Isolation of rat liver microsomes by gel filtration

Tangen, O.; Jonsson, John; Orrenius, Sten

doi:10.1016/0003-2697(73)90392-8

Cited by 119 publications

(25 citation statements)

References 7 publications

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“…1493,1494 More-intense centrifugation of the supernatant at ∼100 000 g pelletizes the microsomal membranes, which are usually of a reddish-brown color because of the high content of the CYP heme. (1495) The vesicles were prepared from the liver, 1496–1501 brain, 1502,1503 lungs,(1504) heart, 1505,1506 kidney, 1505,1507 intestine, 1508,1509 skeletal muscle, 1510,1511 placenta,(1512) leukemic cells,(1513) and other sources. The vesicular structure of the microsomes was demonstrated by electron microscopy.…”

Section: Disposition Of Chemicalsmentioning

confidence: 99%

Modeling Kinetics of Subcellular Disposition of Chemicals

Baláž

2009

Chem. Rev.

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Section: Disposition Of Chemicalsmentioning

confidence: 99%

Modeling Kinetics of Subcellular Disposition of Chemicals

Baláž

2009

Chem. Rev.

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“…Preparation of Rat Liver Microsomes-Rat liver microsomes were prepared according to a previously reported method (25) and either used immediately or divided into aliquots and stored at Ϫ20°C unless otherwise stated. Aliquots of rat liver microsomes thawed for use were washed in double-distilled H 2 O to remove extra-microsomal material and any lumenal contents released by freeze-thawing.…”

Section: Methodsmentioning

confidence: 99%

A Major Fraction of Endoplasmic Reticulum-located Glutathione Is Present as Mixed Disulfides with Protein

Bass

Ruddock

Klappa

et al. 2004

Journal of Biological Chemistry

145

122

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The tripeptide glutathione is the most abundant thiol/ disulfide component of the eukaryotic cell and is known to be present in the endoplasmic reticulum lumen. Accordingly, the thiol/disulfide redox status of the endoplasmic reticulum lumen is defined by the status of glutathione, and it has been assumed that reduced and oxidized glutathione form the principal redox buffer. We have determined the distribution of glutathione between different chemical states in rat liver microsomes by labeling with the thiol-specific label monobromobimane and subsequent separation by reversed phase high performance liquid chromatography. More than half of the microsomal glutathione was found to be present in mixed disulfides with protein, the remainder being distributed between the reduced and oxidized forms of glutathione in the ratio of 3:1. The high proportion of the total population of glutathione that was found to be in mixed disulfides with protein has significant implications for the redox state and buffering capacity of the endoplasmic reticulum and, hence, for the formation of disulfide bonds in vivo. The endoplasmic reticulum (ER)1 lumen of uni-and multicellular eukaryotes is the compartment in which nascent secretory proteins fold to attain their native structure. The luminal environment favors the maturation of secretory proteins by providing chaperones; for example, immunoglobulin heavy chain-binding protein, folding catalysts such as protein disulfide isomerase (PDI), and an oxidizing environment suitable for native disulfide bond formation. There are numerous studies on different aspects of the molecular chaperones and disulfide isomerases (for examples, see Ref. 1-10), but there have been only limited studies directly concerning the redox environment of the ER lumen. This is because of the difficulties of developing suitable methods for characterizing organelle microenvironments. One landmark study (11) using a probe thiol peptide confirmed that glutathione is the principal redox buffer in the ER lumen and reported that the ratio of reduced (GSH) to oxidized (GSSG) glutathione in the secretory pathway is between 1:1 and 3:1. This is considerably more oxidizing than the cytosolic ratio of 30:1-100:1 (11) and similar to the optimum for in vitro folding of disulfide bond-containing proteins; in most cases refolding in vitro is carried out with [GSH] at ϳ2 mM (range, 1-5 mM) and [GSSG] at ϳ0.5 mM (range 0.1-2 mM) after the initial optimization of oxidative refolding of lysozyme (12).The redox environment of the ER lumen is of great importance for the production of secretory proteins, and a role for glutathione in maintaining appropriate redox conditions in this compartment has been conjectured for many years (13-14). There is indirect evidence for the involvement of glutathione in protein disulfide bond formation in vivo (15), but some basic questions still require an answer. First, how are net oxidizing equivalents delivered to the ER lumen to maintain the observed oxidizing environment? The study of Hwang et al. (1...

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“…Crude microsomal vesicles were prepared by differential centrifugation of fresh bovine liver as described by Tangen et al (1973). Crude microsomal pellets were resuspended in minimum volume of 500 mM KCI/50 mM Tris/HCl, pH 7.5, and washed to minimize contamination with bound cytosolic PPI [the accompanying paper (Bose and Freedman (1994) Stoscheck (1990), with BSA as standard.…”

Section: Preparation Of Washed Microsomesmentioning

confidence: 99%

The characterization of a cyclophilin-type peptidyl prolyl cis-trans-isomerase from the endoplasmic-reticulum lumen

Bose

Mücke

Freedman

1994

Biochemical Journal

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A luminally located peptidyl prolyl cis-trans-isomerase (PPI) has been purified from bovine liver microsomes. It has a molecular mass of 20.6 kDa, and N-terminal sequencing demonstrates strong sequence similarity to the sequences of the cyclophilin B family. The enzyme catalyses the isomerization of the standard proline-containing peptide N-succinyl-Ala-Ala-Pro-Phe p-nitroanilide, as well as the refolding of RNAase T1. Kinetic properties, substrate-specificity data and inhibition by cyclosporin A indicate that it is a cyclophilin-type PPI, consistent with the amino-acid-sequence results.

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Isolation of rat liver microsomes by gel filtration

Cited by 119 publications

References 7 publications

Modeling Kinetics of Subcellular Disposition of Chemicals

Modeling Kinetics of Subcellular Disposition of Chemicals

A Major Fraction of Endoplasmic Reticulum-located Glutathione Is Present as Mixed Disulfides with Protein

The characterization of a cyclophilin-type peptidyl prolyl cis-trans-isomerase from the endoplasmic-reticulum lumen

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