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Anaerobic Formation of D-Lactate and Partial Purification and Characterization of a Pyruvate Reductase from Chlamydomonas reinhardtii¹

Department of Biochemistry, Michigan State University, East Lansing, Michigan 48824

D-Lactate accumulation in Chlamydomonas reinhardtii was dependent on anaerobic conditions. As much as 50% of the ¹⁴C after 2 minutes of photosynthetic ¹⁴CO₂ fixation moved into D-lactate from sugar phosphates if the cells became anaerobic for short time periods. No lactate accumulated in the dark until the O₂ concentration decreased to less than 0.1%. Lactate was determined to be of the D-configuration using stereospecific lactate dehydrogenases. D-Lactate produced anaerobically by algae grown on 5% CO₂ was only slowly metabolized aerobically in the light or dark, and in the dark, only a trace of the lactate was excreted.

A pyruvate reductase (D-lactate: diphosphopyridine nucleotide oxidoreductase, EC 1.1.1.28) was partially purified 47-fold from Chlamydomonas. Because this enzyme catalyzes an essentially irreversible reaction in the direction of pyruvate reduction, it is considered to be a pyruvate reductase. The reductase activity in extracts of Chlamydomonas was 30 micromoles per hour per milligram chlorophyll. For the partially purified enzyme, the apparent K_m (pyruvate) was 0.5 millimolar, and the pH optimum was 7.0. Studies with cycloheximide and chloramphenicol indicated that the enzyme was constitutive in aerobic cells. Potassium phosphate stimulated the reductase, and high salt and dithiothreitol were required for stability. The enzyme demonstrated substrate inhibition and was inhibited by ATP. Pyruvate reductase was separated from a hydroxypyruvate reductase by gel filtration chromatography, indicating the presence of separate reductases for these two substrates in Chlamydomonas.