Subcellular distribution of carbonic anhydrase in Solanum tuberosum L. leaves: characterization of two compartment-specific isoforms.
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 Mr of 30,000. The chloroplastic isoenzyme (Mr 220,000) is also an octamer composed of two different subunits with Mr 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 Mr-27,000 subunit was three amino acids shorter than that of the Mr-27,500 subunit. Cytosolic and chloroplastic CA isoenzymes were found to be similarly inhibited by monovalent anions (Cl-, I-, N3- and NO3-) 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.
Rumeau
D
D
CEA-CNRS, Département d'Ecophysiologie Végétale et Microbiologie, UMR-CNRS 163, Centre de Cadarache, Saint-Paul-lez Durance, France.
Cuiné
S
S
Fina
L
L
Gault
N
N
Nicole
M
M
Peltier
G
G
D000595:Amino Acid Sequence; D002256:Carbonic Anhydrases / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D002736:Chloroplasts / Q000201:enzymology; D003600:Cytosol / Q000201:enzymology; D015151:Immunoblotting; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism*; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D018515:Plant Leaves / Q000201:enzymology; D017386:Sequence Homology, Amino Acid; D011198:Solanum tuberosum / Q000201:enzymology*
D007527:Isoenzymes; D002256:Carbonic Anhydrases