Abstract
Peroxisomes are subcellular organelles bound by a single membrane. They are involved in a variety of metabolic processes such as β-oxidation of very long chain fatty acids. Isolated and purified peroxisomes are necessary to obtain a detailed understanding of their structure and function. Here, our three protocols for the isolation of peroxisomes from rat liver, rat hepatoma H4IIE cells and yeast Komagataella phaffii (previously called Pichia pastoris) are presented. Highly pure peroxisomes can be prepared from rat liver by differential centrifugation followed by Nycodenz gradient centrifugation. It is difficult to prepare highly purified peroxisomes from cultured mammalian cells, but the subcellular fractionation of peroxisomes is still a powerful tool to analyze whether a certain protein is localized to peroxisomes. Peroxisomes are potently induced in methylotrophic yeast such as K. phaffii. A large amount of mammalian peroxisomal proteins can be expressed in cells under the control of the alcohol oxidase gene promoter in a medium containing methanol as the only carbon source. Therefore, purified peroxisomes are useful for characterizing mammalian peroxisomal proteins. The protocols for the isolation of peroxisomes by immuno-beads and peroxisomal membranes by a sodium carbonate procedure are also presented.
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- 1.
Centrifugation conditions are express in integrate form; g-min, rather than in the conventional terms of at a × g for b min in the original paper (Leighton et al. 1968). The article considers the force during acceleration and deceleration, and determines the centrifugation condition (g-min) using the following formula; g-min = <InlineEquation ID="IEq1">dt × Rav/981 × 60. Rav is the distance to the middle of the tube of the fluid during centrifugation. In this paper, conventionally, the determination of the centrifugation conditions is made from the plateau speed and the centrifugation time from the starting point as “a × gav for b min”. In this paper, gav is expressed simply as g. The flowchart for the isolation of peroxisomes is shown in Fig. 9.1.
Abbreviations
- ER:
-
Endoplasmic reticulum
- PMP:
-
Peroxisomal membrane protein
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The publication is supported in part by a Grant-in-Aid for Intractable Diseases from the Ministry of Health, Labour and Welfare of Japan. Pacific Edit reviewed the manuscript prior to submission.
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Imanaka, T. (2019). The Isolation of Peroxisomes. In: Imanaka, T., Shimozawa, N. (eds) Peroxisomes: Biogenesis, Function, and Role in Human Disease. Springer, Singapore. https://doi.org/10.1007/978-981-15-1169-1_9
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DOI: https://doi.org/10.1007/978-981-15-1169-1_9
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