Abstract
Abstract Microbodies, designated as peroxisomes because of their enzyme complement, have been isolated from spinach leaves. After grinding leaves in 0.5 m sucrose, the peroxisomes were removed with the broken chloroplast fraction by differential centrifugation. During sucrose density gradient centrifugation, the peroxisomes banded in about 1.9 m sucrose and were separated from mitochondria and chloroplasts. The particles, 0.5 to 1.0 µ in diameter, contained a dense granular stroma surrounded by a single membrane. The leaf peroxisomes contained glycolate oxidase, DPNH-glyoxylate reductase, and catalase. Up to 55% of the activity for these enzymes in spinach leaves have been found in the particulate fractions after the initial centrifugation. The leaf peroxisomes are probably the site of oxygen uptake during photorespiration. No catalase activity was present in chloroplasts after removal of the peroxisomes by density gradient centrifugation. P-Glycolate phosphatase, TPNH-glyoxylate reductase, d-amino acid oxidase, urate oxidase, and peroxidase were not present in leaf peroxisomes.
Highlights
MethodsThe chilled leaves were washed and deribbed, and 40 g of tissue were chopped into small segments before grinding at maximum speed for 10 set in a Waring Blendor with 80 ml of grinding medium
After grinding leaves in 0.5 M sucrose, the peroxisomes were removed with the broken chloroplast fraction by differential centrifugation
Up to 55 % of the activity for these enzymes in spinach leaves have been found in the particulate fractions after the initial centrifugation
Summary
The chilled leaves were washed and deribbed, and 40 g of tissue were chopped into small segments before grinding at maximum speed for 10 set in a Waring Blendor with 80 ml of grinding medium. The homogenate was hand squeezed through six layers of cheesecloth, and the pH of the sap immediately readjusted to 7.5. Particles from 70 ml of this sap were precipitated by differential centrifugation at 0” for 20 min at each step. The second pellet (3,000 x g) was designated as “broken chloroplasts,” it contained some mitochondria and a large part of the peroxisomes. The third pellet (35,000 x g), designated as “mitochondria” contained the remaining microbodies and broken chloroplasts.
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