Determination of plasma membrane lipid mass and composition in cultured Chinese hamster ovary cells using high gradient magnetic affinity chromatography

Warnock, Dale E.; Roberts, Charles W.M.; Lutz, Mallory S.; Blackburn, W A; Young, William W.; Baenziger, Jacques U.

doi:10.1016/s0021-9258(18)82184-6

Cited by 114 publications

(31 citation statements)

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“…An overall mean Ϯ SD of 24.8 Ϯ 1.7 fmol/cell (Ϯ6.9% SD) for the cellular concentration of free cholesterol was obtained (values from precursor ion scan of m/z 97). The resulting ratio of cholesterol to total phospholipids of 0.34 is consistent with data (0.36 and 0.34 mol/mol) from the literature (7,16).…”

Section: Sensitivitysupporting

confidence: 90%

“…Intact Golgi membranes of CHO cells were isolated according to Beckers and Rothman (6). In order to access the degree of enrichment and contamination, the following enzyme activities were determined: cytochrome c oxidoreductase as a marker for endoplasmic reticulum (7), APDE-1 as a marker for plasma membrane (7), ␤ -hexosaminidase as a marker for lysosomes/late endosomes (7), and sphingomyelin synthase as a marker for Golgi organelles (8,9). According to these markers, Golgi membranes in the isolated fraction are enriched 30-fold over the homogenate.…”

Section: Isolation Of Golgi Membranesmentioning

confidence: 99%

“…The concentration of the [3,[4][5][6][7][8][9][10][11][12][13] C 2 ]cholesterol standard solution was calculated from ESI-MS/MS data. Therefore the signal intensities of cholesterol and standard were compared in several samples containing a defined amount of cholesterol from a 99% pure cholesterol solution of known concentration and a defined volume of the standard solution.…”

Section: Standard Solutionmentioning

confidence: 99%

“…Aliquots of subcellular membrane fractions or total CHO cells were pipetted into a 500l micro test tube and water was added to a total volume of 20 l. All dioxane-containing solutions were handled with Hamilton syringes. Appropriate amounts of [3,[4][5][6][7][8][9][10][11][12][13] C 2 ]cholesterol standard from a stock solution, 4 m in 1,4-dioxane, were added and each sample was made up to a final volume of 120 l with 1,4-dioxane. The samples were mixed, sonicated in an ultrasonic bath at 100% (Bransonic 12) for 10-20 sec, and dried in a vacuum concentrator.…”

Section: Membrane Extractionmentioning

confidence: 99%

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Determination of cholesterol at the low picomole level by nano-electrospray ionization tandem mass spectrometry

Sandhoff

Brügger

Jeckel

et al. 1999

Journal of Lipid Research

103

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A mass spectrometric method for the quantification of free cholesterol in cells and subcellular membranes is presented. The method is based on a simple one-step chemical derivatization of cholesterol to cholesterol-3-sulfate by a sulfur trioxide-pyridine complex. Quantification is performed by nano-electrospray ionization tandem mass spectrometry (nanoESI-MS/MS) using a stable isotope labeled internal standard. The determination of free cholesterol is demonstrated in about 250 cells of a Chinese hamster ovary (CHO) cell line. With this method a molar ratio of free cholesterol to total phospholipids of 0.34 mol/mol in CHO cells was determined. In a subcellular membrane fraction enriched in Golgi membranes, a molar ratio of free cholesterol to total phospholipids of 0.57 mol/mol was determined. The method should be of value for quantification of other sterols as demonstrated for ergosterol and stigmasterol.-Sandhoff, R., B. Brügger, D. Jeckel, W. D. Lehmann, and F. T. Wieland. Determination of cholesterol at the low picomole level by nano-electrospray ionization tandem mass spectrometry.

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Section: Sensitivitysupporting

confidence: 90%

Section: Isolation Of Golgi Membranesmentioning

confidence: 99%

Section: Standard Solutionmentioning

confidence: 99%

Section: Membrane Extractionmentioning

confidence: 99%

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Determination of cholesterol at the low picomole level by nano-electrospray ionization tandem mass spectrometry

Sandhoff

Brügger

Jeckel

et al. 1999

Journal of Lipid Research

103

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“…C holesterol is the most abundant sterol in mammalian cells and an essential component of cell membranes. The major pool of membrane cholesterol is present at the plasma membrane (Coleman and Finean 1966;Pfleger et al 1968;DeGrella and Simoni 1982;Lange and Ramos 1983;Lange et al 1989;Warnock et al 1993). It is required for the formation of clathrin-coated pits (Rodal et al 1999) and for the structure and function of caveolae (Rothberg et al 1992;Parton et al 1994;Schnitzer et al 1994;Hailstones et al 1998;Ikonen and Parton 2000).…”

mentioning

confidence: 99%

Immunoelectron Microscopic Localization of Cholesterol Using Biotinylated and Non-cytolytic Perfringolysin O

Möbius

Ohno‐Iwashita

Donselaar

et al. 2002

J Histochem Cytochem.

270

185

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We used a proteolytically modified and biotinylated derivative of the cholesterol-binding Theta-toxin (perfringolysin O) to localize cholesterol-rich membranes in cryosections of cultured human lymphoblastoid cells (RN) by electron microscopy. We developed a fixation and immunolabeling procedure to improve the preservation of membranes and minimize the extraction and dislocalization of cholesterol on thin sections. We also labeled the surface of living cells and applied high-pressure freezing and subsequent fixation of cryosections during thawing. Cholesterol labeling was found at the plasma membrane, with strongest labeling on filopodium-like processes. Strong labeling was also associated with internal vesicles of multivesicular bodies (MVBs) and similar vesicles at the cell surface after secretion (exosomes). Tubulovesicular elements in close vicinity of endosomes and the Golgi complex were often positive as well, but the surrounding membrane of MVBs and the Golgi cisternae appeared mostly negative. Treatment of cells with methyl-beta-cyclodextrin completely abolished the labeling for cholesterol. Our results show that the Theta-toxin derivative, when used in combination with improved fixation and high-pressure freezing, represents a useful tool for the localization of membrane cholesterol in ultrathin cryosections.

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Cholelithiasis induced in the Syrian hamster: Evidence for an intramucinous nucleating process and down regulation of cholesterol 7α-hydroxylase (CYP7) gene by medroxyprogesterone

Gilloteaux

Karkare

Don

et al. 1997

Microsc. Res. Tech.

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This report reviews previously published studies from our laboratory and shows some recent morphological data obtained with scanning and transmission electron microscopy regarding gallstone formation and alteration of the gallbladder epithelium in the Syrian hamster model. Both male and female hamsters were treated with female sex steroids (estradiol alone, estradiol and medroxyprogesterone, medroxyprogesterone alone) during one month. The results show that the Syrian hamster is a good model to study bile changes, gallbladder structure changes, including gallstone formation, and the regulation of cholesterol metabolism at the molecular level. Arguments in favor of this animal model are presented and, during gallstone formation, epithelial cell changes, anionic mucus secretion, and formation of gallbladder luminal deposits can be demonstrated. Recent molecular biology observations related to the effect of female sex steroids on liver cholesterol 7α‐hydroxylase (CYP7) gene suggest that progestin alone or primed by estrogen down regulates CYP7 transcription and activity. In addition, progesterone in cell culture systems has been shown to enhance intracellular accumulation of free cholesterol by increasing its uptake and synthesis and by decreasing its esterification by inhibiting the activity of acylcoenzyme A: cholesterol acyltransferase. Non‐esterified cholesterol is free to migrate to the extracellular spaces and may contribute to nucleation within the bile. It is suggested that these effects of progesterone on cholesterol metabolism combined with the CYP7 gene down regulation, physical changes in the mucus and the hypomotility of the gallbladder and biliary ducts result in hypersaturation of cholesterol in the bile which favors gallstone formation. Microsc. Res. Tech. 39:56–70, 1997. © 1997 Wiley‐Liss, Inc.

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Determination of plasma membrane lipid mass and composition in cultured Chinese hamster ovary cells using high gradient magnetic affinity chromatography

Cited by 114 publications

References 0 publications

Determination of cholesterol at the low picomole level by nano-electrospray ionization tandem mass spectrometry

Determination of cholesterol at the low picomole level by nano-electrospray ionization tandem mass spectrometry

Immunoelectron Microscopic Localization of Cholesterol Using Biotinylated and Non-cytolytic Perfringolysin O

Cholelithiasis induced in the Syrian hamster: Evidence for an intramucinous nucleating process and down regulation of cholesterol 7α-hydroxylase (CYP7) gene by medroxyprogesterone

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