Peroxisomes: Methods for Preparation

Abstract

Peroxisomes are ubiquitous cell organelles particularly enriched in the mammalian liver and kidney. They contain oxidases producing hydrogen peroxide and catalase, which degrades it. The majority of 60 enzymes, so far, localised to mammalian peroxisomes is involved in lipid metabolism. However, they catalyse via β‐oxidation the degradation of saturated and unsaturated long and very long‐chain fatty acids and fatty acid derivatives including complex lipids, such as bile acids, leucotrienes and prostaglandins. On the other, they are engaged in the biosynthesis of lipids, such as polyunsaturated fatty acids, ether glycerolipids and polyisoprenoids. The essential metabolic role of peroxisomes in humans is shown quite plainly by inherited disorders, the so‐called peroxisomopathies, which are partly associated with severe peroxisomal dysfunctions. Commonly, peroxisomes are isolated by classical density gradient centrifugation. Meanwhile, however, advanced alternatives have been established, for example, immunochemical techniques as well as zonal free‐flow electrophoresis.

Key Concepts:

  • Peroxisomes are multipurpose organelles playing a pivotal metabolic role in humans as is revealed by a variety of inherited peroxisomal disorders.

  • They display a remarkable plasticity in both morphology and biochemistry not only between species and tissues but also regarding different regions of the same organ.

  • In mammalian and wild‐type yeast cells, peroxisomes are considered commonly to multiply by growth and division and usually do not form de novo. There is, however, growing evidence that peroxisomes can also arise de novo from the ER, and even new perspectives are provided on the potential contribution of the mitochondria to peroxisomal biogenesis.

  • The overall heterogeneity of peroxisomes and the consistency in the buoyant density of the subpopulations with other cell organelles hamper their purification by classic cell fractionation methods, inevitably calling for adequate techniques based upon alternative physicochemical traits.

Keywords: density gradient centrifugation; immunomagnetic sorting; immune free‐flow electrophoresis; zonal free‐flow electrophoresis; mammalian peroxisomes; liver; rodents

Figure 1.

Flowchart of the procedure used for subfractionating tissue and cell homogenates and isolating highly (>98%) purified peroxisomes. Note that as alternatives to density gradient centrifugation, the stippled fractions also provide the basis for the purification of peroxisomes by immunomagnetic sorting and immune FFE. The values given for the integrated relative centrifugal force (IRCF) imply that the g force in this case is not constant during the centrifugation procedure, due to acceleration and deceleration times. In ultracentrifuges with an advanced design (e.g. Beckman), the IRCF values can be exactly preset on the instrument panel.

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Further Reading

Nissum M, Kuhfuss S, Hauptmann M et al. (2007) Two‐dimensional separation of human plasma proteins using iterative free‐flow electrophoresis. Proteomics 7: 4218–4227.

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Islinger, Markus, Fahimi, H Dariush, and Völkl, Alfred(Feb 2014) Peroxisomes: Methods for Preparation. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0002594.pub3]