caa a22 4500 477101461 CHVBK 20180405111542.0 cr unu---uuuuu 170330e19960501xx s 000 0 eng 10.1007/BF00196884 doi (NATIONALLICENCE)springer-10.1007/BF00196884 Subcellular distribution of carbonic anhydrase in Solanum tuberosum L. leaves [Elektronische Daten] Characterization of two compartment-specific isoforms [Dominique Rumeau, Stephan Cuiné, Laurent Fina, Nathalie Gault, Michel Nicole, Gilles Peltier] The intracellular compartmentation of carbonic anhydrase (CA; EC 4.2.1.1), an enzyme that catalyses the reversible hydration of CO2 to bicarbonate, has been investigated in potato (Solanum tuberosum L.) leaves. Although enzyme activity was mainly located in chloroplasts (87% of total cellular activity), significant activity (13%) was also found in the cytosol. The corresponding CA isoforms were purified either from chloroplasts or crude leaf extracts, respectively. The cytosolic isoenzyme has a molecular mass of 255 000 and is composed of eight identical subunits with an estimated M r of 30000. The chloroplastic isoenzyme (M r 220000) is also an octamer composed of two different subunits with M r estimated at 27 000 and 27 500, respectively. The N-terminal amino acid sequences of both chloroplastic CA subunits demonstrated that they were identical except that the M r-27 000 subunit was three amino acids shorter than that of the M r-27 500 subunit. Cytosolic and chloroplastic CA isoenzymes were found to be similarly inhibited by monovalent anions (Cl−, I−, N 3 - and NO 3 - ) and by sulfonamides (ethoxyzolamide and acetozolamide). Both CA isoforms were found to be dependent on a reducing agent such as cysteine or dithiothreitol in order to retain the catalytic activity, but 2-mercaptoethanol was found to be a potent inhibitor. A polyclonal antibody directed against a synthetic peptide corresponding to the N-terminal amino acid sequence of the chloroplastic CA monomers also recognized the cytosolic CA isoform. This antibody was used for immunocytolocalization experiments which confirmed the intracellular compartmentation of CA: within chloroplasts, CA is restricted to the stroma and appears randomly distributed in the cytosol. Springer-Verlag, 1996 Carbonic anhydrase purification nationallicence Intracellular compartmentation nationallicence Solanum nationallicence BSA : bovine serum albumin nationallicence CA : carbonic anhydrase nationallicence PMSF : phenylmethylsulphonyl fluoride nationallicence BAM : benzamidine nationallicence DTT : dithiothreitol nationallicence 2-ME : 2-mercaptoethanol nationallicence PVDF : polyvinylidene difluoride nationallicence Rumeau Dominique Département d'Ecophysiologie Végétale et Microbiologie, CEA-CNRS, UMR-CNRS 163, F-13108, Saint-Paul-lez-Durance Cedex, France aut Cuiné Stephan Département d'Ecophysiologie Végétale et Microbiologie, CEA-CNRS, UMR-CNRS 163, F-13108, Saint-Paul-lez-Durance Cedex, France aut Fina Laurent Département d'Ecophysiologie Végétale et Microbiologie, CEA-CNRS, UMR-CNRS 163, F-13108, Saint-Paul-lez-Durance Cedex, France aut Gault Nathalie Département d'Ecophysiologie Végétale et Microbiologie, CEA-CNRS, UMR-CNRS 163, F-13108, Saint-Paul-lez-Durance Cedex, France aut Nicole Michel ORSTOM, Laboratoire de Phythopathologie, Centre de Montpellier, 911 Avenue Agropolis, F-34032, Montpellier Cedex, France aut Peltier Gilles Département d'Ecophysiologie Végétale et Microbiologie, CEA-CNRS, UMR-CNRS 163, F-13108, Saint-Paul-lez-Durance Cedex, France aut Planta Springer-Verlag 199/1(1996-05-01), 79-88 0032-0935 199:1<79 1996 199 425 https://doi.org/10.1007/BF00196884 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1007/BF00196884 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Rumeau Dominique Département d'Ecophysiologie Végétale et Microbiologie, CEA-CNRS, UMR-CNRS 163, F-13108, Saint-Paul-lez-Durance Cedex, France aut NATIONALLICENCE 700 1- Cuiné Stephan Département d'Ecophysiologie Végétale et Microbiologie, CEA-CNRS, UMR-CNRS 163, F-13108, Saint-Paul-lez-Durance Cedex, France aut NATIONALLICENCE 700 1- Fina Laurent Département d'Ecophysiologie Végétale et Microbiologie, CEA-CNRS, UMR-CNRS 163, F-13108, Saint-Paul-lez-Durance Cedex, France aut NATIONALLICENCE 700 1- Gault Nathalie Département d'Ecophysiologie Végétale et Microbiologie, CEA-CNRS, UMR-CNRS 163, F-13108, Saint-Paul-lez-Durance Cedex, France aut NATIONALLICENCE 700 1- Nicole Michel ORSTOM, Laboratoire de Phythopathologie, Centre de Montpellier, 911 Avenue Agropolis, F-34032, Montpellier Cedex, France aut NATIONALLICENCE 700 1- Peltier Gilles Département d'Ecophysiologie Végétale et Microbiologie, CEA-CNRS, UMR-CNRS 163, F-13108, Saint-Paul-lez-Durance Cedex, France aut NATIONALLICENCE 773 0- Planta Springer-Verlag 199/1(1996-05-01), 79-88 0032-0935 199:1<79 1996 199 425 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer