Iodixanol (Optiprep), an Improved Density Gradient Medium for the Iso-osmotic Isolation of Rat Liver Peroxisomes

Veldhoven, Paul P. Van; Baumgart, Eveline; Mannaerts, Guy P.

doi:10.1006/abio.1996.0194

Cited by 64 publications

(56 citation statements)

References 21 publications

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“…Glucose-6-phosphatase (Glc-6-Pase) activity was measured by quantifying the release of phosphate from glucose 6-phosphate by an adapted method of Fiske-SubbaRow (see 23). Pyruvate dehydrogenase (PDH) activity was quantified using a PDH enzyme activity kit (MitoSciences) according to the manufacturer's guidelines.…”

Section: Protein Analysismentioning

confidence: 99%

Carbohydrate Metabolism Is Perturbed in Peroxisome-deficient Hepatocytes Due to Mitochondrial Dysfunction, AMP-activated Protein Kinase (AMPK) Activation, and Peroxisome Proliferator-activated Receptor γ Coactivator 1α (PGC-1α) Suppression

Peeters

Fraisl

Berg

et al. 2011

Journal of Biological Chemistry

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Background:It is not known whether peroxisomes influence hepatic carbohydrate metabolism. Results: Carbohydrate metabolism is perturbed in peroxisome-deficient mouse liver through mitochondrial deficits, AMPK, and PGC-1␣. Conclusion: Dysfunctional peroxisome metabolism disrupts carbohydrate homeostasis by indirect mechanisms. Significance: The impact of peroxisome deficiency on liver metabolism is broader than expected.

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Section: Protein Analysismentioning

confidence: 99%

Carbohydrate Metabolism Is Perturbed in Peroxisome-deficient Hepatocytes Due to Mitochondrial Dysfunction, AMP-activated Protein Kinase (AMPK) Activation, and Peroxisome Proliferator-activated Receptor γ Coactivator 1α (PGC-1α) Suppression

Peeters

Fraisl

Berg

et al. 2011

Journal of Biological Chemistry

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“…After centrifugation at 108,000 g (Beckman SW55) during 45 min, the supernatant, containing interfering myelin, was discarded. Marker enzyme and protein measurements were done as described previously for glutamate dehydrogenase (33), catalase (31), acid phosphatase (33), glucose-6-phosphatase (33), carboxylesterase (31), lactate dehydrogenase (33), proteins (31), and CERK.…”

Section: Tissue Distribution and Subcellular Studiesmentioning

confidence: 99%

Further characterization of mammalian ceramide kinase: substrate delivery and (stereo)specificity, tissue distribution, and subcellular localization studies

Overloop

Gijsbers²,

Veldhoven

2006

Journal of Lipid Research

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Recombinant human ceramide kinase (HsCERK) was analyzed with regard to dependence on divalent cations and to substrate delivery, spectrum, specificity, and stereoselectivity. Depending on the chain length of the ceramide, either albumin for short-chain ceramide or a mixed micellar form (octylglucoside/cardiolipin) for long-chain ceramide was preferred for the substrate delivery, the former resulting in higher activities. Bacterially expressed HsCERK was highly dependent on Mg 21 ions, much less on Ca 21 ions. A clear preference for the D-erythro isomer was seen. Various N-acylated amino alcohols were no substrate, but Nhexanoyl-1-O-hexadecyl-2-desoxy-2-amino-sn-glycerol and Ntetradecanoyl-2S-amino-1-butanol were phosphorylated, suggesting that the secondary hydroxy group is not required for recognition. The properties of HsCERK, expressed in CHO cells, were similar to those of the bacterially expressed protein, including the Mg 21 dependence. In mouse, the highest activities were found in testis and cerebellum, and upon subcellular fractionation the activity was recovered mainly in the microsomal fraction. This fits with the plasma membrane localization in CHO cells, which was mediated by the N-terminal putative pleckstrin domain.No evidence for phosphorylation of ceramide by the recently described multiple lipid kinase was found. The latter kinase is localized in the mitochondria, but no firm conclusions with regard to its substrate could be drawn.

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“…We therefore believe that the enzyme catalyses peroxisomal acetoacetyl-CoA synthesis. Although the amount and activity of pTh are low in comparison with those of the other peroxisomal thiolases and in comparison (A) A light mitochondrial fraction (33.9 mg of protein) containing 124 U of catalase (peroxisomal marker enzyme), 3.5 U of glucose 6-phosphatase (marker enzyme for the endoplasmic reticulum), 24 U of glutamate dehydrogenase (mitochondrial marker enzyme) and 7.8 U of acid phosphatase (lysosomal marker enzyme) was subfractionated in a preformed 20±40% (w/v) Optiprep density gradient [27]. Fractions were collected from the bottom and were analysed for protein (A; recovery 118%) and marker enzymes.…”

Section: Discussionmentioning

confidence: 99%

“…Subfractionation of a light mitochondrial fraction by means of an Iodixanol (Optiprep) density gradient and measuring of marker enzyme activities were performed as described [27]. SDS/PAGE in 10±20% (w/v) gradient gels, staining with Coomassie Blue or silver and immunoblotting were carried out as described before [28].…”

Section: Other Methodsmentioning

confidence: 99%

Identification, purification and characterization of an acetoacetyl-CoA thiolase from rat liver peroxisomes

Antonenkov¹,

Croes²,

Waelkens³

et al. 2000

Eur J Biochem

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Acetoacetyl-CoA specific thiolases catalyse the cleavage of acetoacetyl-CoA into two molecules of acetyl-CoA and the synthesis (reverse reaction) of acetoacetyl-CoA. The formation of acetoacetyl-CoA is the first step in cholesterol and ketone body synthesis. In this report we describe the identification of a novel acetoacetyl-CoA thiolase and its purification from isolated rat liver peroxisomes by column chromatography. The enzyme, which is a homotetramer with a subunit molecular mass of 42 kDa, could be distinguished from the cytosolic and mitochondrial acetoacetyl-CoA thiolases by its chromatographic behaviour, kinetic characteristics and partial internal amino-acid sequences. The enzyme did not catalyse the cleavage of medium or long chain 3-oxoacylCoAs. The enzyme cross-reacted with polyclonal antibodies raised against cytosolic acetoacetyl-CoA thiolase. The latter property was exploited to confirm the peroxisomal localization of the novel thiolase in subcellular fractionation experiments.The peroxisomal acetoacetyl-CoA thiolase most probably catalyses the first reaction in peroxisomal cholesterol and dolichol synthesis. In addition, its presence in peroxisomes along with the other enzymes of the ketogenic pathway indicates that the ketogenic potential of peroxisomes needs to be re-evaluated.

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Iodixanol (Optiprep), an Improved Density Gradient Medium for the Iso-osmotic Isolation of Rat Liver Peroxisomes

Cited by 64 publications

References 21 publications

Carbohydrate Metabolism Is Perturbed in Peroxisome-deficient Hepatocytes Due to Mitochondrial Dysfunction, AMP-activated Protein Kinase (AMPK) Activation, and Peroxisome Proliferator-activated Receptor γ Coactivator 1α (PGC-1α) Suppression

Carbohydrate Metabolism Is Perturbed in Peroxisome-deficient Hepatocytes Due to Mitochondrial Dysfunction, AMP-activated Protein Kinase (AMPK) Activation, and Peroxisome Proliferator-activated Receptor γ Coactivator 1α (PGC-1α) Suppression

Further characterization of mammalian ceramide kinase: substrate delivery and (stereo)specificity, tissue distribution, and subcellular localization studies

Identification, purification and characterization of an acetoacetyl-CoA thiolase from rat liver peroxisomes

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