SUBCELLULAR-DISTRIBUTION OF CARBONIC-ANHYDRASE IN SOLANUM-TUBEROSUM LLEAVES - CHARACTERIZATION OF 2 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 bicar bonate, has been investigated in potato (Solanum tuberosum L.) leaves. Although enzyme activity was mainly located in chloroplasts (87% of t otal cellular activity), significant activity (13%) was also found in the cytosol. The corresponding CA isoforms were purified either from c hloroplasts or crude leaf extracts, respectively. The cytosolic isoenz yme has a molecular mass of 255 000 and is composed of eight identical subunits with an estimated M(r) of 30 000. The chloroplastic isoenzym e (M(r) 220 000) is also an octamer composed of two different subunits with M(r) estimated at 27 000 and 27 500, respectively. The N-termina l 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 a nd chloroplastic CA isoenzymes were found to be similarly inhibited by monovalent anions (Cl-,I-, N-3(-) and NO3-) and by sulfonamides (etho xyzolamide and acetozolamide). Both CA isoforms were found to be depen dent on a reducing agent such as cysteine or dithiothreitol in order t o retain the catalytic activity, but 2-mercaptoethanol was found to be a potent inhibitor. A polyclonal antibody directed against a syntheti c peptide corresponding to the N-terminal amino acid sequence of the c hloroplastic CA monomers also recognized the cytosolic CA isoform. Thi s antibody was used for immunocytolocalization experiments which confi rmed the intracellular compartmentation of CA: within chloroplasts, CA is restricted to the stroma and appears randomly distributed in the c ytosol.