Rapid separation of the chloroplast and cytoplasmic fractions from intact leaf protoplasts

Intact protoplasts are ruptured by rapid centrifugation through a narrow-aperture nylon mesh and the intact chloroplasts are then separated from the cytoplasm by sedimentation through a layer of silicone oil below the mesh. Within 6 to 8 s of starting the centrifuge, 90% of the chloroplasts are separated into the pellet fraction which contains only 10 to 15% contamination by mitochondria and peroxisomes and less than 5% contamination by soluble cytoplasm as judged by the distribution of marker enzymes. This technique should allow determination of the distribution of metabolites between the chloroplast and cytoplasmic compartments of intact protoplasts.