Improved horizontal two‐dimensional electrophoresis with hybrid isoelectric focusing in immobilized pH gradients in the first dimension and laying‐on transfer to the second dimension

Görg, Angelika; Postel, Wilhelm; Günther, Siegfried; Weser, Johann

doi:10.1002/elps.1150061206

Cited by 111 publications

(35 citation statements)

References 21 publications

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“…2D gel electrophoresis was performed as described by Gorg et al (29,30). In the first dimension, 50 or 500 g of protein were isoelectrically focused in 7-or 18-cm immobilized nonlinear pH 3-10 gradients at 6.5 or 12 kV-h (Amersham Biosciences Multiphor II).…”

Section: Methodsmentioning

confidence: 99%

The Lipolytic Proteome of Mouse Adipose Tissue

Birner‐Gruenberger

Susani-Etzerodt

Waldhuber

et al. 2005

Molecular & Cellular Proteomics

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In animals, plant seeds, and fungi, excessive amounts of lipids are stored in the form of intracellular triacylglycerol (TG) 1 and steryl ester deposits. Mammals store TG in adipose tissue as the primary source of energy during periods of starvation and increased energy demand. The balance of lipid storage and mobilization is tightly regulated to ensure whole body energy homeostasis. Stored fat is mobilized by activation of lipolytic enzymes, which degrade adipose TG and cholesterol esters and release non-esterified (free) fatty acids into the circulation. Variation in the concentration of circulating free fatty acids is an established risk factor for the development of insulin resistance in type 2 diabetes and related disorders (1-4). Cholesteryl esters represent the stored esterified form of cholesterol, which is an important constituent of membranes and a precursor of bile acid and steroid hormones. TG lipases and cholesteryl esterases are key enzymes in lipid mobilization, and respective lipolytic activities have been described in various tissues. However, only some of them have been identified on the molecular level. Many characterized lipases are secretory proteins (5-8), whereas the hormonesensitive lipase (HSL) (9), the monoglyceride lipase (MGL) (10), and the triacylglycerol hydrolase (TGH) (11) are intracellular enzymes. HSL was shown to hydrolyze TG and cholesteryl esters and was thought to catalyze the rate-limiting step in TG hydrolysis in adipose tissue. Yet in HSL knock-out mice, TG hydrolysis in adipose tissue as well as cholesteryl ester hydrolysis in macrophages were not abolished (9). MGL is ubiquitously expressed and rather specific for monoacylglycerol hydrolysis (10). TGH appears to be involved in TG mobilization in liver and is also expressed in adipose tissue where its role is not clear (11,12).The present study was aimed at the molecular identification of the lipolytic proteome of mouse adipose tissue using activity labels designed in our laboratory (Fig. 1). Basically an activity label is a molecule consisting of (i) a recognition site targeting a certain enzyme species, (ii) a properly positioned reactive site that forms a covalent bond with the target, and (iii) a tag for visualization and/or purification of the covalently bound target (13-16). The active site for lipases and esterases typically consists of a catalytic triad formed by Ser-His-(Asp/ Glu), which is common for most serine hydrolases (17). A feature specific for many lipases and esterases, however, is the ␣/␤-hydrolase fold consisting of a series of parallel ␤-sheets and a number of helices that flank the sheets on both sides (18,19). Most lipases contain a lid controlling the access of substrates to the hydrophobic active site. The same structural features are found in esterases except for the lid. Therefore, in contrast to lipases, most esterases do not show activation at lipid/water interfaces. However, it has to be emphasized that the borderline between lipases and esterFrom the ‡Institute

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Section: Methodsmentioning

confidence: 99%

The Lipolytic Proteome of Mouse Adipose Tissue

Birner‐Gruenberger

Susani-Etzerodt

Waldhuber

et al. 2005

Molecular & Cellular Proteomics

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“…Polyacrylamide gel electrophoresis was done under nondenaturing conditions or in the presence of 1% SDS in gradient slab gels (9.5 -22.6%) of0.5 mm thickness [lo]. Twodimensional electrophoresis followed a recently described procedure [16]. Gels were stained with Coomassie brilliant blue R-250.…”

Section: Methodsmentioning

confidence: 99%

Type 1, blue copper proteins constitute a respiratory nitrite‐reducing system in Pseudomonas aureofaciens

Zumft

Gotzmann

Kroneck

1987

European Journal of Biochemistry

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Pseudomonas aureofaciens truncates the respiratory reduction of nitrate (denitrification) at the level of N 2 0 . The nitrite reductase from this organism was purified to apparent electrophoretic homogeneity and found to be a blue copper protein. The enzyme contained 2 atoms of copper/85 kDa, both detectable by electron paramagnetic resonance (EPR) spectroscopy. The protein was dimeric, with subunits of identical size (40 3 kDa). Its PI was 6.05.The EPR spectrum showed an axial signal with 8 1 1 at 2.21(8) and g, at 2.04(5). The magnitude of the hyperfine splitting ( A 11 = 6.36 mT) indicated the presence of type 1 copper only. The electronic spectrum had maxima at 280 nm, 474 nm and 595 nm ( E = 7.0 mM-' cm-'), and a broad shoulder around 780 nm.A copper protein of low molecular mass (15 kDa), with properties similar to azurin, was also isolated from P. aureofaciens. The electronic spectrum of this protein showed a maximum at 624 nm in the visible range ( E = 2.5 mM-' cm-') and pronounced structures in the ultraviolet region. The EPR parameters were gll = 2.26(6) and g, = 2.05(6), with A II = 5.8 mT. The reduced azurin transferred electrons efficiently to nitrite reductase; the product of nitrite reduction was nitric oxide.The specific nitrite-reducing activity with ascorbate-reduced phenazine methosulfate as electron donor was 1 pmol substrate min-' mg protein-'. The reaction product again was nitric oxide. Nitrous oxide was the reaction product from hydroxylamine and nitrite and from dithionite-reduced methyl viologen and nitrite. No 'oxidase' activity could be demonstrated for the enzyme. Our data disprove the presumed exclusiveness of cytochrome cdl as nitrite reductase within the genus Pseudomonas.

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“…Sample solutions were applied to 18 cm immobilized pH gradient strips (Immobiline DryStrip pH 3-10 NL; Amersham Biosciences) (32) by rehydration loading (33,34). Isoelectric focusing and SDS-PAGE were performed as described previously (35,36) using 10% polyacrylamide gels. After isoelectric focusing, Immobiline Dry Strip pH 3-10 NL strips were stored at 270jC.…”

Section: Two-dimensional Gel Electrophoresis Of Labeled Proteinsmentioning

confidence: 99%

Import and fate of fluorescent analogs of oxidized phospholipids in vascular smooth muscle cells

Moumtzi

Trenker

Flicker

et al. 2007

Journal of Lipid Research

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Lipid oxidation is now thought to be an initiating and sustaining event in atherogenesis. Oxidatively fragmented phospholipids, namely 1-palmitoyl-2-glutaroylsn-glycero-3-phosphocholine (PGPC) and 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC), present in minimally modified LDL and atherosclerotic lesions, have been reported to elicit a wide range of pathophysiological responses in the cells of the vascular wall. Nevertheless, the question of their potential sites of action and their primary molecular targets remains open. To address this issue, a series of fluorescently labeled analogs, which differ with regard to structure and binding site of the fluorophore, were synthesized and used as tools for studying the uptake, intracellular stability, and distribution of PGPC and POVPC in vascular smooth muscle cells (VSMCs). We demonstrate that in accordance with their lysophospholipid-like structure, these highly similar molecules transferred rapidly either from aqueous phospholipid dispersions or preloaded native LDL into VSMCs, producing disparate fluorescence patterns irrespective of the attached fluorophore. PGPC derivatives were translocated to the lysosomes. In sharp contrast, POVPC analogs were initially captured in the plasma membrane, most likely in consequence of the formation of covalent adducts with free amino and sulfhydryl groups of proteins and phospholipids.

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Improved horizontal two‐dimensional electrophoresis with hybrid isoelectric focusing in immobilized pH gradients in the first dimension and laying‐on transfer to the second dimension

Cited by 111 publications

References 21 publications

The Lipolytic Proteome of Mouse Adipose Tissue

The Lipolytic Proteome of Mouse Adipose Tissue

Type 1, blue copper proteins constitute a respiratory nitrite‐reducing system in Pseudomonas aureofaciens

Import and fate of fluorescent analogs of oxidized phospholipids in vascular smooth muscle cells

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