Carbonic anhydrase isolation and characterization from the marine diatom Phaeodactylum tricornutum

The carbonic anhydrase (CA) isozymes have been identified and isolated in three strains of Phaeodactylum tricornutum (UTEX 640, B31 and 1052/1A). External and internal CA activity was detected by potentiometric assay of intact cells and cell homogenates of air and high CO₂-grown cells. External CA was detected for the UTEX 640 strain under both high CO ₂ and CO₂ limiting conditions, but the CA_ext activity was only present in trace amounts when cells were grown on high CO₂. Internal CA activity was detected potentiometrically in all three strains when grown under air and high CO₂ conditions. CA isozymes for all three strains were detected using cellulose acetate electrophoresis. The B31 and 1052/1A strains had two CA bands on cellulose acetate plates, while the UTEX 640 had three bands. The two bands in UTEX 640 corresponded to the two bands detected in B31 and 1052/1A and were the internal CA isozymes, while the fast running band present only in air-grown UTEX 640 was the external CA isozyme.; CA isozymes of UTEX 640 were isolated using 35% to 65% ammonium sulfate fractionation, and purified on DEAE-Sephacell and p-AMBS-Sepharose affinity columns. The CA isoforms were shown to have molecular weights of 28, 25 and 24 kDa on 15% SDS-PAGE. The larger CAs (28 & 25 kDa) were the internal CA isozymes, while the lighter CA (24 kDa) was the external isoform as indicated by comparison of air and high CO₂-grown cells. The CA isozymes were low-CO₂-inducible proteins, since expression was reduced when cells were grown on high CO₂ (5% CO₂) as opposed to air (0.034% CO₂). Polyclonal antibodies were raised in rabbits against the CA isoforms. Western blot analysis indicated that CA expression was repressed in high CO₂-grown cells. The CA and polyclonal antibody were unique to the Phaeodactylum tricornutum, since there was no apparent cross-reaction between the anti CA-antiserum and the CAs of two other algal species Isochrysis galbana and Chlorella saccharophila.; The anti-CA antiserum and subsequently immunogold labelling was used to detect the CA isozymes in ultra-thin mycrotom sections of air and high CO₂ cells. The CA_int was detected in the cytosol, mitochondria and the chloroplast, while the CA_ext was detected around the cell wall and cell membrane, suggesting a role for the external CA in the CCM.