Separation Of Lysosomes Research Articles

The Preparation of Subcellular Organelles from Mouse Liver in Self-Generated Gradients of Iodixanol

This paper reports the use of a new density gradient compound, Iodixanol, for the resolution of the major organelles from mouse liver. A major advantage of Iodixanol over other iodinated density gradient media is its ready ability to form self-generated gradients. Gradient-forming conditions have been modulated to provide optimal recoveries of Golgi membranes, lysosomes, mitochondria, and peroxisomes. The organelles were isolated in high yield (80-90% of gradient input) and high purity. Nycodenz and Iodixanol were compared using preformed gradients. Iodixanol provided resolution superior to that of Nycodenz, notably of peroxisomes and mitochondria and the separation of lysosomes from endoplasmic reticulum. Because Iodixanol does not interfere significantly with marker enzyme activities, gradient fractions can be analyzed without removal of the gradient medium.

Separation of lysosomes and autophagosomes by means of glycyl-phenylalanine-naphthylamide, a lysosome-disrupting cathepsin-C substrate.

In density-gradient analyses of autophagic vacuoles from isolated rat hepatocytes, autophagosomes could be recognized by the presence of an autophagically sequestered cytosolic enzyme, lactate dehydrogenase (LDH). Lysosomes were identified by marker enzymes such as acid phosphatase, or by degradation products from 125I-tyramine-cellobiose-asialoorosomucoid (125I-TC-AOM) loaded into the lysosomes by an intravenous injection in vivo 18 h prior to cell isolation. Autophagosomes and lysosomes showed similar, largely overlapping, density distributions both in hypertonic sucrose gradients and in isotonic Nycodenz gradients. As a step towards the purification of autophagosomes, we investigated the possibility of using lysosomal enzyme substrates to achieve selective destruction of lysosomes by swelling. Hepatocytes were first incubated for 2 h at 37 degrees C with vinblastine (50 microM) to obtain an accumulation of autophagosomes (to 3-5-times above the control level). The cells were then electrodisrupted and the disruptates incubated with a variety of substrates for lysosomal enzymes. Among these, glycyl-phenylalanine-2-naphthylamide (GPN), a cathepsin-C substrate, and methionine-O-methylester (MetOMe), an esterase substrate, turned out to induce extensive rupture of lysosomes, as measured by a strongly reduced sedimentability of acid phosphatase and a nearly complete loss of 125I-TC-AOM sedimentability in substrate-treated preparations from control or vinblastine-treated cells. The lysosomes of cells treated with leupeptin or asparagine were largely resistant to the action of GPN, probably as a result of interference with cathepsin-C activity or lysosomal function in general. Autophagosomes were partially destroyed by MetOMe, as indicated by a reduction in sedimentable LDH, but GPN had no effect on either autophagosomes or mitochondria. The ability of GPN to selectively destroy lysosomes without affecting the autophagosomes of vinblastine-treated cells should make GPN treatment a useful aid in the purification of rat liver autophagosomes.

Isolation of rat liver lysosomes by a single two-phase partition on dextran/polyethylene glycol

Crude mitochondria from liver rats were added to a two-phase system containing dextran and polyethylene glycol. The polymer and ionic concentration values of the two-phase system were changed in order to separate lysosomes from mitochondria. The best separation of lysosomes and mitochondria was obtained at 6.6-6.6% ( w w ) dextran-polyethylene glycol and 5 mmol/kg ammonium chloride as shown by enzyme assays. This procedure showed good reproducibility, and lysosomes were never contaminated with more than 16% mitochondria, as determined by succinate dehydrogenase activity, and β- d-galactosidase and acid phosphatase activities were enriched five- to sixfold. The lipid composition profile of lysosomes was quite similar to that obtained by means of free carrier electrophoresis, considered a reference method.

抗生物質の腎毒性に関する基礎的研究

In order to assess the potential nephrotoxicity of antibiotics, effects of these agents on rat kidney lysosomal membrane were investigated in various conditions. Antibiotics were given to Wistar rats for 5 successive days. After nephrectomy rat lysosomes were separated and their membrane stability was examined by measuring the activities of acid-phosphatase. In addition, after separation of lysosomes from normal untreated Wistar rats, antibiotics were added in the incubation system to assess the in vitro effect of antibiotics. Effects of renal ischemia and the lysosomal membrane stabilizer (cortisol) were also examined. Aminoglycosides antibiotics (streptomycin, kanamycin, gentamycin), doxytetracycline, chloramphenicol, and cephems (cephalothin, cephaloridine, ceftezol, latamoxef) were used for this purpose. It was clearly pointed out that aminoglycosides interfered with the lysosomal stability in vivo and in vitro. After a 60 minutes ischemia of the rat kidneys by clamping the renal arteries, effects of antibiotics on administration of 5 successive days on rat lysosomal stability were investigated. It was demonstrated that aminoglycosides also made the lysosomal membrane more unstable. Effects of the lysosomal stabilizer, cortisol, on rat kidney lysosomes were examined. Use of cortisol simultaneously with an antibiotic was more effective than that before and after it.

Liver autophagy in the influenza B virus model of Reye's syndrome in mice.

Biochemical evidence is presented for the autophagic destruction of liver mitochondria in the influenza B virus model of Reye's syndrome in mice. Separation of lysosomes and autophagic vacuoles from mitochondria was accomplished by prior treatment of the mice with Triton WR-1339, resulting in uptake of detergent by these organelles (tritosomes), reducing their densities. The organelles were banded in a discontinuous sucrose gradient. Total protein in the heavy tritosomal fraction increased from 1-2% in controls to 7-8% in virus-treated animals. Ornithine carbamoyl transferase (OCTase), a mitochondrial marker, increased from 2-3% (controls) to 11-15% (virus-treated), and glucose-6-phosphatase, a marker for endoplasmic reticulum, increased from 1-2% (controls) to 8-10% (virus-treated). beta-Galactosidase, a soluble enzyme in the lysosome, and OCTase also increase in the cell extract fraction following virus treatment, indicating that there was turnover of heavy lysosomal contents.

Tissue and subcellular distribution of 252Cf and 241Am in the seastar Marthasterias glacialis

Seastars, which are known to accumulate transuranium nuclides to relatively high levels, were used in laboratory radiotracer experiments (April–May, 1982) designed to determine the subcellular distribution of tissue-incorporated californium-252 and americium-241. Mediterranean asteroids (Marthasterias glacialis), collected in April 1982 and fed prey contaminated with 252Cf and 241Am, accumulated and retained large fractions of these radionuclides (252Cf, 91 to 98%; 241Am, 82 to 88%) in their pyloric caeca, the glandular complex involved with digestive and food storage processes. Differential centrifugation demonstrated an association of 252Cf and 241Am with the lysosome-mitochondrial fraction of plyoric caeca cells. Partial separation of lysosomes and mitochondria by isopycnic gradient centrifugation suggests a somewhat different subcellular fractionation of the two transuranics; 252Cf is associated primarily with the mitochondria, whereas 241Am is accumulated in both lysosomes and mitochondria. From comparison with analogous studies in mammalian systems, it is hypothesized that the long biological half-lives reported for ingested transuranics in seastars result in large part from the radionuclide association with mitochondria in the pyloric caeca.

Further 237Pu experiments with the plaice Pleuronectes platessa: Subcellular distribution of plutonium in the liver

The subcellular distribution of 237Pu in the livers of the plaice Pleuronectes platessa has been studied by differential and isopycnic gradient centrifugation. It has been shown that the 237Pu is particularly accumulated in pellets sedimenting out at 3000 and 6000 g which, by deduction from marker enzyme tests, contain a mixture of mitochondria and lysosomes. Separation of lysosomes from mitochondria was achieved by using a non-lytic detergent injected into the fish 3 d prior to removal of the livers; it was found that 237Pu was associated with the lysosomal fraction. In this respect, the metabolism of plutonium of the plaice resembles that of the rat.

Translocation of Inducible Tyrosine Aminotransferase to the Mitochondrial Fraction: FACILITATION BY ACUTE UREMIA AND OTHER CONDITIONS

Abstract Acute uremia induced both soluble and mitochondrial tyrosine aminotransferase activity, EC 2.6.1.5, in rat liver. Such induction was demonstrated to be independent of glucocorticoid action. Mitochondrial aspartate aminotransferase, EC 2.6.1.1, although identical with mitochondrial tyrosine aminotransferase, was not elevated during acute uremia. Purified antibody specific against cytosol tyrosine aminotransferase cross-reacted significantly with the mitochondrial enzyme from uremic rats, suggesting that the elevation of activity was due to translocation of the induced cytosol enzyme form to the mitochondria. Considerable translocation was also caused by a combination of fasting together with some factor known to increase urea synthesis in the liver, such as high nitrogen loads or intraperitoneal injection of hydrocortisone. Intravenous injection of rats with Triton WR-1339 resulted in the separation of lysosomes from mitochondria during sucrose density gradient centrifugation. All of the tyrosine aminotransferase activity was associated with the mitochondria and not with the lysosomes. Treatment of intact mitochondria with the antisoluble tyrosine aminotransferase antibody resulted in inhibition of about 50% of the total inhibitable mitochondrial activity, suggesting that some of the translocated cytosol enzyme may not be exposed and thus may be unavailable for antibody cross-reaction. In vitro crossover experiments using induced cytosol tyrosine aminotransferase with normal mitochondria resulted in an elevation of the mitochondrial activity, whereas incubation of noninduced cytosol enzyme with mitochondria from induced rats did not. Consequently, the translocation phenomenon is a function of altered cytosol tyrosine aminotransferase rather than altered mitochondria.

Catalase particles in the epithelial cells of the guinea-pig small intestine.

Purified preparations of epithelial cells have been made from the guinea-pig small intestine. Homogenates of these preparations have been analysed by centrifugation in a zonal rotor. The results confirm the presence of lysosomes in these cells and indicate the existence of catalase particles which equilibrate in a sucrose gradient at a density of between 1.21 and 1.23 and which have a different distribution from other subcellular particles except lysosomes. Injection of Triton WR-1339 into fasting animals enables the separation of lysosomes and catalase particles.