A Simple Procedure for the Isolation of Highly Purified Lysosomes from Normal Rat Liver

Yamada, Haruo; Hayashi, Hisashi; Natori, Yasuo

doi:10.1093/oxfordjournals.jbchem.a134704

Cited by 53 publications

(24 citation statements)

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“…Future studies could utilize the methods we have established herein to assess cell lines and animals models that are known to have impaired lysosome function due to, for example, accumulation of the age pigment lipofuscin, substrate accumulation in various lysosomal storage diseases, or accumulation of -amyloid (Zhao et al, 2011). Based on the fact that Cbl deficiency includes a neurological phenotype (Baik and Russell, 1999;Calvaresi and Bryan, 2001;Healton et al, 1991) (Graham, 2001;Koenig, 1974;Yamada et al, 1984)) may yield similar results.…”

Section: Discussionmentioning

confidence: 97%

Analysis of subcellular [57Co] cobalamin distribution in SH-SY5Y neurons and brain tissue

Zhao¹,

Ruberu²,

Li³

et al. 2013

Journal of Neuroscience Methods

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Cobalamin (Cbl) utilization as a cofactor for methionine synthase and methylmalonyl-CoA mutase is dependent on the transport of Cbl through lysosomes and its subsequent delivery to the cytosol and mitochondria. We speculated that neuropathological conditions that impair lysosomal function (e.g., agerelated lipofuscinosis and specific neurodegenerative diseases) might impair lysosomal Cbl transport. To address this question, an appropriate method to quantify intracellular Cbl transport in neuronal cell types and brain tissue is required. Thus, we developed methods to measure [ 57 Co] Cbl levels in lysosomes, mitochondria and cytosol obtained from in vitro and in vivo sources. Human SH-SY5Y neurons or HT1080 fibroblasts were labeled with [ 57 Co] Cbl and homogenized using a ball-bearing homogenizer, and the lysates were separated into 10 fractions using ultracentrifugation in an OptiPrep density gradient. Lysosomes were recovered from the top of the gradient (fractions 1-5), which were clearly separated from mitochondria (fractions 7-9) on the basis of the expression of the marker proteins, LAMP2 and VDAC1. The isolated lysosomes were intact based on their colocalization with acid phosphatase activity. The lysosomal and mitochondrial fractions were free of the cytosolic markers beta-actin and methionine synthase. The relative distribution of [ 57 Co] Cbl in both neurons and fibroblasts was as follows: 6% in the lysosomes, 14% in the mitochondria and 80% in the cytosol. This technique was also used to fractionate organelles from mouse brain, where marker proteins were detected in the gradient at positions similar to those observed for the cell lines, and the relative distribution of [ 57 Co] Cbl was as follows: 12% in the lysosomes, 15% in the mitochondria and 73% in the cytosol. These methods provide a useful tool for the investigation of intracellular Cbl trafficking in a neurobiological setting. fractions using ultracentrifugation on an OptiPrep density gradient. Lysosomes were recovered at the top of the gradient (fractions 1 to 5) and were clearly separated from mitochondria (fractions 7 to 9) based on marker proteins LAMP2 and VDAC1, respectively.The isolated lysosomes were intact based on colocalisation with acid phosphatase activity.The lysosomal and mitochondrial fractions were free of cytosolic markers beta-actin and methionine synthase.

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Section: Discussionmentioning

confidence: 97%

Analysis of subcellular [57Co] cobalamin distribution in SH-SY5Y neurons and brain tissue

Zhao¹,

Ruberu²,

Li³

et al. 2013

Journal of Neuroscience Methods

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show abstract

“…Cytosolic extracts (S-100) were prepared from mouse hepatocytes using the approach described by Yang et al (40). Highly purified lysosomes free of mitochondria were isolated from mouse liver as described elsewhere (41,42). The lysosomal fraction was free of mitochondrial contamination as verified by electron microscopy and by immunoblot analysis for cytochrome c oxidase (data not shown).…”

Section: Isolation and Culture Of Mouse Hepatocytes; Culture Ofmentioning

confidence: 99%

Cathepsin B contributes to TNF-α–mediated hepatocyte apoptosis by promoting mitochondrial release of cytochrome c

Guicciardi¹,

Deussing²,

Miyoshi³

et al. 2000

J. Clin. Invest.

671

666

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“…Lysosomes were isolated by isopycnic density centrifugation according to a modification ofthe method ofYamada et al (1,24). Briefly, livers were weighed and minced before homogenization in iced 250 mM sucrose-3 mM imidazole-0.…”

Section: Introductionmentioning

confidence: 99%

“…A postnuclear supernatant (E fraction) containing cellular organelles and cytosol was prepared as previously described (15). The E fraction was then incubated with 1 mM CaCl2 for 5 min at 370C; incubation with CaCl2 causes mitochondria to swell and become less dense, a maneuver which permits nearly complete separation of lysosomes from mitochondria (1,24). The E fraction was then layered on a self-forming gradient of Percoll (Pharmacia LKB Biotechnology Inc., Piscataway, NJ) and centrifuged at 80,000 gfor 15 min using a TI 50.2 rotor in an ultracentrifuge (LZ-65B; Beckman Instruments, Inc., Fullerton, CA).…”

Section: Introductionmentioning

confidence: 99%

Alterations in the structure, physicochemical properties, and pH of hepatocyte lysosomes in experimental iron overload.

Myers

Prendergast²,

Holman³

et al. 1991

J. Clin. Invest.

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While hemochromatosis is characterized by sequestration of iron-protein complexes in hepatocyte lysosomes, little is known about the effects of excess iron on these organelles. Therefore, we studied the effects of experimental iron overload on hepatocyte lysosomal structure, physicochemical properties, and function in rats fed carbonyl iron. A sixfold increase (P < 0.0001) in hepatic iron and a fivefold increase in lysosomal iron (P < 0.01) was observed after iron loading; as a result, hepatocyte lysosomes became enlarged and misshapen. These lysosomes displayed increased (P < 0.0001) fragility, moreover, the fluidity of lysosomal membranes isolated from livers of iron-loaded rats was decreased (P < 0.0003) as measured by fluorescence polarization. Malondialdehyde, an end product of lipid peroxidation, was increased by 73% (P < 0.008) in lysosomal membranes isolated from livers of iron-overloaded rats. While amounts of several individual fatty acids in isolated lysosomal membranes were altered after iron overload, cholesterol/ phospholipid ratios, lipid/protein ratios, double-bond index, and total saturated and unsaturated fatty acids remained unchanged. The pH of lysosomes in hepatocytes isolated from livers of iron-loaded rats and measured by digitized video microscopy was increased (control, 4.70±0.05; iron overload, 5.21 ±0.10; P < 0.01). Our results demonstrate that experimental iron overload causes marked alterations in hepatocyte lysosomal morphology, an increase in lysosomal membrane fragility, a decrease in lysosomal membrane fluidity, and an increase in intralysosomal pH. Iron-catalyzed lipid peroxidation is likely the mechanism of these structural, physicochemical, and functional disturbances. (J. Clin. Invest. 1991. 88:1207-1215

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A Simple Procedure for the Isolation of Highly Purified Lysosomes from Normal Rat Liver

Cited by 53 publications

References 0 publications

Analysis of subcellular [57Co] cobalamin distribution in SH-SY5Y neurons and brain tissue

Analysis of subcellular [57Co] cobalamin distribution in SH-SY5Y neurons and brain tissue

Cathepsin B contributes to TNF-α–mediated hepatocyte apoptosis by promoting mitochondrial release of cytochrome c

Alterations in the structure, physicochemical properties, and pH of hepatocyte lysosomes in experimental iron overload.

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