A simple procedure for the isolation of rat kidney lysosomes

Kawashima, Akira; Sato, Atsushi; Kawashima, Megumi; Nitta, Kosaku; Yumura, Wako; Sugino, Nobuhiro; Nihei, Hiroshi; Natori, Yasuo

doi:10.1046/j.1523-1755.1998.00958.x

Cited by 21 publications

(32 citation statements)

References 21 publications

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“…The cell membranes and subcellular organelle fractions (mitochondria, ER, Golgi) were recovered from the cold or radiolabeled cells as described earlier [40]- [43] [46] [47] [54]- [58]. The ER and Golgi organelles sediment, remaining after separation of nuclei, mitochondria, endosomes and lysosomes, and cell cytosol, was suspended in the buffer containing 0.2 M PIPES (pH 6.9), 2.0 M glycerol, 1 mM EGTA and 1.0 mM magnesium acetate and applied on the top of discontinuous gradient of 2.0/1.5/1.3/1.0 M sucrose and centrifuged at 100,000 xg for 16 h. The cell membranes were recovered from 1.0 M sucrose, ER from 1.3 M and 1.5 M sucrose and Golgi from the top of the 2.0 M sucrose.…”

Section: Preparation Of Cellular Organelles and Membranesmentioning

confidence: 99%

“…The mitochondria and lysosomes were purified from 10,000 xg spun fraction [54]- [56] [58]. From the initial fraction, the fluffy layer of broken mitochondria and ER microsomes that covered the crude mitochondrial pellet was suctioned off, the pellet was gently resuspended in five volumes of medium consisting of 70 mM sucrose, 0.2 M mannitol, 0.1 mM disodium EDTA, and 1 mM TRIS, pH 7.2, and the suspension was spun for 10 min at low speed (500 xg), the supernatant was recovered and spun for 10 min at 9000 xg [43] [53]- [56].…”

Section: Preparation Of Cellular Organelles and Membranesmentioning

confidence: 99%

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Concluding Step in Cell Restitution Cycle: ER Transport Vesicles with Sphingolipids in the Outer Leaflet of the Membrane Restore Lysosomes

Slomiany¹,

Slomiany²

2014

ABC

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Restitution of the cell organelles and the membrane implicates serine palmitoyltransferase (SPT) in signal-specific and selective assembly of the transport vesicles. Here, we reveal that SPT, embedded in the outer leaflet (OL) of endoplasmic reticulum (ER), is engaged in the synthesis of ER transport vesicles that recondition cell organelles, and the inner leaflet (IL) SPT in the restitution of the cell membrane. The OL SPT impacts assembly of sphingomyelinase (SMase)-susceptible ER vesicles but not the SMase-resistant and sphingolipid (SPhL) core-carrying vesicles that refurbish the cell membrane. The investigation of the SPT-initiated differences in the placement of SPhL in vesicular membranes by utilizing ER depleted of OL SPT, allows us to conclude that the restitution of endosomal and lysosomal membranes is achieved with the involvement of OL SPT, whereas the IL SPT is involved in formation of the lipid core for glycosphingolipids (GSL) and sphingomyelin (SM) of the apical and basolateral cell membrane. These findings along with our previously published report (Slomiany and Slomiany, Advances in Biological Chemistry, 2013, 3, 275-287), provide a clear distinction between the processes that renovate cell membrane and its organelles from that of the endocytotic cell debridement, and show that vesicles are navigated to the specific organelles and the cell membrane by the biomembrane constituents programmed in ER.

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Section: Preparation Of Cellular Organelles and Membranesmentioning

confidence: 99%

Section: Preparation Of Cellular Organelles and Membranesmentioning

confidence: 99%

Concluding Step in Cell Restitution Cycle: ER Transport Vesicles with Sphingolipids in the Outer Leaflet of the Membrane Restore Lysosomes

Slomiany¹,

Slomiany²

2014

ABC

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“…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 purified according to the method Kawashima et al [15] from human small intestine. 500g of human small intestine were minced with scissors, and suspended in 6 volumes of cold 0.3M sucrose bubbled with nitrogen gas and containing 50 g/ml leupeptin (a protease inhibitor).…”

Section: Purification Of Lysosomesmentioning

confidence: 99%

A Novel Method For Purifying a DNase From Lysosomal Fraction From Human Small Intestine

Shikara¹,

Al-jaff²

2012

Turk J Bioch

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Aims:The purification of an acid DNase related DNase II family from lysosomes of human small intestine (jejunum). Materials and Methods: Two different methods: a column chromatography series, including phosphocellulose, CM-cellulose and Sephadex G-200 and a novel procedure that use immunoabsorbent technique were used in the purification. Results:The purified DNase is an endonuclease that consisted of one polypeptide chain (monomer) with a molecular mass of 28-32kDa with an optimal pH and temperature of 6.0 and 30°C, respectively. The enzyme prefers native DNA on denatured DNA Discussion: The catalytic properties of the purified enzyme are essentially the same as those of DNase II family such as independency of divalent ions which inhibited its activity at 10mM. The enzyme acts on dsDNA and ssDNA and generates 3'-phosphate and 5'-OH termini which was a characteristic of DNase II. The functions of the intestinal purified DNase from lysosomes were unclear, but lysosomes contained a set of enzymes required for degradation of food, and the enzyme may be necessary for degradation of nucleic acids within food, but small amounts of the enzyme were found in the nuclei and cytoplasm were detected. Several researchers suggested that DNase II family may be active in apoptosis and played a role as a barrier to transfection. The purified was designated SIDNase. Key words: DNase II, small intestine, immunoabsorption, lysosomes, DLAD Conflict of interests: There is no conflict in interest. ÖZETAmaç: İnsan ince bağırsağından (jejunum) asit DNaz II ailesi ile ilintili DNaz izolasyonu. Materyal ve Metod: İzolasyonlar için iki farklı metod uygulanmıştır: Fosfoselüloz CMselüloz ve Sephadex G-200 den oluşan bir kolon serisi ve immünabsorbsiyon tekniği kullanılarak geliştirilen yeni bir prosedür. Sonuçlar: İzole edilen DNaz tek bir polipeptit zincirinden (monomer) oluşan bir endonükleaz olup, moleküler ağırlığı 28-32 kDa, fonksiyonu için optimal ısısı 30°C, pH ise 6.0 dır. Bu enzim natif DNA'yı denatüre DNA'ya tercih etmektedir. Tartışma: İzole edilen enzimin katalitik özellikleri DNaz II ailesine ait enzimler ile temelde aynıdır. Mesela iki değerlikli iyonlardan bağımsız aktivite göstermesi, aktivitesinin 10mM da inhibe olması gibi. Bu enzim dsDNA ve ssDNA üzerinde DNaz II gibi 3'-fosfat ve 5'-OH terminali oluşturur. Bağırsaklardan izole edilen lizozomal DNaz'ın fonksiyonları tam olarak nitelenememiştir ancak lizozomler gıda parçalanması için bir set enzim içermektedir ve gıdada bulunan nükleik asitlerin parçalanması için gerekli olabilirler. Az miktarda enzim çekirdek ve sitoplazmada bulunmuştur. Bazı araştırmacılar DNAz II ailesinin apoptozda aktif olabileceğini ve görevinin transfeksiyonda bariyer oluşturmak olabileceğini öne sürmüşlerdir. İzolat SIDNaz olarak adlandırılmsiyonıştır.

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A simple procedure for the isolation of rat kidney lysosomes

Abstract: We believe that this procedure for isolating kidney lysosome will be useful in the study of the mechanisms of specific modification, processing and catabolism of proteins.

Cited by 21 publications

References 21 publications

Concluding Step in Cell Restitution Cycle: ER Transport Vesicles with Sphingolipids in the Outer Leaflet of the Membrane Restore Lysosomes

Concluding Step in Cell Restitution Cycle: ER Transport Vesicles with Sphingolipids in the Outer Leaflet of the Membrane Restore Lysosomes

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

A Novel Method For Purifying a DNase From Lysosomal Fraction From Human Small Intestine

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