Search

An intracellular carbonic anhydrase (CA; EC 4.2.1.1) was purified and characterised from the unicellular green alga Coccomyxa sp. Initial studies showed that cultured Coccomyxa cells contain an intracellular CA activity around 100 times higher than that measured in high-CO-grown cells of Chlamydomonas reinhardtii CW 92. Purification of a protein extract containing the CA activity was carried out using ammonium-sulphate precipitation followed by anion-exchange chromatography. Proteins were then separated by native (non-dissociating) polyacrylamide gel electrophoresis, with each individual protein band excised and assayed for CA activity. Measurements revealed CA activity associated with two discrete protein bands with similar molecular masses of 80 +5 kDa. Dissociation by denaturing polyacrylamide gel electrophoresis showed that both proteins contained a single polypeptide of 26 kDa, suggesting that each 80-kDa native protein was a homogeneous trimer. Isoelectric focusing of the 80-kDa proteins also produced a single protein band at a pH of 6.5. Inhibition studies on the purified CA extract showed that 50% inhibition of CA activity was obtained using 1 μM azetazolamide. Polyclonal antibodies against the 26-kDa CA were produced and shown to have a high specific binding to a single polypeptide in soluble protein extracts from Coccomyxa cells. The same antiserum, however, failed to cross-react with soluble proteins isolated from two different species of green algae, Chlamydomonas reinhardtii and Chlorella vulgaris. Correspondingly, antisera directed against pea chloroplastic CA, extracellular CA from C. reinhardtii and human CAII, showed no cross-hybridisation to the 26-kDa polypeptide in Coccomyxa. The 26-kDa protein was confirmed as being a CA by N-terminal sequencing of two internal polypeptide fragments and alignment of these sequences with that of previously identified CA proteins from several different species. [ABSTRACT FROM AUTHOR]

We have assessed the effect of vanadate as an inhibitor of plasma-membrane ATPase on photosynthesis and the ATP/ADP ratio in Chlamydomonas reinhardtii CW-92 (a mutant strain lacking a cell wall). This effect was compared in low-CO-adapted cells grown in media bubbled with air containing 400 or 70 μL · L CO. Evidence is presented indicating that cells grown at 70 μL · L CO have a higher rate of photosynthetic O evolution than cells grown at 400 μL · L CO, at limiting carbon concentrations. Extracellular and intracellular carbonic-anhydrase activities were, however, similar in cells grown in both of the low-carbon conditions. Vanadate inhibited, to a different extent, the HCO-dependent O evolution in cells grown at 400 and 70 μL · L CO. At 400 μM vanadate, inhibition reached 70-75% in cells grown at 400 μL · L but only 50% in those grown at 70 μL · L CO. The ATP/ADP ratios determined with and without vanadate at limiting concentrations of dissolved inorganic carbon indicated that more ATP was hydrolysed in algae grown at 70 μL · L than in those grown at 400 μL · L CO. We conclude that the maximal capacity to accumulate dissolved inorganic carbon is inversely related to the CO concentration in the medium. Activation and - or synthesis of vanadate-sensitive ATPase may be the major explanation for the higher capacity for HCO-dependent O evolution in cells grown under limited CO concentrations. [ABSTRACT FROM AUTHOR]