Isolation of Glyoxysomes from Pumpkin Cotyledons

Nicola Harrison‐Lowe1, Laura J. Olsen1

1 University of Michigan, Ann Arbor, Michigan
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 3.19
DOI:  10.1002/0471143030.cb0319s29
Online Posting Date:  January, 2006
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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.

Keywords: glyoxysomes; peroxisomes; protein import

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Table of Contents

  • Basic Protocol 1: Isolation of Glyoxysomes
  • Support Protocol 1: Growing Seedlings for Glyoxysome Isolation
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Isolation of Glyoxysomes

  • 5‐ to 7‐day, dark‐grown pumpkin seedlings ( protocol 2)
  • 1× grinding buffer working solution (see recipe)
  • 2 M sucrose
  • 28% (v/v) Percoll/resuspension buffer solution (see recipe)
  • 1× resuspension buffer working solution (see recipe)
  • 250‐ml beakers
  • Balance accurate to 0.1 g
  • Waring blender (2‐speed) and 500‐ml blender cup, precooled to 4°C
  • Miracloth (Calbiochem)
  • Glass funnel, precooled to 4°C
  • 50‐ml round‐bottom polypropylene centrifuge tubes
  • Sorvall RC5C centrifuge with HB‐6 swinging‐bucket rotor (or equivalent refrigerated centrifuge and rotor)
  • 15‐ml Corex tubes, cooled to 4°C
  • Small, soft paintbrush (natural hair)
  • UV spectrophotometer
NOTE: Keep all equipment and all solutions cold (4°C) for the duration of the procedure.

Support Protocol 1: Growing Seedlings for Glyoxysome Isolation

  • Pumpkin seeds (or other plant seeds depending on desired organelle source)
  • Medium‐coarse vermiculite (available from gardening supply stores)
  • Flat for germination (available from gardening supply stores)
  • Plastic wrap
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Literature Cited

Literature Cited
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