PURIFICATION AND CHARACTERIZATION OF AN INTRACELLULAR CARBONIC-ANHYDRASE FROM THE UNICELLULAR GREEN-ALGA COCCOMYXA

An intracellular carbonic anhydrase (CA; EC 4.2.1.1) was purified and characterised from the unicellular green alga Coccomyxa sp. Initial st udies showed that cultured Coccomyxa cells contain an intracellular CA activity around 100 times higher than that measured in high-CO2-grown cells of Chlamydomonas reinhardtii CW 92. Purification of a protein e xtract containing the CA activity was carried out using ammonium-sulph ate precipitation followed by anion-exchange chromatography. Proteins were then separated by native (non-dissociating) polyacrylamide gel el ectrophoresis, with each individual protein band excised and assayed f or 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 t hat 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 a t a pH of 6.5. Inhibition studies on the purified CA extract showed th at 50% inhibition of CA activity was obtained using 1 mu M azetazolami de. Polyclonal antibodies against the 26-kDa CA were produced and show n to have a high specific binding to a single polypeptide in soluble p rotein extracts from Coccomyxa. cells. The same antiserum, however, fa iled to cross-react with soluble proteins isolated from two different species of green algae, Chlamydomonas reinhardtii and Chlorella vulgar is. Correspondingly, antisera directed against pea chloroplastic CA, e xtracellular CA from C. reinhardtii and human CAII, showed no cross-hy bridisation 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 pr eviously identified CA proteins from several different species.