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Articles

Cellular Fractionation of Chlamydomonas reinhardii with Emphasis on the Isolation of the Chloroplast ¹

Institute for Photobiology of Cells and Organelles, Brandeis University, Waltham, Massachusetts 02254, Department of Botany and Plant Sciences, University of California, Riverside, California 92521

A method for cellular fractionation of Chlamydomonas reinhardii, SAG 11-32/b, and isolation of intact chloroplasts from synchronized cells of the alga is described. The procedure for cell fractionation comprises essentially four steps: (1) protoplast production with autolysine; (2) lysis of the protoplasts with digitonin; (3) aggregation of broken protoplasts; and (4) separation of organelles by differential centrifugations.

Replacing the differential centrifugations (step 4) by Percoll cushion centrifugations yields intact chloroplasts. Starting with 100 milliliters of an algal culture containing 3000 micrograms chlorophyll, intact chloroplasts with 100 to 200 micrograms of chlorophyll can be isolated. Envelope integrity is about 90% (ferricyanide assay). Examination of the chloroplasts by electron microscopy and marker enzyme activities indicated some mitochondrial and cytoplasmic contamination.

³ Supported by a grant from the Ministry of Education, People's Republic of China. Permanent address: Department of Biology, Hunan Teachers College, Changsha, People's Republic of China.

¹ Supported by Department of Energy DE-AC02-76-ER03231 and National Science Foundation PCM 79-22612.

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