Purification Of Peroxisomes Research Articles

Quantitative mass spectrometry reveals a role for the GTPase Rho1p in actin organization on the peroxisome membrane.

We have combined classical subcellular fractionation with large-scale quantitative mass spectrometry to identify proteins that enrich specifically with peroxisomes of Saccharomyces cerevisiae. In two complementary experiments, isotope-coded affinity tags and tandem mass spectrometry were used to quantify the relative enrichment of proteins during the purification of peroxisomes. Mathematical modeling of the data from 306 quantified proteins led to a prioritized list of 70 candidates whose enrichment scores indicated a high likelihood of them being peroxisomal. Among these proteins, eight novel peroxisome-associated proteins were identified. The top novel peroxisomal candidate was the small GTPase Rho1p. Although Rho1p has been shown to be tethered to membranes of the secretory pathway, we show that it is specifically recruited to peroxisomes upon their induction in a process dependent on its interaction with the peroxisome membrane protein Pex25p. Rho1p regulates the assembly state of actin on the peroxisome membrane, thereby controlling peroxisome membrane dynamics and biogenesis.

Purification of peroxisomes in a preformed iodixanol gradient in a fixed-angle rotor.

In iodixanol, peroxisomes are the densest organelle in the light mitochondrial fraction and are therefore easily separated from the other components (lysosomes, mitochondria, etc.) in a preformed isosmotic continuous gradient. The resolution of the peroxisomes is far superior than that in sucrose and, unlike in Percoll® there is no contamination from endoplasmic reticulum.

Purification of peroxisomes using a density barrier in a swinging-bucket rotor.

In iodixanol, peroxisomes are the densest organelle in the light mitochondrial fraction and are therefore easily separated from the other components (lysosomes, mitochondria, etc.) in a preformed isosmotic continuous gradient. Because of the large difference in density between peroxisomes and the next densest organelle (mitochondria), a density barrier is effective. The resolution of the peroxisomes is far superior than that in sucrose and, unlike in Percoll®, there is no contamination from endoplasmic reticulum.

Purification of peroxisomes in a self-generated gradient.

In iodixanol, peroxisomes are the densest organelle in the light mitochondrial fraction and are therefore easily separated from the other components (lysosomes, mitochondria, etc.) in a self-generated gradient. Self-generated gradients make sample handling very easy and are highly reproducible but need to be formed in either a vertical, near-vertical, or small volume high-performance fixed-angle rotor. The resolution of the peroxisomes is far superior than that in sucrose and, unlike in PercollⓇ there is no contamination from endoplasmic reticulum.

Peroxisomes in molluscs, characterization by subcellular fractionation combined with western blotting, immunohistochemistry, and immunocytochemistry.

Peroxisomes of the digestive glands of mussels, Mytilus galloprovincialis Lmk, were investigated by immunoblotting and immunohistochemistry using rabbit antibodies against several mammalian hepatic peroxisomal proteins. Western blot analysis of main subcellular fractions revealed immunoreactive polypeptides with molecular weights comparable to those of the corresponding mammalian hepatic proteins. They could be localized to the peroxisomal matrix in the case of catalase, multifunctional enzyme (PH), and palmitoyl-CoA oxidase (AOX), and to the peroxisomal membrane in respect to PMP 70. The purification of peroxisomes by metrizamide density gradient centrifugation revealed the existence of two subpopulations with densities of 1.16 and 1.20 g cm(-3) exhibiting different protein compositions. In paraffin sections, positive immunolabeling for catalase was distributed along the apical cytoplasm of the epithelia of digestive ducts and stomach and throughout the cytoplasm of digestive tubule cells. The peroxisomal beta-oxidation enzymes, AOX and PH, also appeared predominantly in the ducts and the stomach epithelia with a weaker immunolabeling in the tubules. At the electron microscopic level a clear labeling with gold particles was observed in the peroxisomal matrix with the anti-guinea pig catalase antibody. In addition to peroxisomes, the anti-PH antibody also labeled the mitochondria. The similarity in the protein composition of molluscan and mammalian peroxisomes as revealed by the present study indicates that those proteins have been well conserved in evolution suggesting that functionally peroxisomes in molluscs could also be involved in the metabolism of lipids and in detoxification of xenobiotics. Thus, the antibodies tested could provide useful tools for detection of peroxisomal induction in molluscan biomonitoring programs for the assessment of aquatic environmental pollution.

Purification of peroxisomes and subcellular distribution of enzyme activities for activation and oxidation of very-long-chain fatty acids in rat brain

Brain contains high amounts of very-long-chain (VLC) fatty acids (> C22). Since mitochondria from liver and skin fibroblasts lack lignoceroyl-CoA ligase, in liver and skin fibroblasts fatty acids are exclusively oxidized in peroxisomes. Findings by Poulos and associates [9]suggested that contrary to liver and cultured skin fibroblasts brain mitochondria contain lignoceroyl-CoA ligase and can oxidize lignoceric acid. The present study was undertaken to develop a procedure for the isolation of subcellular organelles of higher purity from brain and to get a better understanding of the subcellular localization of the oxidation of VLC fatty acids in brain. The enzyme activities for activation and oxidation of palmitic and lignoceric acids were determined in peroxisomes, mitochondria, microsomes and a myelin fraction from rat brain and peroxisomes, mitochondria and microsomes purified from rat liver. Like in liver, brain lignoceroyl-CoA ligase activity in microsomes and peroxisomes was approx. 9 times higher than in mitochondria. In addition to palmitoyl-CoA ligase the antibodies against palmitoyl-CoA ligase inhibited the residual mitochondrial lignoceroyl-CoA ligase activity, meaning that lignoceroyl-CoA ligase activity in mitochondria was derived from palmitoyl-CoA ligase. Accordingly, in peroxisomes lignoceric acid was oxidized at 7 times higher rate than in mitochondria. Mitochondria were able to oxidize lignoceric acid efficiently when supplemented with lignoceroyl-CoA ligase activity from microsomes or myelin. These results show that in brain lignoceric acid is oxidized in peroxisomes and that lignoceroyl-CoA ligase activity is localized in peroxisomes and microsomes, but not in mitochondria. Peroxisomes and microsomes contain both lignoceroyl-CoA and palmitoyl-CoA ligases. Similar to peroxisomes and microsomes, the antibodies against palmitoyl-CoA ligase inhibited only the palmitoyl-CoA ligase activity in myelin but not the lignoceroyl-CoA ligase activity. These results suggest that in addition to palmitoyl-CoA ligase, myelin also contaies lignoceroyl-CoA ligase.

Purification of peroxisomes from livers of normal and clofibrate-treated mice

A method for the isolation of peroxisomes from livers of normal and clofibrate-treated mice is described. The method utilizes glutaraldehyde to stabilize peroxisomal membranes, and isopyenic centrifugation of a light mitochondrial fraction through a linear metrizamide gradient to achieve optimal resolution from other organelles. On the basis of the biochemical and morphological data, the peroxisomal preparations are indicated as of high purity: contamination by mitochondria, lysosomes, and plasma membranes is negligible, and the level of contaminating microsomes is around 5% for normal peroxisomes and 8% for peroxisomes from clofibratetreated mice. Peroxisomal membranes prepared by carbonate extraction contain two major polypeptides of approximately 70,000 Da, and show 2 and 8% contamination by microsomal membrane protein for the preparations from normal and clofibrate-treated mice, respectively.

The localization of serine hydroxymethyltransferase in leaves of C3 and C4 species

The intracellular distribution of serine hydroxymethyltransferase (EC 2.1.2.1) was studied in young wheat (Triticum aestivum L. cv. Starke II) leaves by fractionation of protoplasts and further purification of peroxisomes and chloroplasts. Essentially all of the activity in wheat leaves was located in the mitochondria. Within the mitochondria the enzyme was mainly in the matrix as shown by centrifugation of sonicated wheat mitochondria. In the C4 plants, Zea mays (L. cv. Earliking), Panicum miliaceum and Panicum maximum (cv. Australia) belonging to different C4 types, serine hydroxymethyltransferase was almost exclusively found in bundle sheath cells. The location of this enzyme in leaves is consistent with its role relative to glycine decarboxylation during photorespiration.

Purification of Peroxisomes and Mitochondria from Spinach Leaf by Percoll Gradient Centrifugation

A procedure was developed to purify simultaneously peroxisomes and mitochondria from spinach (Spinacia oleracea L.) leaf under isoosmotic and low viscosity conditions. This method involved differential centrifugation and density gradient centrifugation on four layers of Percoll. Chlorophyll-free preparations of highly intact and active organelles were obtained and cross-contamination was negligible. Both organelles were stable for several hours, even if they remained in Percoll. Purified mitochondria were able to carry out the oxidation of different substrates with excellent respiratory control and ADP:O ratios. The method described in the present work was also suitable to purify mitochondria and peroxisomes from potato (Solanum tuberosum L.) tubers.