Isolation of Glyoxysomes from Pumpkin Cotyledons

Peroxisomes are single‐membrane‐bound organelles found in virtually all eukaryotes. In plants, there are several classes of peroxisomes. Glyoxysomes are found in germinating seedlings and contain enzymes specific for the glyoxylate cycle, including isocitrate lyase and malate synthase. After seedlings become photosynthetic, leaf peroxisomes participate in reactions of the photorespiration pathway and contain characteristic enzymes such as glycolate oxidase and hydroxypyruvate reductase. As leaves begin to senesce, leaf peroxisomes are transformed back into glyoxysomes. Root peroxisomes in the nodules of legumes, for example, sequester enzymes such as allantoinase and uricase, which contribute to nitrogen metabolism in these tissues. Thus, peroxisomes participate in many metabolic pathways and contain specific enzyme complements, depending on the tissue source. All peroxisomes contain catalase to degrade hydrogen peroxide and enzymes to accomplish β‐oxidation of fatty acids. Glyoxysomes can be isolated from pumpkin cotyledons by standard differential centrifugation and density separation, as described in this article.