Biology Reference
In-Depth Information
Chapter 36
Isolation of Leaf Peroxisomes from Arabidopsis
for Organelle Proteome Analyses
Sigrun Reumann and Rajneesh Singhal
Abstract
The isolation of cell organelles from model organisms in high purity is important for biochemical analyses
of single proteins, entire metabolic pathways, and protein complexes and is absolutely essential for organ-
elle proteome analyses. The effi cient enrichment of nearly all cell organelles is more diffi cult from
Arabidopsis as compared to traditional model plants and especially challenging for peroxisomes. Leaf per-
oxisomes are generally very instable in aqueous solution due to the presence of a single membrane and
(para-)crystalline inclusions in the matrix. Leaf peroxisomes from Arabidopsis are particularly fragile and,
moreover, strongly physically adhere to chloroplasts and mitochondria for largely unknown reasons. Here,
we provide a detailed protocol for the isolation of Arabidopsis leaf peroxisomes by Percoll followed by
sucrose density gradient centrifugation that yields high purity suitable for proteome analyses. Diverse
enzymatic and immuno-biochemical methods are summarized to assess purity and intactness.
Key words
Peroxisome, Arabidopsis, Organelle purity, Proteomics
Abbreviations
CE Crude extract
FW Fresh weight
GB Grinding buffer
HPR Hydroxypyruvate reductase
LP-P1/2 First/second purifi ed leaf peroxisome fraction
TE
Tricine-EDTA
1
Introduction
Our knowledge of the metabolic and regulatory functions of
plant peroxisomes is far from complete because fundamental
research is made diffi cult by (1) high peroxisome fragility, (2) low
peroxisome abundance in most tissues and organs, (3) pronounced
peroxisome adherence to plastids and mitochondria, particularly in
