Studies on the intracellular distributions of soluble epoxide hydrolase and of catalase by digitonin‐permeabilization of hepatocytes isolated from control and clofibrate‐treated mice

Eriksson, Anna Messing; Zetterqvist, Mary-Ann; Lundgren, Bo; Andersson, Karin; Beije, Brita; DePierre, Joseph W.

doi:10.1111/j.1432-1033.1991.tb16037.x

Cited by 34 publications

(6 citation statements)

References 29 publications

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“…After the initial reports of sEH activity, sEH was detected in mitochondrial, microsomal and cytosolic fractions by activity or in the peroxisomes alone by immunohistochemistry (Waechter et al, 1983; Kaur and Gill, 1986; Hollinshead and Meijer, 1988). Eventually it was determined that the activity in the mitochondrial fractions was an artifact from peroxisomal contamination, and the same enzyme was responsible for the activity in the cytosol and peroxisomes (Chang and Gill, 1991; Eriksson et al, 1991). The small amount of sEH either trapped in the microsomes or tightly associated with them can be differentiated with selective substrates or inhibitors.…”

Section: Mammalian Gene Expressionmentioning

confidence: 99%

Soluble epoxide hydrolase: Gene structure, expression and deletion

Harris

Hammock

2013

Gene

192

184

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Mammalian soluble epoxide hydrolase (sEH) converts epoxides to their corresponding diols through the addition of a water molecule. sEH readily hydrolyzes lipid signaling molecules, including the epoxyeicosatrienoic acids (EETs), epoxidized lipids produced from arachidonic acid by the action of cytochrome p450s. Through its metabolism of the EETs and other lipid mediators, sEH contributes to the regulation of vascular tone, nociception, angiogenesis and the inflammatory response. Because of its central physiological role in disease states such as cardiac hypertrophy, diabetes, hypertension, and pain sEH is being investigated as a therapeutic target. This review begins with a brief introduction to sEH protein structure and function. sEH evolution and gene structure are then discussed before human small nucleotide polymorphisms and mammalian gene expression are described in the context of several disease models. The review ends with an overview of studies that have employed the sEH knockout mouse model.

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Section: Mammalian Gene Expressionmentioning

confidence: 99%

Soluble epoxide hydrolase: Gene structure, expression and deletion

Harris

Hammock

2013

Gene

192

184

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“…The cells were washed sequentially with warm Puck's saline followed by cytomix at 37°C, permeabilized using the optimized conditions noted, and chased in 5 ml of DMEM (without serum and additives) containing 10 mM ATP and 10 mM MgCl 2 at pH 7.0. Our methods were developed from published procedures (36). Permeabilized cells were maintained at 37°C in the incubator during the chase.…”

Section: Cellsmentioning

confidence: 99%

Turnover of the Acyl Phosphates of Human and Murine Prothymosin α in Vivo

Wang

Tao

Trumbore

et al. 1997

Journal of Biological Chemistry

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Prothymosin ␣ is a small, highly acidic, abundant, nuclear, mammalian protein which is essential for cell growth. Our laboratory has recently shown that primate prothymosin ␣ contains stoichiometric amounts of phosphate on the glutamyl groups of the protein and that in vitro the phosphate undergoes rapid hydrolysis or transfer to a nearby serine residue. Here an assay for the presence of acyl phosphates in vivo has been developed by measuring stable phosphoserine and phosphothreonine in vitro. The assay was used to determine the half-life of the acyl phosphates on prothymosin ␣ in vivo by pulse-labeling HeLa cells with [ 32 P]orthophosphate and chasing using three different techniques: permeabilization with digitonin to allow extracellular ATP to equilibrate with the intracellular pool; electroporation in the presence of ATP to reduce the specific activity of Prothymosin ␣ is a small, highly acidic (1, 2), nuclear (3-6) protein found in virtually all mammalian tissues (7-11). Under conditions of rapid growth, a cultured cell accumulates upwards of 17 million molecules, a number roughly equivalent to that of histone cores (12). When cells are disrupted with detergents, the protein readily leaks out of the nucleus, suggesting that stable interactions with nucleosomes or with the nuclear matrix are not an inherent part of its activity (2, 3). Its precise function is unknown. Nevertheless, a role in cell proliferation has been proposed based on the following observations: prothymosin ␣ mRNA is plentiful only in rapidly dividing cells (13)(14)(15); the level of the protein declines 10-fold in cells forced to subsist in stationary phase (12); the amount of prothymosin ␣ is directly proportional to the proliferative activity of the tissue from which it is isolated (13); and the uptake of antisense oligodeoxyribonucleotides directed toward various locations in prothymosin ␣ mRNA prevents synchronized human myeloma cells from entering mitosis (16). Hence, a deficiency in prothymosin ␣ is associated with failure to complete the cell cycle.Prothymosin ␣ has several unusual features. The human protein, which is almost identical to that of all other mammals (17), has 109 amino acids, nearly 50% of which are acidic (1, 2). A potent nuclear localization signal consisting of five basic amino acids has been identified near the carboxyl terminus (3, 6), while a second cluster of five basic residues near the amino terminus has no unambiguous role (3,18). The absence of all aromatic residues renders the protein transparent at 280 nm; it also lacks methionine, cysteine, and histidine. Based on biophysical data, it is believed to have an unfolded structure (19), a conclusion consistent with the presence of only seven widely dispersed hydrophobic residues in the human protein. Prothymosin ␣ does not bind SDS and stains anomalously with silver stain (2). The protein and the peptides derived from it exhibit poor immunogenicity. However, due to yet another aberrant property, the ability to partition quantitatively into the aqueous phase ...

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“…At low concentrations the detergent digitonin affects the permeability of the plasma membrane without having any detectable effects on the integrity of the peroxisomal membrane [37]. As a result, cytosolic proteins are released from cells during incubation with digitonin but there is no release of peroxisomal proteins.…”

Section: Resultsmentioning

confidence: 99%

Farnesyl diphosphate synthase, the target for nitrogen-containing bisphosphonate drugs, is a peroxisomal enzyme in the model system Dictyostelium discoideum

Nuttall

Hettema

Watts

2012

Biochemical Journal

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NBP (nitrogen-containing bisphosphonate) drugs protect against excessive osteoclast-mediated bone resorption. After binding to bone mineral, they are taken up selectively by the osteoclasts and inhibit the essential enzyme FDPS (farnesyl diphosphate synthase). NBPs inhibit also growth of amoebae of Dictyostelium discoideum in which their target is again FDPS. A fusion protein between FDPS and GFP (green fluorescent protein) was found, in D. discoideum, to localize to peroxisomes and to confer resistance to the NBP alendronate. GFP was also directed to peroxisomes by a fragment of FDPS comprising amino acids 1–22. This contains a sequence of nine amino acids that closely resembles the nonapeptide PTS2 (peroxisomal targeting signal type 2): there is only a single amino acid mismatch between the two sequences. Mutation analysis confirmed that the atypical PTS2 directs FDPS into peroxisomes. Furthermore, expression of the D. discoideum FDPS–GFP fusion protein in strains of Saccharomyces cerevisiae defective in peroxisomal protein import demonstrated that import of FDPS into peroxisomes was blocked in a strain lacking the PTS2-dependent import pathway. The peroxisomal location of FDPS in D. discoideum indicates that NBPs have to cross the peroxisomal membrane before they can bind to their target.

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Studies on the intracellular distributions of soluble epoxide hydrolase and of catalase by digitonin‐permeabilization of hepatocytes isolated from control and clofibrate‐treated mice

Cited by 34 publications

References 29 publications

Soluble epoxide hydrolase: Gene structure, expression and deletion

Soluble epoxide hydrolase: Gene structure, expression and deletion

Turnover of the Acyl Phosphates of Human and Murine Prothymosin α in Vivo

Farnesyl diphosphate synthase, the target for nitrogen-containing bisphosphonate drugs, is a peroxisomal enzyme in the model system Dictyostelium discoideum

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