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Isolation and Purification of Intact Peroxisomes from Green Leaf Tissue ¹

Department of Horticulture, University of Wisconsin, Madison, Wisconsin 53706, Department of Botany, University of Wisconsin, Madison, Wisconsin 53706, Department of Botany, Washington State University, Pullman, Washington 99164-4230

Intact peroxisomes were prepared from green leaves of a number of C₃ species, both monocots and dicots. A protoplast extract from which chloroplasts have been removed by a 1-minute 10,000g centrifugation was applied to a step gradient of 5, 15, 30, and 45% Percoll containing 0.5 molar sucrose, 0.1% BSA, and 25 millimolar Hepes-KOH (pH 7.5). After centrifugation, a peroxisomal fraction with low contamination by chloroplastic and mitochondrial markers was recovered from the 30/45% Percoll interface. This fraction was passed through a Sepharose 2B column to remove the Percoll which resulted in a peroxisomal preparation exhibiting high intactness (estimated from enzyme latency) and stability.

¹ Supported by the Science and Education Administration of the United States Department of Agriculture under Grant 59-2531-01-1-516-0 from the Competitive Research Grants Office, and by the College of Agriculture and Life Sciences, University of Wisconsin, Madison, Wisconsin.