tagged WT/non-His-tagged R396H, His-tagged R194C/non-His-tagged R396C, and His-tagged L311V/non-His-tagged R396H enzyme pairs. We generated various hybrids by denaturing pairs of enzymes in 1 M guanidinium chloride and renaturing them by removing the denaturant. The hybrids were separated on a nickel−nitrilotriacetic acid−agarose column based on the number of His tags present in the enzyme tetramer. Analytical ultracentrifuge data indicate that the hybrids have predominant amounts of heterotetramers. Analysis of the V max values of the hybrids indicates that most of the subunits behave independently; however, the hybrid tetramers retain weak positive cooperativity, indicating that there is some interaction between the different subunit types. The interactions between WT and mutant subunits may be advantageous to the parents of ASL deficient patients, while the interactions between some mutant subunits may assist heterozygote ASL deficient patients.</description><subject>Adenylosuccinate Lyase - chemistry</subject><subject>Adenylosuccinate Lyase - genetics</subject><subject>Adenylosuccinate Lyase - isolation & purification</subject><subject>Adenylosuccinate Lyase - metabolism</subject><subject>Amino Acid Sequence</subject><subject>Aminoimidazole Carboxamide - analogs & derivatives</subject><subject>Aminoimidazole Carboxamide - metabolism</subject><subject>Area Under Curve</subject><subject>Electrophoresis, Polyacrylamide Gel</subject><subject>Enzyme Stability</subject><subject>Histidine</subject><subject>Humans</subject><subject>Kinetics</subject><subject>Metabolism, Inborn Errors - enzymology</subject><subject>Metabolism, Inborn Errors - genetics</subject><subject>Models, Molecular</subject><subject>Molecular Sequence Data</subject><subject>Mutagenesis, Site-Directed</subject><subject>Mutant Proteins - chemistry</subject><subject>Mutant Proteins - genetics</subject><subject>Mutant Proteins - isolation & purification</subject><subject>Mutant Proteins - metabolism</subject><subject>Mutation</subject><subject>Protein Conformation</subject><subject>Protein Denaturation</subject><subject>Protein Engineering - methods</subject><subject>Protein Renaturation</subject><subject>Recombinant Fusion Proteins - chemistry</subject><subject>Recombinant Fusion Proteins - genetics</subject><subject>Recombinant Fusion Proteins - isolation & purification</subject><subject>Recombinant Fusion Proteins - metabolism</subject><subject>Ribonucleotides - metabolism</subject><subject>Succinic Acid - chemistry</subject><subject>Succinic Acid - metabolism</subject><subject>Temperature</subject><issn>0006-2960</issn><issn>1520-4995</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2011</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNptkclOwzAQhi0EglI48ALIFw4cAuMlSy9IVVmlIg5sx2jiOGDU2MVOkMKd9ya0UIHEyR7933wjewjZY3DEgLPjwjBgqZCva2TAYg6RHI3idTIAgCTiowS2yHYIL30pIZWbZIszziBlYkA-rix9MI139LIrvCnNOzbGWYq2pLd6jn5Zuuo7D4trW6Ol41LbbuZCq5Sx2Gg67TBoWnlX00czK6O7bq4XolMTdB9F4xCcMj1a0uu2QdvQM_ve1TrskI0KZ0Hvfp9Dcn9-dje5jKY3F1eT8TRCCbKJkqSKk0oCl4ojyDTGTGCcqLKCWCY8FSBilaWoMuCCCVYUVZqJNIu_GrJSiCE5WXrnbVHrUmnbeJzlc29q9F3u0OR_E2ue8yf3lguQgsmsFxwuBcq7ELyuVr0M8q9d5Ktd9Oz-72Er8ufze-BgCaAK-Ytrve3f_o_oE9tpkiQ</recordid><startdate>20110301</startdate><enddate>20110301</enddate><creator>De Zoysa Ariyananda, Lushanti</creator><creator>Antonopoulos, Christina</creator><creator>Currier, Jenna</creator><creator>Colman, Roberta F</creator><general>American Chemical Society</general><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>5PM</scope></search><sort><creationdate>20110301</creationdate><title>In Vitro Hybridization and Separation of Hybrids of Human Adenylosuccinate Lyase from Wild-Type and Disease-Associated Mutant Enzymes</title><author>De Zoysa Ariyananda, Lushanti ; Antonopoulos, Christina ; Currier, Jenna ; Colman, Roberta F</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-a404t-66f56f4024c2a0475a83a56cdf0546273035c87ac8023131bbf78378524c28d33</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2011</creationdate><topic>Adenylosuccinate Lyase - chemistry</topic><topic>Adenylosuccinate Lyase - genetics</topic><topic>Adenylosuccinate Lyase - isolation & purification</topic><topic>Adenylosuccinate Lyase - metabolism</topic><topic>Amino Acid Sequence</topic><topic>Aminoimidazole Carboxamide - analogs & derivatives</topic><topic>Aminoimidazole Carboxamide - metabolism</topic><topic>Area Under Curve</topic><topic>Electrophoresis, Polyacrylamide Gel</topic><topic>Enzyme Stability</topic><topic>Histidine</topic><topic>Humans</topic><topic>Kinetics</topic><topic>Metabolism, Inborn Errors - enzymology</topic><topic>Metabolism, Inborn Errors - genetics</topic><topic>Models, Molecular</topic><topic>Molecular Sequence Data</topic><topic>Mutagenesis, Site-Directed</topic><topic>Mutant Proteins - chemistry</topic><topic>Mutant Proteins - genetics</topic><topic>Mutant Proteins - isolation & purification</topic><topic>Mutant Proteins - metabolism</topic><topic>Mutation</topic><topic>Protein Conformation</topic><topic>Protein Denaturation</topic><topic>Protein Engineering - methods</topic><topic>Protein Renaturation</topic><topic>Recombinant Fusion Proteins - chemistry</topic><topic>Recombinant Fusion Proteins - genetics</topic><topic>Recombinant Fusion Proteins - isolation & purification</topic><topic>Recombinant Fusion Proteins - metabolism</topic><topic>Ribonucleotides - metabolism</topic><topic>Succinic Acid - chemistry</topic><topic>Succinic Acid - metabolism</topic><topic>Temperature</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>De Zoysa Ariyananda, Lushanti</creatorcontrib><creatorcontrib>Antonopoulos, Christina</creatorcontrib><creatorcontrib>Currier, Jenna</creatorcontrib><creatorcontrib>Colman, Roberta F</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Biochemistry (Easton)</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>De Zoysa Ariyananda, Lushanti</au><au>Antonopoulos, Christina</au><au>Currier, Jenna</au><au>Colman, Roberta F</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>In Vitro Hybridization and Separation of Hybrids of Human Adenylosuccinate Lyase from Wild-Type and Disease-Associated Mutant Enzymes</atitle><jtitle>Biochemistry (Easton)</jtitle><addtitle>Biochemistry</addtitle><date>2011-03-01</date><risdate>2011</risdate><volume>50</volume><issue>8</issue><spage>1336</spage><epage>1346</epage><pages>1336-1346</pages><issn>0006-2960</issn><eissn>1520-4995</eissn><abstract>Human adenylosuccinate lyase (ASL) deficiency is an inherited metabolic disease in which the majority of the patients are compound heterozygotes for the mutations that occur in the ASL gene. Starting with purified wild-type (WT) and single-mutant human ASL, we generated in vitro hybrids that mimic compound heterozygote ASL. For this study, we used His-tagged WT/non-His-tagged WT, His-tagged WT/non-His-tagged R396C, His-tagged WT/non-His-tagged R396H, His-tagged R194C/non-His-tagged R396C, and His-tagged L311V/non-His-tagged R396H enzyme pairs. We generated various hybrids by denaturing pairs of enzymes in 1 M guanidinium chloride and renaturing them by removing the denaturant. The hybrids were separated on a nickel−nitrilotriacetic acid−agarose column based on the number of His tags present in the enzyme tetramer. Analytical ultracentrifuge data indicate that the hybrids have predominant amounts of heterotetramers. Analysis of the V max values of the hybrids indicates that most of the subunits behave independently; however, the hybrid tetramers retain weak positive cooperativity, indicating that there is some interaction between the different subunit types. The interactions between WT and mutant subunits may be advantageous to the parents of ASL deficient patients, while the interactions between some mutant subunits may assist heterozygote ASL deficient patients.</abstract><cop>United States</cop><pub>American Chemical Society</pub><pmid>21210713</pmid><doi>10.1021/bi101734q</doi><tpages>11</tpages><oa>free_for_read</oa></addata></record> |
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| ispartof | Biochemistry (Easton), 2011-03, Vol.50 (8), p.1336-1346 |
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| source | MEDLINE; ACS Publications |
| subjects | Adenylosuccinate Lyase - chemistry Adenylosuccinate Lyase - genetics Adenylosuccinate Lyase - isolation & purification Adenylosuccinate Lyase - metabolism Amino Acid Sequence Aminoimidazole Carboxamide - analogs & derivatives Aminoimidazole Carboxamide - metabolism Area Under Curve Electrophoresis, Polyacrylamide Gel Enzyme Stability Histidine Humans Kinetics Metabolism, Inborn Errors - enzymology Metabolism, Inborn Errors - genetics Models, Molecular Molecular Sequence Data Mutagenesis, Site-Directed Mutant Proteins - chemistry Mutant Proteins - genetics Mutant Proteins - isolation & purification Mutant Proteins - metabolism Mutation Protein Conformation Protein Denaturation Protein Engineering - methods Protein Renaturation Recombinant Fusion Proteins - chemistry Recombinant Fusion Proteins - genetics Recombinant Fusion Proteins - isolation & purification Recombinant Fusion Proteins - metabolism Ribonucleotides - metabolism Succinic Acid - chemistry Succinic Acid - metabolism Temperature |
| title | In Vitro Hybridization and Separation of Hybrids of Human Adenylosuccinate Lyase from Wild-Type and Disease-Associated Mutant Enzymes |
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