Rapid purification and thermostability of the cytoplasmic aspartate aminotransferase from carrot suspension cultures
Several isoenzymic forms of aspartate aminotransferase (AAT) have been identified in protein extracts from carrot (Daucus carota) cell suspension cultures. The cellular location of the major form (form I) of AAT in carrot suspension cultures was determined by heat inactivation, subcellular fractiona...
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| Veröffentlicht in: | Plant physiology (Bethesda) 1991-10, Vol.97 (2), p.606-612 |
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| creator | Turano, F.J. (USDA, ARS, Climate Stress Laboratory, Beltsville, MD) Wilson, B.J Matthews, B.F |
| description | Several isoenzymic forms of aspartate aminotransferase (AAT) have been identified in protein extracts from carrot (Daucus carota) cell suspension cultures. The cellular location of the major form (form I) of AAT in carrot suspension cultures was determined by heat inactivation, subcellular fractionation, and amino acid sequence analysis. in mammalian systems, there are two forms of AAT, a heat-stable cytoplasmic form and a heat-labile form in the mitochondria. The thermostability of three isoenzymes of carrot AAT was examined, and the results showed that form I was more thermostable than forms II or III. Organelles were separated in sucrose gradients by isopynic centrifugation. Activity for form I was identified in the soluble fractions and not in fractions containing peroxisomes, proplastids, or mitochondria. Form I was purified to homogeneity and endoproteolytically cleaved, and the peptide fragments were separated by reverse phase chromatography. Analysis of the sequence data from two of the polypeptides showed that the amino acid identity of form I is more conserved to the animal cytoplasmic AAT than to animal mitochondrial AAT sequences. These data strongly suggest that form I of AAT from carrot is the cytoplasmic isoenzyme. Additionally, a rapid purification scheme for form I of AAT from carrot is presented using selective heat denaturation and anion-exchange chromatography |
| doi_str_mv | 10.1104/pp.97.2.606 |
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(USDA, ARS, Climate Stress Laboratory, Beltsville, MD) ; Wilson, B.J ; Matthews, B.F</creator><creatorcontrib>Turano, F.J. (USDA, ARS, Climate Stress Laboratory, Beltsville, MD) ; Wilson, B.J ; Matthews, B.F</creatorcontrib><description>Several isoenzymic forms of aspartate aminotransferase (AAT) have been identified in protein extracts from carrot (Daucus carota) cell suspension cultures. The cellular location of the major form (form I) of AAT in carrot suspension cultures was determined by heat inactivation, subcellular fractionation, and amino acid sequence analysis. in mammalian systems, there are two forms of AAT, a heat-stable cytoplasmic form and a heat-labile form in the mitochondria. The thermostability of three isoenzymes of carrot AAT was examined, and the results showed that form I was more thermostable than forms II or III. Organelles were separated in sucrose gradients by isopynic centrifugation. Activity for form I was identified in the soluble fractions and not in fractions containing peroxisomes, proplastids, or mitochondria. Form I was purified to homogeneity and endoproteolytically cleaved, and the peptide fragments were separated by reverse phase chromatography. Analysis of the sequence data from two of the polypeptides showed that the amino acid identity of form I is more conserved to the animal cytoplasmic AAT than to animal mitochondrial AAT sequences. These data strongly suggest that form I of AAT from carrot is the cytoplasmic isoenzyme. Additionally, a rapid purification scheme for form I of AAT from carrot is presented using selective heat denaturation and anion-exchange chromatography</description><identifier>ISSN: 0032-0889</identifier><identifier>EISSN: 1532-2548</identifier><identifier>DOI: 10.1104/pp.97.2.606</identifier><identifier>PMID: 16668442</identifier><identifier>CODEN: PPHYA5</identifier><language>eng</language><publisher>Rockville, MD: American Society of Plant Physiologists</publisher><subject>ACTIVIDAD ENZIMATICA ; ACTIVITE ENZYMATIQUE ; Amino acids ; ASPARTATE AMINOTRANSFERASE ; ASPARTATO AMINOTRANSFERASA ; Biological and medical sciences ; Cell culture techniques ; Centrifugation ; Chemical suspensions ; Chromatography ; CITOPLASMA ; CULTIVO DE CELULAS ; CULTURE DE CELLULE ; Cultured cells ; CYTOPLASME ; DAUCUS CAROTA ; Enzymes ; EXPRESION GENICA ; EXPRESSION DES GENES ; Fundamental and applied biological sciences. Psychology ; Gels ; ISOENZIMAS ; ISOENZYME ; Metabolism ; Metabolism and Enzymology ; Organelles ; Plant physiology and development ; PROTEINAS ; PROTEINE ; PURIFICACION ; PURIFICATION ; TECHNIQUE ANALYTIQUE ; TECNICAS ANALITICAS ; Thermal stability</subject><ispartof>Plant physiology (Bethesda), 1991-10, Vol.97 (2), p.606-612</ispartof><rights>Copyright 1991 American Society of Plant Physiologists</rights><rights>1992 INIST-CNRS</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c482t-ee93e54f8198e4ee82479a0c63ffef19ffb2fb7630832e3bfa7e12fa992a56443</citedby></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.jstor.org/stable/pdf/4273878$$EPDF$$P50$$Gjstor$$H</linktopdf><linktohtml>$$Uhttps://www.jstor.org/stable/4273878$$EHTML$$P50$$Gjstor$$H</linktohtml><link.rule.ids>230,314,777,781,800,882,27905,27906,57998,58231</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=5112324$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/16668442$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Turano, F.J. (USDA, ARS, Climate Stress Laboratory, Beltsville, MD)</creatorcontrib><creatorcontrib>Wilson, B.J</creatorcontrib><creatorcontrib>Matthews, B.F</creatorcontrib><title>Rapid purification and thermostability of the cytoplasmic aspartate aminotransferase from carrot suspension cultures</title><title>Plant physiology (Bethesda)</title><addtitle>Plant Physiol</addtitle><description>Several isoenzymic forms of aspartate aminotransferase (AAT) have been identified in protein extracts from carrot (Daucus carota) cell suspension cultures. The cellular location of the major form (form I) of AAT in carrot suspension cultures was determined by heat inactivation, subcellular fractionation, and amino acid sequence analysis. in mammalian systems, there are two forms of AAT, a heat-stable cytoplasmic form and a heat-labile form in the mitochondria. The thermostability of three isoenzymes of carrot AAT was examined, and the results showed that form I was more thermostable than forms II or III. Organelles were separated in sucrose gradients by isopynic centrifugation. Activity for form I was identified in the soluble fractions and not in fractions containing peroxisomes, proplastids, or mitochondria. Form I was purified to homogeneity and endoproteolytically cleaved, and the peptide fragments were separated by reverse phase chromatography. Analysis of the sequence data from two of the polypeptides showed that the amino acid identity of form I is more conserved to the animal cytoplasmic AAT than to animal mitochondrial AAT sequences. These data strongly suggest that form I of AAT from carrot is the cytoplasmic isoenzyme. Additionally, a rapid purification scheme for form I of AAT from carrot is presented using selective heat denaturation and anion-exchange chromatography</description><subject>ACTIVIDAD ENZIMATICA</subject><subject>ACTIVITE ENZYMATIQUE</subject><subject>Amino acids</subject><subject>ASPARTATE AMINOTRANSFERASE</subject><subject>ASPARTATO AMINOTRANSFERASA</subject><subject>Biological and medical sciences</subject><subject>Cell culture techniques</subject><subject>Centrifugation</subject><subject>Chemical suspensions</subject><subject>Chromatography</subject><subject>CITOPLASMA</subject><subject>CULTIVO DE CELULAS</subject><subject>CULTURE DE CELLULE</subject><subject>Cultured cells</subject><subject>CYTOPLASME</subject><subject>DAUCUS CAROTA</subject><subject>Enzymes</subject><subject>EXPRESION GENICA</subject><subject>EXPRESSION DES GENES</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Gels</subject><subject>ISOENZIMAS</subject><subject>ISOENZYME</subject><subject>Metabolism</subject><subject>Metabolism and Enzymology</subject><subject>Organelles</subject><subject>Plant physiology and development</subject><subject>PROTEINAS</subject><subject>PROTEINE</subject><subject>PURIFICACION</subject><subject>PURIFICATION</subject><subject>TECHNIQUE ANALYTIQUE</subject><subject>TECNICAS ANALITICAS</subject><subject>Thermal stability</subject><issn>0032-0889</issn><issn>1532-2548</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1991</creationdate><recordtype>article</recordtype><recordid>eNpVkUuLFDEURgtRnHZ05U5EshBcSLd5VSXZCDL4ggFBnXW4nb6ZyVBViUlK6H9vmm5aXSXkO5zc5Ou654xuGKPyXUobozZ8M9DhQbdiveBr3kv9sFtR2vZUa3PRPSnlnlLKBJOPuws2DIOWkq-6-h1S2JG05OCDgxriTGDekXqHeYqlwjaMoe5J9Icj4vY1phHKFByBkiBXqEhgCnOsGebiMUNB4nOciIOcYyVlKQnnchC7ZaxLxvK0e-RhLPjstF52N58-_rz6sr7-9vnr1YfrtZOa1zWiEdhLr5nRKBE1l8oAdYPwHj0z3m-536pBUC04iq0HhYx7MIZDP0gpLrv3R29athPuHM5tyNGmHCbIexsh2P-TOdzZ2_jbMqoZ7WkTvDkJcvy1YKl2CsXhOMKMcSlWCSGNopw38u2RdDmWktGfb2HUHmqyKVmjLLetpka_-newv-yplwa8PgFQHIy-_a0L5cz1jHHBDw98ecTuS435HEuuhFa6xS-OsYdo4TY3w80Pw3rVcy3-AA_PsTM</recordid><startdate>19911001</startdate><enddate>19911001</enddate><creator>Turano, F.J. (USDA, ARS, Climate Stress Laboratory, Beltsville, MD)</creator><creator>Wilson, B.J</creator><creator>Matthews, B.F</creator><general>American Society of Plant Physiologists</general><scope>FBQ</scope><scope>IQODW</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7X8</scope><scope>5PM</scope></search><sort><creationdate>19911001</creationdate><title>Rapid purification and thermostability of the cytoplasmic aspartate aminotransferase from carrot suspension cultures</title><author>Turano, F.J. (USDA, ARS, Climate Stress Laboratory, Beltsville, MD) ; Wilson, B.J ; Matthews, B.F</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c482t-ee93e54f8198e4ee82479a0c63ffef19ffb2fb7630832e3bfa7e12fa992a56443</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1991</creationdate><topic>ACTIVIDAD ENZIMATICA</topic><topic>ACTIVITE ENZYMATIQUE</topic><topic>Amino acids</topic><topic>ASPARTATE AMINOTRANSFERASE</topic><topic>ASPARTATO AMINOTRANSFERASA</topic><topic>Biological and medical sciences</topic><topic>Cell culture techniques</topic><topic>Centrifugation</topic><topic>Chemical suspensions</topic><topic>Chromatography</topic><topic>CITOPLASMA</topic><topic>CULTIVO DE CELULAS</topic><topic>CULTURE DE CELLULE</topic><topic>Cultured cells</topic><topic>CYTOPLASME</topic><topic>DAUCUS CAROTA</topic><topic>Enzymes</topic><topic>EXPRESION GENICA</topic><topic>EXPRESSION DES GENES</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>Gels</topic><topic>ISOENZIMAS</topic><topic>ISOENZYME</topic><topic>Metabolism</topic><topic>Metabolism and Enzymology</topic><topic>Organelles</topic><topic>Plant physiology and development</topic><topic>PROTEINAS</topic><topic>PROTEINE</topic><topic>PURIFICACION</topic><topic>PURIFICATION</topic><topic>TECHNIQUE ANALYTIQUE</topic><topic>TECNICAS ANALITICAS</topic><topic>Thermal stability</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Turano, F.J. (USDA, ARS, Climate Stress Laboratory, Beltsville, MD)</creatorcontrib><creatorcontrib>Wilson, B.J</creatorcontrib><creatorcontrib>Matthews, B.F</creatorcontrib><collection>AGRIS</collection><collection>Pascal-Francis</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Plant physiology (Bethesda)</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Turano, F.J. (USDA, ARS, Climate Stress Laboratory, Beltsville, MD)</au><au>Wilson, B.J</au><au>Matthews, B.F</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Rapid purification and thermostability of the cytoplasmic aspartate aminotransferase from carrot suspension cultures</atitle><jtitle>Plant physiology (Bethesda)</jtitle><addtitle>Plant Physiol</addtitle><date>1991-10-01</date><risdate>1991</risdate><volume>97</volume><issue>2</issue><spage>606</spage><epage>612</epage><pages>606-612</pages><issn>0032-0889</issn><eissn>1532-2548</eissn><coden>PPHYA5</coden><abstract>Several isoenzymic forms of aspartate aminotransferase (AAT) have been identified in protein extracts from carrot (Daucus carota) cell suspension cultures. The cellular location of the major form (form I) of AAT in carrot suspension cultures was determined by heat inactivation, subcellular fractionation, and amino acid sequence analysis. in mammalian systems, there are two forms of AAT, a heat-stable cytoplasmic form and a heat-labile form in the mitochondria. The thermostability of three isoenzymes of carrot AAT was examined, and the results showed that form I was more thermostable than forms II or III. Organelles were separated in sucrose gradients by isopynic centrifugation. Activity for form I was identified in the soluble fractions and not in fractions containing peroxisomes, proplastids, or mitochondria. Form I was purified to homogeneity and endoproteolytically cleaved, and the peptide fragments were separated by reverse phase chromatography. Analysis of the sequence data from two of the polypeptides showed that the amino acid identity of form I is more conserved to the animal cytoplasmic AAT than to animal mitochondrial AAT sequences. These data strongly suggest that form I of AAT from carrot is the cytoplasmic isoenzyme. Additionally, a rapid purification scheme for form I of AAT from carrot is presented using selective heat denaturation and anion-exchange chromatography</abstract><cop>Rockville, MD</cop><pub>American Society of Plant Physiologists</pub><pmid>16668442</pmid><doi>10.1104/pp.97.2.606</doi><tpages>7</tpages><oa>free_for_read</oa></addata></record> |
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| source | Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals; Jstor Complete Legacy; Alma/SFX Local Collection |
| subjects | ACTIVIDAD ENZIMATICA ACTIVITE ENZYMATIQUE Amino acids ASPARTATE AMINOTRANSFERASE ASPARTATO AMINOTRANSFERASA Biological and medical sciences Cell culture techniques Centrifugation Chemical suspensions Chromatography CITOPLASMA CULTIVO DE CELULAS CULTURE DE CELLULE Cultured cells CYTOPLASME DAUCUS CAROTA Enzymes EXPRESION GENICA EXPRESSION DES GENES Fundamental and applied biological sciences. Psychology Gels ISOENZIMAS ISOENZYME Metabolism Metabolism and Enzymology Organelles Plant physiology and development PROTEINAS PROTEINE PURIFICACION PURIFICATION TECHNIQUE ANALYTIQUE TECNICAS ANALITICAS Thermal stability |
| title | Rapid purification and thermostability of the cytoplasmic aspartate aminotransferase from carrot suspension cultures |
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