Determination of microgram quantities of protein in the presence of milligram levels of lipid with amido black 10B1

Kaplan, Ronald S.; Pedersen, Peter L.

doi:10.1016/0003-2697(85)90445-2

Cited by 229 publications

(90 citation statements)

References 14 publications

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“…Cell cultures were grown, and the expression of UCP2, induced by isopropyl 1-thio-␤-D-galactopyranoside and including the inclusion bodies preparation, was performed as previously described (45). Protein content was assayed by the Amido Black method (52).…”

Section: Methodsmentioning

confidence: 99%

“…Reconstitution involved supplementing the extract by additional lipids (solubilized in octylpentaoxyethylene and adjusted to total 40 mg of egg yolk lecithin, type XI-E, supplemented with 5% cardiolipin), detergent removal on Bio-Beads SM2, and external probe removal on Sephadex G50 -300. The protein content of proteoliposomes was estimated by the Amido Black method (52). Usually, a lipid-to-protein ratio of about 200 or higher was obtained.…”

Section: Methodsmentioning

confidence: 99%

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Hydroperoxy Fatty Acid Cycling Mediated by Mitochondrial Uncoupling Protein UCP2

Jabůrek

Miyamoto

Mascio

et al. 2004

Journal of Biological Chemistry

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Using Escherichia coli-expressed UCP2 reconstituted into liposomes we found that LAOOH induced purine nucleotide-sensitive H ؉ uniport in UCP2-proteoliposomes with higher affinity than LA (K m values 97 M for LAOOH and 275 M for LA). In UCP2-proteoliposomes LAOOH also induced purine nucleotide-sensitive K ؉ influx balanced by anionic charge transfer, indicating that LAOOH was also transported as an anion with higher affinity than linoleate anion, the K m values being 90 and 350 M, respectively. These data suggest that hydroperoxy fatty acids are transported via UCP2 by a fatty acid cycling mechanism. This may alternatively explain the observed activation of UCP2 by the externally generated superoxide. The ability of LAOOH to induce UCP2-mediated H ؉ uniport points to the essential role of superoxide reaction products, such as hydroperoxyl radical, hydroxyl radical, or peroxynitrite, initiating lipoperoxidation, the released products of which support the UCP2-mediated uncoupling and promote the feedback down-regulation of mitochondrial reactive oxygen species production.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Hydroperoxy Fatty Acid Cycling Mediated by Mitochondrial Uncoupling Protein UCP2

Jabůrek

Miyamoto

Mascio

et al. 2004

Journal of Biological Chemistry

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“…Pellets (approximately 4.0 mg) were dissolved in approximately 600 mL of isoelectric focusing (IEF) buffer containing 8 M urea, 2 M thiourea, 2% CHAPS, 2% deionized Triton X-100, 50 mM dithiothreitol (DTT), and 0.5% pH 3 to 10 ampholytes. An Amido black assay (Kaplan and Pedersen, 1985) was used to determine concentration of protein. One to three milligrams of protein was extracted per gram of crown fresh weight.…”

Section: Sample Preparation For 2dementioning

confidence: 99%

Proteomic Study of Low-Temperature Responses in Strawberry Cultivars (Fragaria×ananassa) That Differ in Cold Tolerance

Koehler

Wilson²,

Goodpaster

et al. 2012

Plant Physiology

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To gain insight into the molecular basis contributing to overwintering hardiness, a comprehensive proteomic analysis comparing crowns of octoploid strawberry (Fragaria 3 ananassa) cultivars that differ in freezing tolerance was conducted. Four cultivars were examined for freeze tolerance and the most cold-tolerant cultivar ('Jonsok') and least-tolerant cultivar ('Frida') were compared with a goal to reveal how freezing tolerance is achieved in this distinctive overwintering structure and to identify potential cold-tolerance-associated biomarkers. Supported by univariate and multivariate analysis, a total of 63 spots from twodimensional electrophoresis analysis and 135 proteins from label-free quantitative proteomics were identified as significantly differentially expressed in crown tissue from the two strawberry cultivars exposed to 0-, 2-, and 42-d cold treatment. Proteins identified as cold-tolerance-associated included molecular chaperones, antioxidants/detoxifying enzymes, metabolic enzymes, pathogenesis-related proteins, and flavonoid pathway proteins. A number of proteins were newly identified as associated with cold tolerance. Distinctive mechanisms for cold tolerance were characterized for two cultivars. In particular, the 'Frida' cold response emphasized proteins specific to flavonoid biosynthesis, while the more freezing-tolerant 'Jonsok' had a more comprehensive suite of known stress-responsive proteins including those involved in antioxidation, detoxification, and disease resistance. The molecular basis for 'Jonsok'-enhanced cold tolerance can be explained by the constitutive level of a number of proteins that provide a physiological stress-tolerant poise. Strawberry (Fragaria 3 ananassa) cultivation predominates in regions with mild winters. In colder climates, overwintering hardiness is an essential trait for strawberry cultivation. Freezing injury of strawberry plants is one of the greatest factors in reducing crop yield and quality in temperate regions. Winter damage in Norway, for example, on average causes losses of 20%. Thus, production of cultivars with improved freezing hardiness is one of Norway's major objectives for their strawberry breeding programs. Improvement of cold hardiness is desirable for securing economic sustainability of the existing crops, and for expanding the growing regions of temperate fruit crops. Because strawberry is a representative species for the Rosaceae crops (e

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“…in the 3-2400 s range). Following preincubation with a given concentration of MTS reagent, transport was subsequently triggered via the addition of 21.5 l of [1,[5][6][7][8][9][10][11][12][13][14] C]citrate (Amersham Pharmacia Biotech; specific radioactivity ϳ2.4 ϫ 10 4 dpm/nmol; concentration in reaction mix, 1.04 mM). Reactions were allowed to proceed for a time interval defined by the intrinsic activity of a given mutant.…”

Section: Measurement Of the Effect Of Mts Reagents On Reconstituted Cmentioning

confidence: 99%

The Yeast Mitochondrial Citrate Transport Protein

Kaplan¹,

Mayor²,

Brauer³

et al. 2000

Journal of Biological Chemistry

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The mitochondrial citrate transport protein (CTP) has been investigated by replacing 22 consecutive residues within transmembrane domain IV, one at a time, with cysteine. A cysteine-less CTP retaining wild-type functional properties served as the starting template. The single Cys CTP variants were overexpressed in Escherichia coli, isolated, and functionally reconstituted in a liposomal system. The accessibility of each single Cys mutant to three methanethiosulfonate reagents was evaluated by determining the pseudo first order rate constants for inhibition of CTP function. These rate constants varied by seven orders of magnitude. With three independent data sets we observed peaks and troughs in the rate constant data at identical amino acid positions and a periodicity of four was observed from residues 177-193. Based on the pattern of accessibility we conclude that residues 177-193 exist as an ␣-helix. The citrate transport protein (i.e. CTP) 1 from mammalian mitochondria catalyzes an electroneutral exchange across the inner mitochondrial membrane of a tricarboxylate (i.e. citrate, isocitrate, cis-aconitate) plus a proton, for either another tricarboxylate-H ϩ , a dicarboxylate (i.e. malate, succinate), or phosphoenolpyruvate (1). Following the efflux of citrate from the mitochondrial matrix via the CTP, cytoplasmic citrate serves as both a carbon precursor for the fatty acid and the sterol biosynthetic pathways, as well as a source of NAD ϩ (via the concerted actions of ATP-citrate lyase and malate dehydrogenase) for the glycolytic pathway (2-5). Because of the central role of the CTP in higher eukaryotic metabolism, the transporter has been extensively studied. Thus, it has been purified (6, 7), kinetically characterized (8), cloned (9), and overexpressed (10). More recently we have extended our studies of the CTP to the yeast homologue of the higher eukaryotic protein (11). An advantage afforded by the yeast mitochondrial CTP is that following overexpression in Escherichia coli and subsequent isolation, CTP function can be reconstituted with high specific activity. Thus, the yeast CTP represents ideal material with which to conduct a comprehensive structure/function analysis. With this goal in mind, we recently constructed a Cys-less CTP that displays near native functional properties. Moreover, we have demonstrated that upon isolation, both the isolated wild-type and Cys-less CTPs exist as functional dimers.3 Based on hydropathy analysis, each monomer of the homodimer is predicted to contain six membrane-spanning domains (11).Within the CTP, transmembrane domain IV (i.e. TMDIV) is of particular interest for several reasons. First, this domain contains two arginine residues (Arg-181 and -189). We have previously demonstrated that CTP function requires the presence of positive charge at both sites.2 Second, TMDIV can be modeled as an ␣-helix with both arginines residing on the same face of the helix, thereby providing a polar domain, which might conceivably be accessible to citrate and provide a partial neutra...

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Determination of microgram quantities of protein in the presence of milligram levels of lipid with amido black 10B1

Cited by 229 publications

References 14 publications

Hydroperoxy Fatty Acid Cycling Mediated by Mitochondrial Uncoupling Protein UCP2

Hydroperoxy Fatty Acid Cycling Mediated by Mitochondrial Uncoupling Protein UCP2

Proteomic Study of Low-Temperature Responses in Strawberry Cultivars (Fragaria×ananassa) That Differ in Cold Tolerance

The Yeast Mitochondrial Citrate Transport Protein

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