Carbonic anhydrase in subchloroplast particles of pea plants

Abstract

The partitioning of carbonic anhydrase (CA) activity in chloroplasts isolated from 10–14-day-old pea (Pisum sativum L.) seedlings was investigated. The effect of CA inhibitors on the kinetics of chlorophyll fluorescence in photosystem II (PSII) preparations was also studied. The activity of CA was detected in fractions of soluble proteins and in the polypeptide complexes of the PSI and PSII. Isolated particles of photosystems retained a high photochemical activity similar to that of intact chloroplasts and the high level of polyunsaturated fatty acids. The association of CA with the particles of PSII (PSII-CA) was also tested by Western-blot analysis using antibodies against PSII-CA (Cah3) from Chlamydomonas reinhardtii. The PSII particles isolated with Triton X-100 (T-20) showed a higher activity of the enzyme as calculated on a protein basis than the DT-20 particles isolated with digitonin and Triton X-100. This difference seems to be related to the higher degree of nativity of the chloroplast T-20 fragments as compared to DT-20 particles. The higher level of chlorophyll per reaction center as well as the higher content of chlorophyll b and lipid fatty acids as calculated on protein basis, in particular of E-16:1ω13 acid, which stabilizes the oligomeric structure of the light-harvesting complex of the PSII, also confirms this suggestion. The activity of CA was not detected in the DT-20 preparations treated with Tris–HCl to eliminate manganese ions. This is likely to indicate that one of the extrinsic polypeptides of PSII exhibits CA activity. Specific inhibitors of CA (acetazolamide and imidazole) inhibited the photoinduced yield of chlorophyll fluorescence (ΔF). This might be determined by damaging the water-oxidizing system or its interaction with the PSII reaction centers. The functional role of PSII-CA for СО2-concentrating in carboxylation sites as well as its role in the coupling of light and dark reactions in chloroplasts is discussed.