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Carbonic anhydrase (CA) (EC 4.2.1.1) enzymes catalyze the reversible hydration of CO,, a reaction that is important in many physiological processes. We have cloned and sequenced a full length cDNA encoding an intracellular P-CA from the unicellular green alga Coccomyxa. Nucleotide sequence data show that the isolated cDNA contains an open reading frame encoding a polypeptide of 227 amino acids. The predicted polypeptide is similar to beta-type CAs from Escherichia coli and higher plants, with an identity of 26% to 30%. The Coccomyxa cDNA was overexpressed in E. coli, and the enzyme was purified and biochemically characterized. The mature protein is a homotetramer with an estimated molecular mass of 100 kD. The CO2-hydration activity of the Coccomyxa enzyme is comparable with that of the pea homolog. However, the activity of Coccomyxa CA is largely insensitive to oxidative conditions, in contrast to similar enzymes from most higher plants. Fractionation studies further showed that Coccomyxa CA is extrachloroplastic.

A cDNA coding for glutathione S-transferase (GST) was cloned and sequenced from the unicellular green algae Coccomyxa sp. The predicted 215 amino acid polypeptide (23.9 kDa, pI 5.3) is most similar to the theta-type GSTs found in a variety of different eukaryotic organisms. Within this sub-class, the Coccomyxa GST is 42% identical (63% similar) to the flatfish Pleuronectes platessa homologue, and 24 to 35% (49-57%) to other theta-type GST's.

An intracellular carbonic anhydrase (CA; EC 4.2.1.1) was purified to homogeneity from a mutant strain of Chlamydomonas reinhardtii (CW 92) lacking a cell wall. Intact cells were washed to remove periplasmic CA and were lysed and fractionated into soluble and membrane fractions by sedimentation. All of the CA activity sedimented with the membrane fraction and was dissociated by treatment with a buffer containing 200 mM KCI. Solubilized proteins were fractionated by ammonium sulfate precipitation, anionic exchange chromatography, and hydrophobic interaction chromatography. The resulting fraction had a specific activity of 1260 Wilbur-Anderson units/mg protein and was inhibited by acetazolamide (50% inhibition concentration, 12 nM). Final purification was accomplished by the specific absorption of the enzyme to a Centricon-10 microconcentrator filter. A single, 29.5-kD polypeptide was eluted from the filter with sodium dodecyl sulfate-polyacrylamide gel electrophoresis sample buffer, and a 1.5 M ammonium sulfate eluate contained CA activity. In comparison with human CA isoenzyme II, the N-terminal and internal amino acid sequences from the 29.5-kD polypeptide were 40% identical with the N-terminal region and 67% identical with an internal conserved region. Based on this evidence, we postulate that the 29.5-kD polypeptide is an internal CA in C. reinhardtii and that the enzyme is closely related to the alpha-type CAs observed in animal species.

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-CO2-adapted cells grown in media bubbled with air containing 400 or 70 muL . L-1 CO2. Evidence is presented indicating that cells grown at 70 muL . L-1 CO2 have a higher rate of photosynthetic O2 evolution than cells grown at 400 muL . L-1 CO2, 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 HCO3--dependent O2 evolution in cells grown at 400 and 70 muL . L-1 CO2. At 400 muM vanadate, inhibition reached 70-75 % in cells grown at 400 muL . L-1 but only 50 % in those grown at 70 muL . L-1 CO2. 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 muL . L-1 than in those grown at 400 muL . L-1 CO2. We conclude that the maximal capacity to accumulate dissolved inorganic carbon is inversely related to the CO2 concentration in the medium. Activation and - or synthesis of vanadate-sensitive ATPase may be the major explanation for the higher capacity for HCO3--dependent O2 evolution in cells grown under limited CO2 concentrations.