Summary
Peroxisomes, lysosomes, and mitochondria have been purified from rat liver by sucrose density gradient centrifugation without prior treatment of the animals with Triton WR-1339 or other detergents which cause hyperlipidemia. A crude organelle fraction was first prepared by differential centrifugation of a rat liver homogenate, this fraction contained approximately 70% of the mitochondrial, 40% of the peroxisomal, and 30% of the lysosomal marker enzymes measured in the homogenate. The crude organelle fraction was applied to the top of a sucrose density gradient and centrifuged. A clear separation of the organelles was obtained only when dextran was present in the gradients. Success or failure of the method was found to depend on the particular preparation of dextran used in the gradients. A method for subfractionating dextran was developed which yields dextran fractions that make the separations completely reproducible. Starting with a crude organelle fraction derived from 12 g of liver, approximately 85% of the mitochondrial, 70% of the peroxisomal, and 50% of the lysosomal activities were obtained as pure fractions. The organelle separation takes less than five hours to complete, it represents a substantial improvement over previous methods.
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References
Appelkvist EL, Brunk U, Dallner G (1981) Isolation of peroxisomes from rat liver using sucrose and Percoll gradients. J Biochem Biophys Methods 5: 203–217
Arnett EM, Harvey N, Johnson EA, Johnson DS, Chapman D (1986) No phospholipid monolayer-sugar interactions. Biochemistry 25: 5239–5242
Barber EJ (1966) Calculation of density and viscosity of sucrose solutions as a function of concentration and temperature. In: Anderson NG (ed) Development of zonal centrifuges and ancillary systems for tissue fractionation and analysis. National Cancer Institute Monograph No. 21. US Government Printing Office, Washington, pp 219–239
Baudhuin P (1974) Isolation of rat liver peroxisomes. Methods Enzymol 31: 356–368
Beers RF Jr, Sizer IW (1952) A spectrophotometric method for measuring the breakdown of hydrogen peroxide by catalase. J Biol Chem 195: 133–140
Bronfman M, Inestrosa NC, Leighton F (1979) Fatty acid oxidation by human liver peroxisomes. Biochem Biophys Res Commun 88: 1030–1036
Brooks DE (1973) The effect on neutral polymers on the electrokinetic potential of cells and other charged particles. J Colloid Interface Sci 43: 714–725
Bucher NL (1959) Alterations of cholesterol biosynthesis in liver cell fractions from rats in various experimental conditions. In: Wolsterholme GEW, O'Connor M (eds) Ciba Foundation Symposium on the biosynthesis of terpenes and sterols. Little, Brown and Co, Boston, pp 46–60
Cooperstein SJ, Lazarow A (1951) A microspectro-photometric method for the determination of cytochrome oxidase. J Biol Chem 189: 665–670
Crowe LM, Crowe JH, Chapman D (1985) Interactions of carbohydrates with dry dipalmitoylphosphatidylcholine. Arch Biochem Biophys 236: 289–296
de Duve C, Pressman BC, Gianetto R, Wattiaux R, Appelmans F (1955) Intracellular distribution patterns of enzymes in rat-liver tissue. Biochem J 60: 604–617
Frantz ID, Hinkelman BT (1955) Acceleration of hepatic cholesterol synthesis by Triton WR-1339. J Exp Med 101: 225–232
Gekko K (1981) Solution properties of Dextran and its ionic derivatives. In: Brant DA (ed) Solution properties of polysaccharides. American Chemical Society, Washington, pp 415–438
Gekko K, Noguchi H (1971) Physicochemical studies of Oligodextran. I. Molecular weight dependence of intrinsic viscosity, partial specific compressibility and hydrated water. Biopolymers 10: 1513–1524
Harms E, Kern H, Schneider JA (1980) Human lysosomes can be purified from diploid skin fibroblasts by free-flow electrophoresis. Proc Natl Acad Sci USA 77: 6139–6143
Hayashi H, Suga T (1978) Some characteristics of peroxisomes in the slime moldDictyostelium discoideum. J Biochem (Tokyo) 84: 513–520
Ishii H, Suga T, Hayashi H, Ninobe S (1979) Effect of Triton WR-1339 on peroxisomal enzymes. Biochim Biophys Acta 582: 1157–1175
Kase F, Björkem I, Pedersen JI (1983) Formation of cholic acid from 3α, 7α, 12α-trihydroxy-5β-cholestanoic acid by rat liver peroxisomes. J Lipid Res 24: 1560–1567
Jeanes A, Haynes WC, Rankin JC, Melvin EH, Austin MJ, Cluskey JE, Fisher BE, Tsuchiya HM, Rist CE (1954) Characterization and classification of dextrans from ninety-six strains of bacteria. J Am Chem Soc 76: 5041–5052
Lardeux B, Gouhot B, Forestier M (1983) Improved recovery of rat liver fractions enriched in lysosomes by specific alteration of the sedimentation of mitochondria. Anal Biochem 131: 160–165
Leighton F, Poole B, Beaufay H, Baudhuin P, Coffey JW, Fowler S, de Duve C (1968) The large-scale separation of peroxisomes, mitochondria, and lysosomes from the livers of rats injected with Triton WR-1339. J Cell Biol 37: 482–513
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1952) Protein measurement with the folin-phenol reagent. J Biol Chem 193: 265–175
MacDonald RI (1985) Membrane fusion due to dehydration by polyethylene glycol, dextran, or sucrose. Biochemistry 24: 4058–4066
Mahler HR, Hubscher G, Baum H (1955) Studies on uriease. J Biol Chem 216: 625–641
Minetti M, Aducci P, Viti V (1979) Interaction of neutral polysaccharides with phosphatidylcholine multilamellar liposomes. Phase transitions studied by the binding of fluorescein-conjugated dextrans. Biochemistry 18: 2541–2548
Neat CE, Thomassen MS, Osmundsen H (1980) Induction of peroxisomal β-oxidation in rat liver by high-fat diets. Biochem J 186: 369–371
— — — (1981) Effects of high fat diets on hepatic fatty acid oxidation in the rat. Biochem J 196: 149–159
Osmundsen H (1982) Peroxisomal β-oxidation of long fatty acids: Effects of high fat diets. Ann NY Acad Sci 386: 13–29
Otway S, Robinson DS (1967) The use of a non-ionic detergent (Triton WR-1339) to determine rates of triglyceride entry into the circulation of rat under different physiological conditions. J Physiol 190: 321–332
Sanui H (1974) Measurement of inorganic orthophosphate in biological materials: extraction properties of butylacetate. Anal Biochem 60: 489–504
Scanu A, Oriente P (1961) Triton hyperlipemia in dogs. J Exp Med 113: 735–757
Schmidt E, Schmidt FW (1983) Glutamate dehydrogenase. In: Bergmeyer HU (ed) Methods of enzymatic analysis, vol 3. Verlag Chemie, Weinheim, pp 216–227
Trouet A (1964) Immunisation de lapins par des lysosomes hépatiques de rats traités au Triton WR-1339. Arch Int Physiol Biochim 72: 698–700
— (1974) Isolation of modified liver lysosomes. Methods Enzymol 31: 323–329
Trout JJ, Viles JM (1978) Some changes associated with Triton WR-1339 accumulation in rat hepatocytes. Virchows Arch [B] 28: 279–285
Van Den Bosch J, Evrard E, Billiau A, de Somer P, Joossens JV (1961) The influence of a new P-tert-octylphenol derivative on the clearing factor and on the plasma lipids of rabbits. J Atheroscler Res 1: 148–162
Völkl A, Fahimi D (1985) Isolation and characterization of peroxisomes from liver of normal untreated rats. Eur J Biochem 149: 257–265
Wattiaux R, Wibo M, Baudhuin P (1963) Influence of the injection of Triton WR-1339 on the properties of rat liver lysosomes. In: de Reuck AVS, Cameron MP (eds) Ciba Foundation Symposium on lysosomes. Little, Brown and Co, Boston, pp 176–196
—, Wattiaux-de Connick S, Ronveaux-Dupal M, Du Bois F (1978) Isolation of rat liver lysosomes by isopycnic centrifugation in a Metrizamide gradient. J Cell Biol 78: 349–368
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Sephton, G.B., Buechler, K.F. & Lowenstein, J.M. An improved procedure for the simultaneous purification in high yield of peroxisomes, lysosomes, and mitochondria from rat liver. Protoplasma 153, 127–135 (1990). https://doi.org/10.1007/BF01353996
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DOI: https://doi.org/10.1007/BF01353996