Carbonic anhydrase activity in isolated chloroplasts of wild-type and high-CO2-dependent mutants of Chlamydomonas reinhardtii as studied by a new assay
In an assay of carbonic anhydrase (CA), NaH14CO3 solution at the bottom of a sealed vessel releases 14CO2, which diffuses to the top of the vessel to be assimilated by photosynthesizing Chlamydomonas reinhardtii cells that have been adapted to a low-CO2 environment The assay is initiated by illumina...
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| Veröffentlicht in: | Plant physiology (Bethesda) 1994-08, Vol.105 (4), p.1197-1202 |
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| creator | Katzman, G.L. (Methodist Hospital of Indiana, Indianapolis, IN.) Carlson, S.J Marcus, Y Moroney, J.V Togasaki, R.K |
| description | In an assay of carbonic anhydrase (CA), NaH14CO3 solution at the bottom of a sealed vessel releases 14CO2, which diffuses to the top of the vessel to be assimilated by photosynthesizing Chlamydomonas reinhardtii cells that have been adapted to a low-CO2 environment The assay is initiated by illuminating the cells and is stopped by turning the light off and killing the cells with acid. Enzyme activity was estimated from acid-stable radioactivity. With bovine CA, 1.5 Wilbur-Anderson units (WAU) was consistently measured at 5- to 6-fold above background. Sonicated whole cells of air-adapted wild-type C reinhardtii had 740 +/- 12.4 WAU/mg chlorophyll (Chl). Sonicated chloroplasts from a mixotrophically grown wall-less strain, cw-15, had 35.5 +/- 2.6 WAU/mg Chl, whereas chloroplasts from wall-less external CA mutant strain cia5/cw-15 had 33.8 +/- 1.9 WAU/mg Chl. Sonicated chloroplasts from the wall-less mutant strain cia-3/cw- 15, believed to lack an internal CA, had 2.8 +/- 3.2 WAU/mg Chl. Sonicated whole cells from cia3/cw-15 had 2.8 +/- 7.8 WAU/mg Chl. Acetazolamide, ethoxyzolamide, and p-aminomethylbenzene sulfonamide (Mafenide) at 100 micromolar inhibited CA in sonicated chloroplasts from cia-5/cw-15. Treatment at 80-C for 10 min inhibited this CA activity by 90.8 +/- 3.6%. Thus, a sensitive 14C assay has confirmed the presence of a CA in cw-15 and cia-5/cw-15 chloroplasts and the lack of a CA in cia-3/cw-15 chloroplasts. Our results indicate that HCO3- is the inorganic carbon species that is accumulated by chloroplasts of Chlamydomonas and that chloroplastic CA is responsible for the majority of internal CA activity |
| doi_str_mv | 10.1104/pp.105.4.1197 |
| format | Article |
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(Methodist Hospital of Indiana, Indianapolis, IN.) ; Carlson, S.J ; Marcus, Y ; Moroney, J.V ; Togasaki, R.K</creator><creatorcontrib>Katzman, G.L. (Methodist Hospital of Indiana, Indianapolis, IN.) ; Carlson, S.J ; Marcus, Y ; Moroney, J.V ; Togasaki, R.K</creatorcontrib><description>In an assay of carbonic anhydrase (CA), NaH14CO3 solution at the bottom of a sealed vessel releases 14CO2, which diffuses to the top of the vessel to be assimilated by photosynthesizing Chlamydomonas reinhardtii cells that have been adapted to a low-CO2 environment The assay is initiated by illuminating the cells and is stopped by turning the light off and killing the cells with acid. Enzyme activity was estimated from acid-stable radioactivity. With bovine CA, 1.5 Wilbur-Anderson units (WAU) was consistently measured at 5- to 6-fold above background. Sonicated whole cells of air-adapted wild-type C reinhardtii had 740 +/- 12.4 WAU/mg chlorophyll (Chl). Sonicated chloroplasts from a mixotrophically grown wall-less strain, cw-15, had 35.5 +/- 2.6 WAU/mg Chl, whereas chloroplasts from wall-less external CA mutant strain cia5/cw-15 had 33.8 +/- 1.9 WAU/mg Chl. Sonicated chloroplasts from the wall-less mutant strain cia-3/cw- 15, believed to lack an internal CA, had 2.8 +/- 3.2 WAU/mg Chl. Sonicated whole cells from cia3/cw-15 had 2.8 +/- 7.8 WAU/mg Chl. Acetazolamide, ethoxyzolamide, and p-aminomethylbenzene sulfonamide (Mafenide) at 100 micromolar inhibited CA in sonicated chloroplasts from cia-5/cw-15. Treatment at 80-C for 10 min inhibited this CA activity by 90.8 +/- 3.6%. Thus, a sensitive 14C assay has confirmed the presence of a CA in cw-15 and cia-5/cw-15 chloroplasts and the lack of a CA in cia-3/cw-15 chloroplasts. Our results indicate that HCO3- is the inorganic carbon species that is accumulated by chloroplasts of Chlamydomonas and that chloroplastic CA is responsible for the majority of internal CA activity</description><identifier>ISSN: 0032-0889</identifier><identifier>EISSN: 1532-2548</identifier><identifier>DOI: 10.1104/pp.105.4.1197</identifier><identifier>PMID: 12232275</identifier><identifier>CODEN: PPHYA5</identifier><language>eng</language><publisher>Rockville, MD: American Society of Plant Physiologists</publisher><subject>ACTIVIDAD ENZIMATICA ; ACTIVITE ENZYMATIQUE ; Biological and medical sciences ; Carbon dioxide ; CARBONATE DESHYDRATASE ; CARBONATO DESHIDRATASA ; Cell growth ; Cell walls ; CHLOROPHYCEAE ; CHLOROPLASTE ; Chloroplasts ; CLOROPLASTO ; DIOXIDO DE CARBONO ; DIOXYDE DE CARBONE ; EXPERIMENTACION EN LABORATORIO ; EXPERIMENTATION EN LABORATOIRE ; Fundamental and applied biological sciences. Psychology ; Lamps ; Metabolism ; Metabolism and Enzymology ; METHODE ; METODOS ; MUTANT ; MUTANTES ; Photosynthesis ; Photosynthesis, respiration. Anabolism, catabolism ; Plant cells ; Plant physiology and development ; Plants ; Radiocarbon ; TECHNIQUE ANALYTIQUE ; TECNICAS ANALITICAS ; Ungulates ; VARIACION GENETICA ; VARIATION GENETIQUE</subject><ispartof>Plant physiology (Bethesda), 1994-08, Vol.105 (4), p.1197-1202</ispartof><rights>Copyright 1994 American Society of Plant Physiologists</rights><rights>1994 INIST-CNRS</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c490t-f53f8b3f1c68cc83eddb0a2e94617012d314415763e97a4aa1cab9875ae8dee03</citedby></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.jstor.org/stable/pdf/4275970$$EPDF$$P50$$Gjstor$$H</linktopdf><linktohtml>$$Uhttps://www.jstor.org/stable/4275970$$EHTML$$P50$$Gjstor$$H</linktohtml><link.rule.ids>230,314,780,784,803,885,27924,27925,58017,58250</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=4259638$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/12232275$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Katzman, G.L. (Methodist Hospital of Indiana, Indianapolis, IN.)</creatorcontrib><creatorcontrib>Carlson, S.J</creatorcontrib><creatorcontrib>Marcus, Y</creatorcontrib><creatorcontrib>Moroney, J.V</creatorcontrib><creatorcontrib>Togasaki, R.K</creatorcontrib><title>Carbonic anhydrase activity in isolated chloroplasts of wild-type and high-CO2-dependent mutants of Chlamydomonas reinhardtii as studied by a new assay</title><title>Plant physiology (Bethesda)</title><addtitle>Plant Physiol</addtitle><description>In an assay of carbonic anhydrase (CA), NaH14CO3 solution at the bottom of a sealed vessel releases 14CO2, which diffuses to the top of the vessel to be assimilated by photosynthesizing Chlamydomonas reinhardtii cells that have been adapted to a low-CO2 environment The assay is initiated by illuminating the cells and is stopped by turning the light off and killing the cells with acid. Enzyme activity was estimated from acid-stable radioactivity. With bovine CA, 1.5 Wilbur-Anderson units (WAU) was consistently measured at 5- to 6-fold above background. Sonicated whole cells of air-adapted wild-type C reinhardtii had 740 +/- 12.4 WAU/mg chlorophyll (Chl). Sonicated chloroplasts from a mixotrophically grown wall-less strain, cw-15, had 35.5 +/- 2.6 WAU/mg Chl, whereas chloroplasts from wall-less external CA mutant strain cia5/cw-15 had 33.8 +/- 1.9 WAU/mg Chl. Sonicated chloroplasts from the wall-less mutant strain cia-3/cw- 15, believed to lack an internal CA, had 2.8 +/- 3.2 WAU/mg Chl. Sonicated whole cells from cia3/cw-15 had 2.8 +/- 7.8 WAU/mg Chl. Acetazolamide, ethoxyzolamide, and p-aminomethylbenzene sulfonamide (Mafenide) at 100 micromolar inhibited CA in sonicated chloroplasts from cia-5/cw-15. Treatment at 80-C for 10 min inhibited this CA activity by 90.8 +/- 3.6%. Thus, a sensitive 14C assay has confirmed the presence of a CA in cw-15 and cia-5/cw-15 chloroplasts and the lack of a CA in cia-3/cw-15 chloroplasts. Our results indicate that HCO3- is the inorganic carbon species that is accumulated by chloroplasts of Chlamydomonas and that chloroplastic CA is responsible for the majority of internal CA activity</description><subject>ACTIVIDAD ENZIMATICA</subject><subject>ACTIVITE ENZYMATIQUE</subject><subject>Biological and medical sciences</subject><subject>Carbon dioxide</subject><subject>CARBONATE DESHYDRATASE</subject><subject>CARBONATO DESHIDRATASA</subject><subject>Cell growth</subject><subject>Cell walls</subject><subject>CHLOROPHYCEAE</subject><subject>CHLOROPLASTE</subject><subject>Chloroplasts</subject><subject>CLOROPLASTO</subject><subject>DIOXIDO DE CARBONO</subject><subject>DIOXYDE DE CARBONE</subject><subject>EXPERIMENTACION EN LABORATORIO</subject><subject>EXPERIMENTATION EN LABORATOIRE</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Lamps</subject><subject>Metabolism</subject><subject>Metabolism and Enzymology</subject><subject>METHODE</subject><subject>METODOS</subject><subject>MUTANT</subject><subject>MUTANTES</subject><subject>Photosynthesis</subject><subject>Photosynthesis, respiration. Anabolism, catabolism</subject><subject>Plant cells</subject><subject>Plant physiology and development</subject><subject>Plants</subject><subject>Radiocarbon</subject><subject>TECHNIQUE ANALYTIQUE</subject><subject>TECNICAS ANALITICAS</subject><subject>Ungulates</subject><subject>VARIACION GENETICA</subject><subject>VARIATION GENETIQUE</subject><issn>0032-0889</issn><issn>1532-2548</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1994</creationdate><recordtype>article</recordtype><recordid>eNpVkU2PFCEQhjtG446rRy_GGA4evPQIDUzDwYOZ-JVssgfdM6kGeppNN7RA76Z_iX9XJjNZ1xNVvE9VvVBV9ZrgLSGYfZznLcF8y0om2yfVhnDa1A1n4mm1wbjEWAh5Ub1I6RZjTChhz6sL0jS0aVq-qf7sIXbBO43AD6uJkCwCnd2dyytyHrkURsjWID2MIYZ5hJQTCj26d6Op8zoX3Bs0uMNQ76-b2tjZemN9RtOSwZ_Y_TDCtJowBQ8JRev8ANFk51BJU16MKwO6FQHy9r7cJVhfVs96GJN9dT4vq5uvX37tv9dX199-7D9f1ZpJnOue0150tCd6J7QW1BrTYWisZDvSYtKY8l5GeLujVrbAAIiGToqWgxXGWkwvq0-nvvPSTdbo4jzCqOboJoirCuDU_4p3gzqEO0W4ZEyU-g_n-hh-LzZlNbmk7TiCt2FJigguaSt4Iwtan1AdQ0rR9g9TCFbHXap5LiFXTB13Wfh3j639o8_LK8D7MwBJw9hH8NqlB441XO7o0eLbE3abcoiP5JbL9vgDb05yD0HBIZYONz8lpzuJKf0LUpu88g</recordid><startdate>19940801</startdate><enddate>19940801</enddate><creator>Katzman, G.L. (Methodist Hospital of Indiana, Indianapolis, IN.)</creator><creator>Carlson, S.J</creator><creator>Marcus, Y</creator><creator>Moroney, J.V</creator><creator>Togasaki, R.K</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>19940801</creationdate><title>Carbonic anhydrase activity in isolated chloroplasts of wild-type and high-CO2-dependent mutants of Chlamydomonas reinhardtii as studied by a new assay</title><author>Katzman, G.L. (Methodist Hospital of Indiana, Indianapolis, IN.) ; Carlson, S.J ; Marcus, Y ; Moroney, J.V ; Togasaki, R.K</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c490t-f53f8b3f1c68cc83eddb0a2e94617012d314415763e97a4aa1cab9875ae8dee03</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1994</creationdate><topic>ACTIVIDAD ENZIMATICA</topic><topic>ACTIVITE ENZYMATIQUE</topic><topic>Biological and medical sciences</topic><topic>Carbon dioxide</topic><topic>CARBONATE DESHYDRATASE</topic><topic>CARBONATO DESHIDRATASA</topic><topic>Cell growth</topic><topic>Cell walls</topic><topic>CHLOROPHYCEAE</topic><topic>CHLOROPLASTE</topic><topic>Chloroplasts</topic><topic>CLOROPLASTO</topic><topic>DIOXIDO DE CARBONO</topic><topic>DIOXYDE DE CARBONE</topic><topic>EXPERIMENTACION EN LABORATORIO</topic><topic>EXPERIMENTATION EN LABORATOIRE</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>Lamps</topic><topic>Metabolism</topic><topic>Metabolism and Enzymology</topic><topic>METHODE</topic><topic>METODOS</topic><topic>MUTANT</topic><topic>MUTANTES</topic><topic>Photosynthesis</topic><topic>Photosynthesis, respiration. Anabolism, catabolism</topic><topic>Plant cells</topic><topic>Plant physiology and development</topic><topic>Plants</topic><topic>Radiocarbon</topic><topic>TECHNIQUE ANALYTIQUE</topic><topic>TECNICAS ANALITICAS</topic><topic>Ungulates</topic><topic>VARIACION GENETICA</topic><topic>VARIATION GENETIQUE</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Katzman, G.L. (Methodist Hospital of Indiana, Indianapolis, IN.)</creatorcontrib><creatorcontrib>Carlson, S.J</creatorcontrib><creatorcontrib>Marcus, Y</creatorcontrib><creatorcontrib>Moroney, J.V</creatorcontrib><creatorcontrib>Togasaki, R.K</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>Katzman, G.L. (Methodist Hospital of Indiana, Indianapolis, IN.)</au><au>Carlson, S.J</au><au>Marcus, Y</au><au>Moroney, J.V</au><au>Togasaki, R.K</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Carbonic anhydrase activity in isolated chloroplasts of wild-type and high-CO2-dependent mutants of Chlamydomonas reinhardtii as studied by a new assay</atitle><jtitle>Plant physiology (Bethesda)</jtitle><addtitle>Plant Physiol</addtitle><date>1994-08-01</date><risdate>1994</risdate><volume>105</volume><issue>4</issue><spage>1197</spage><epage>1202</epage><pages>1197-1202</pages><issn>0032-0889</issn><eissn>1532-2548</eissn><coden>PPHYA5</coden><abstract>In an assay of carbonic anhydrase (CA), NaH14CO3 solution at the bottom of a sealed vessel releases 14CO2, which diffuses to the top of the vessel to be assimilated by photosynthesizing Chlamydomonas reinhardtii cells that have been adapted to a low-CO2 environment The assay is initiated by illuminating the cells and is stopped by turning the light off and killing the cells with acid. Enzyme activity was estimated from acid-stable radioactivity. With bovine CA, 1.5 Wilbur-Anderson units (WAU) was consistently measured at 5- to 6-fold above background. Sonicated whole cells of air-adapted wild-type C reinhardtii had 740 +/- 12.4 WAU/mg chlorophyll (Chl). Sonicated chloroplasts from a mixotrophically grown wall-less strain, cw-15, had 35.5 +/- 2.6 WAU/mg Chl, whereas chloroplasts from wall-less external CA mutant strain cia5/cw-15 had 33.8 +/- 1.9 WAU/mg Chl. Sonicated chloroplasts from the wall-less mutant strain cia-3/cw- 15, believed to lack an internal CA, had 2.8 +/- 3.2 WAU/mg Chl. Sonicated whole cells from cia3/cw-15 had 2.8 +/- 7.8 WAU/mg Chl. Acetazolamide, ethoxyzolamide, and p-aminomethylbenzene sulfonamide (Mafenide) at 100 micromolar inhibited CA in sonicated chloroplasts from cia-5/cw-15. Treatment at 80-C for 10 min inhibited this CA activity by 90.8 +/- 3.6%. Thus, a sensitive 14C assay has confirmed the presence of a CA in cw-15 and cia-5/cw-15 chloroplasts and the lack of a CA in cia-3/cw-15 chloroplasts. Our results indicate that HCO3- is the inorganic carbon species that is accumulated by chloroplasts of Chlamydomonas and that chloroplastic CA is responsible for the majority of internal CA activity</abstract><cop>Rockville, MD</cop><pub>American Society of Plant Physiologists</pub><pmid>12232275</pmid><doi>10.1104/pp.105.4.1197</doi><tpages>6</tpages><oa>free_for_read</oa></addata></record> |
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| ispartof | Plant physiology (Bethesda), 1994-08, Vol.105 (4), p.1197-1202 |
| issn | 0032-0889 1532-2548 |
| language | eng |
| recordid | cdi_pubmedcentral_primary_oai_pubmedcentral_nih_gov_159448 |
| source | JSTOR Archive Collection A-Z Listing; EZB-FREE-00999 freely available EZB journals; Alma/SFX Local Collection |
| subjects | ACTIVIDAD ENZIMATICA ACTIVITE ENZYMATIQUE Biological and medical sciences Carbon dioxide CARBONATE DESHYDRATASE CARBONATO DESHIDRATASA Cell growth Cell walls CHLOROPHYCEAE CHLOROPLASTE Chloroplasts CLOROPLASTO DIOXIDO DE CARBONO DIOXYDE DE CARBONE EXPERIMENTACION EN LABORATORIO EXPERIMENTATION EN LABORATOIRE Fundamental and applied biological sciences. Psychology Lamps Metabolism Metabolism and Enzymology METHODE METODOS MUTANT MUTANTES Photosynthesis Photosynthesis, respiration. Anabolism, catabolism Plant cells Plant physiology and development Plants Radiocarbon TECHNIQUE ANALYTIQUE TECNICAS ANALITICAS Ungulates VARIACION GENETICA VARIATION GENETIQUE |
| title | Carbonic anhydrase activity in isolated chloroplasts of wild-type and high-CO2-dependent mutants of Chlamydomonas reinhardtii as studied by a new assay |
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