An intracellular carbonic anhydrase (CA; EC 4.2.1 .I) was purified to homogeneity from a mutant strain of Chlamydomonas reinhardtii (CW 92) lacking a cell wall. lntact 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. l h e resulting fraction had a specific activity of 1260 WilburAnderson 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. I n 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 i s an internal CA in C. reinhardtii and that the enzyme is closely related to the a-type CAs observed i n animal species.

Carbonic anhydrase (CA) (EC 4.2.1.1) enzymes catalyze the reversible hydration of CO 2 , a reaction that is important in many physiological processes. We have cloned and sequenced a fulllength cDNA encoding an intracellular ␤-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 ␤-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 CO 2 -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.

The existence of an oxygen dependent pH-gradient across the thylakoid membrane in the dark led to the hypothesis of a chlororespiration in Pleurochloris meirmgensis. Measuring flash-induced absorbanee changes at different wavelength and mathematical fitting of the transient spectra absorbance changes around 515 nm were attributed to an electrochromic shit~ in these chl-b and hitein-less organisms. Adding 2 mM KCN increased the slow phase of the electrochromic absorbance change. The enhanced activity of the cytochrome (cyt) f due to KCN was further demonstrated by the absorbance changes at 553 nm, where a stronger oxidation of the cyt as well as an accelerated reduction became visible favouring the idea of an oxidase competing with the cytf for electrons. The KCN-coneentration needed to induce this effect was 0.25 mM for half saturation, wheareas mitochondrial respiration was completely blocked adding 0.1 raM. Therefore we conclude that there is a KCN-sensitive oxidase located in the thylakoids ofP. meiringensis.