INTRACELLULAR CARBONIC-ANHYDRASE OF CHLAMYDOMONAS-REINHARDTII

An intracellular carbonic anhydrase (CA; EC 4.2.1.1) was purified to h omogeneity from a mutant strain of Chlamydomonas reinhardtii (CW 92) l acking 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 fr action and was dissociated by treatment with a buffer containing 200 m M KCI. Solubilized proteins were fractionated by ammonium sulfate prec ipitation, anionic exchange chromatography, and hydrophobic interactio n chromatography. The resulting fraction had a specific activity of 12 60 Wilbur-Anderson units/mg protein and was inhibited by acetazolamide (50% inhibition concentration, 12 nM). Final purification was accompl ished by the specific absorption of the enzyme to a Centricon-10 micro concentrator 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 activ ity. In comparison with human CA isoenzyme II, the N-terminal and inte rnal amino acid sequences from the 29.5-kD polypeptide were 40% identi cal with the N-terminal region and 67% identical with an internal cons erved region. Based on this evidence, we postulate that the 29.5-kD po lypeptide is an internal CA in C. reinhardtii and that the enzyme is c losely related to the alpha-type CAs observed in animal species.