The rapid preparation of peroxisomes from rat liver by using a vertical rotor.
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ABSTRACT:
SUBMITTER: Neat CE
PROVIDER: S-EPMC1161073 | biostudies-other | 1979 May
REPOSITORIES: biostudies-other
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Project description:1. Rat liver peroxisomal fractions were isolated in iso-osmotic Percoll gradients by using vertical-rotor centrifugation. The fractions obtained with rats given various dietary treatments were characterized. 2. The effect on peroxisomal beta-oxidation of feeding 15% by wt. of dietary fat for 3 weeks was investigated. High-fat diets caused induction of peroxisomal beta-oxidation, but diets rich in very-long-chain mono-unsaturated fatty acids produced a more marked induction. 3. Peroxisomal beta-oxidation induced by diets rich in very-long-chain mono-unsaturated fatty acids can oxidize such acids. Trans-isomers of mono-unsaturated fatty acids are oxidized at rates that are faster than, or similar to, those obtained with corresponding cis-isomers. 4. Rates of oxidation of [14-14C]erucic acid by isolated rat hepatocytes isolated from rats fed on high-fat diets increased with the time on those diets in a fashion very similar to that previously reported for peroxisomal beta-oxidation [see Neat, Thomassen & Osmundsen (1980) Biochem, J. 186, 369-371]. 5. Total liver capacities for peroxisomal beta-oxidation (expressed as acetyl groups produced per min) were estimated to range from 10 to 30% of mitochondrial capacities, depending on dietary treatment and fatty acid substrate. A role is proposed for peroxisomal beta-oxidation in relation to the metabolism of fatty acids that are poorly oxidized by mitochondrial beta-oxidation, and, in general, as regards oxidation of fatty acids during periods of sustained high hepatic influx of fatty acids.
| S-EPMC1162977 | biostudies-other
Project description:A technique for the rapid preparation of mitochondria from rat liver is described. Tissue fractionation is performed by a single centrifugation step with a discontinuous Percoll density gradient. Total preparation times of 5--6 min are achieved by using this method. The mitochondrial fraction obtained is relatively free of contaminating organelles, as judged by marker-enzyme activity determinations. Mitochondria isolated by Percoll-density-gradient centrifugation differ from mitochondria obtained by differential centrifugation [Taylor, Prpić, Exton & Bygrave (1980) Biochem. J. 188, 443--450] in that the former exhibit a higher acceptor control ratio and a higher calcium content. Values obtained for the protonmotive force are not significantly different between the two preparations. The technique described may be widely applicable for studies requiring the rapid preparation of functionally intact and relatively uncontaminated mitochondria.
| S-EPMC1158413 | biostudies-other
Project description:BackgroundDespite major recent advances in the understanding of peroxisomal functions and how peroxisomes arise, only scant information is available regarding this organelle in cellular aging. The aim of this study was to characterize the changes in the protein expression profile of aged versus young liver and kidney peroxisome-enriched fractions from mouse and to suggest possible mechanisms underlying peroxisomal aging. Peroxisome-enriched fractions from 10 weeks, 18 months and 24 months C57bl/6J mice were analyzed by quantitative proteomics.ResultsPeroxisomal proteins were enriched by differential and density gradient centrifugation and proteins were separated by two-dimensional electrophoresis (2-DE), quantified and identified by mass spectrometry (MS). In total, sixty-five proteins were identified in both tissues. Among them, 14 proteins were differentially expressed in liver and 21 proteins in kidney. The eight proteins differentially expressed in both tissues were involved in beta-oxidation, alpha-oxidation, isoprenoid biosynthesis, amino acid metabolism, and stress response. Quantitative proteomics, clustering methods, and prediction of transcription factors, all indicated that there is a decline in protein expression at 18 months and a recovery at 24 months.ConclusionThese results indicate that some peroxisomal proteins show a tissue-specific functional response to aging. This response is probably dependent on their differential regeneration capacity. The differentially expressed proteins could lead several cellular effects: such as alteration of fatty acid metabolism that could alert membrane protein functions, increase of the oxidative stress and contribute to decline in bile salt synthesis. The ability to detect age-related variations in the peroxisomal proteome can help in the search for reliable and valid aging biomarkers.
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Project description:Peroxisomes are metabolic organelles that perform a diverse array of critical functions in human physiology. Traditional isolation methods for peroxisomes can take more than 1 h to complete and can be laborious to implement. To address this, we have now extended our prior work on rapid organellar isolation to peroxisomes via the development of a peroxisomally localized 3XHA epitope tag ("PEROXO-Tag") and associated immunoprecipitation ("PEROXO-IP") workflow. Our PEROXO-IP workflow has excellent reproducibility, is easy to implement, and achieves highly rapid (~10 min post homogenization) and specific isolation of human peroxisomes, which we characterize here via proteomic profiling. By offering speed, specificity, reproducibility, and ease of use, the PEROXO-IP workflow should facilitate studies on the biology of peroxisomes.
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Project description:We have investigated the localization of palmitoyl-CoA (hexadecanoyl-CoA) synthetase (EC 6.2.1.3) and cerotoyl-CoA (hexacosanoyl-CoA) synthetase in peroxisomes isolated from rat liver. Palmitoyl-CoA and cerotoyl-CoA synthetases, like acyl-CoA: dihydroxyacetone phosphate acyltransferase (EC 2.3.1.42), are present in the peroxisomal membrane. Trypsin treatment of intact peroxisomes led to the disappearance of both palmitoyl-CoA and cerotoyl-CoA synthetase activities but had little, if any, effect on L-alpha-hydroxy-acid oxidase (EC 1.1.3.15), D-amino acid oxidase (EC 1.4.3.3) or acyl-CoA:dihydroxyacetone phosphate acyltransferase. The latter three enzymes were inactivated if the trypsin treatment was preceeded by disruption of the peroxisomes by sonication. These results show that the active site, or at least domains essential for the activity of cerotoyl-CoA synthetase, like that of palmitoyl-CoA synthetase, is located on the cytosolic face of the peroxisomal membrane.
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Project description:In this work, a swarm behaviour for multi-rotor Unmanned Aerial Vehicles (UAVs) deployment will be presented. The main contribution of this behaviour is the use of a virtual device for quantitative sematectonic stigmergy providing more adaptable behaviours in complex environments. It is a fault tolerant highly robust behaviour that does not require prior information of the area to be covered, or to assume the existence of any kind of information signals (GPS, mobile communication networks …), taking into account the specific features of UAVs. This behaviour will be oriented towards emergency tasks. Their main goal will be to cover an area of the environment for later creating an ad-hoc communication network, that can be used to establish communications inside this zone. Although there are several papers on robotic deployment it is more difficult to find applications with UAV systems, mainly because of the existence of various problems that must be overcome including limitations in available sensory and on-board processing capabilities and low flight endurance. In addition, those behaviours designed for UAVs often have significant limitations on their ability to be used in real tasks, because they assume specific features, not easily applicable in a general way. Firstly, in this article the characteristics of the simulation environment will be presented. Secondly, a microscopic model for deployment and creation of ad-hoc networks, that implicitly includes stigmergy features, will be shown. Then, the overall swarm behaviour will be modeled, providing a macroscopic model of this behaviour. This model can accurately predict the number of agents needed to cover an area as well as the time required for the deployment process. An experimental analysis through simulation will be carried out in order to verify our models. In this analysis the influence of both the complexity of the environment and the stigmergy system will be discussed, given the data obtained in the simulation. In addition, the macroscopic and microscopic models will be compared verifying the number of predicted individuals for each state regarding the simulation.
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