A method for isolating intact chloroplasts from Chlamydomonas reinhardtii F-60 was developed from the Klein, Chen, Gibbs, Platt-Aloia procedure ( Plant Physiol 72: 481-487). Protoplasts, generated by treatment with autolysine, were lysed with a solution of digitonin and fractionated on Percoll step gradients. The chloroplasts were assessed to be 90% intact (ferricyanide assay) and free from cytoplasmic contamination (NADP isocitrate dehydrogenase activity) and to range from 2 to 5% in mitochondrial contamination (cytochrome c oxidase activity). About 25% of the cellular succinate dehydrogenase activity (21.6 micromoles per milligram chlorophyll per hour, as determined enzymically) was placed within the chloroplast. Chloroplastic succinate dehydrogenase had a Km for succinate of 0.55 millimolar and was associated with the thylakoidal material derived from the intact chloroplasts. This same thylakoidal material, with an enzymic assay of 21.6 micromoles per milligram chlorophyll per hour was able to initiate a light-dependent uptake of oxygen at a rate of 16.4 micromoles per milligram chlorophyll per hour when supplied with succinate and methyl viologen. Malonate was an apparent competitive inhibitor of this reaction. The succinate dehydrogenase activity present in the chloroplast was sufficient to account for the photoanaerobic rate of acetate dissimilation in H2 adapted Chlamydomonas (M Gibbs, RP Gfeller, C Chen  Plant Physiol 82: 160-166).
ASJC Scopus subject areas
- Plant Science