ISSN:
1432-2048
Keywords:
Carbonic anhydrase purification
;
Intracellular compartmentation
;
Solanum
Source:
Springer Online Journal Archives 1860-2000
Topics:
Biology
Notes:
Abstract 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.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1007/BF00196884
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