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Phosphoglycolate phosphatase (PGPase), a key en¬zyme of photorespiration in photosynthetic organisms, was purified from Chlamydomonas reinhardtii. The en¬zyme was an ~65-kDa homodimer with a pI value of 5.1 composed of ~32-kDa subunits not connected by any S–S bridges. It was also highly specific for phosphoglycolate with a Km value of 140 μM and an optimal pH between 8 and 9. The activity was strongly inhibited by CaCl2, and it recovered competitively following the addition of MgCl2 or EGTA. A mobility shift was observed in SDS-polyacrylamide gel electrophoresis by the addition of CaCl2, indicating that the enzyme binds to Ca2+ . The N-terminal region of amino acid sequence deduced from cDNA sequence that was not contained in the purified PGPase had similar characteristics to those of typical stroma-targeting transit peptides in C. reinhardtii. The following region of the deduced sequence containing 302 amino acid residues was similar to p-nitrophe¬nylphosphatase-like proteins, although the purified PG-Pase did not hydrolyze p-nitrophenylphosphate. Genomic DNA fragments from wild type containing the sequence homologous to the cDNA for PGPase comple¬mented the PGPase-deficient mutant pgp1. Possible reg¬ulatory mechanisms during adaptation to limiting CO2 were discussed based on the characteristics of the puri¬fied PGPase and the deduced amino acid sequence.