hilic column  (Agilent technologies)

 
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    Name:
    AdvanceBio Glycan Mapping
    Description:
    AdvanceBio Glycan Mapping columns are designed and manufactured to deliver fast high resolution reproducible glycan identification using HILIC chromatography Two UHPLC configurations are available 2 7 µm superficially porous for high resolution and lower backpressure and 1 8 µm for highest resolution AdvanceBio Glycan Mapping columns leverage technology that optimizes results for MS and fluorescence detection They can deliver glycan maps in less than 10 minutes 40 less time than competing products and can be ordered with a selection of standards for performance testing and retention mapping of labeled and unlabeled glycans
    Catalog Number:
    685775-913
    Price:
    None
    Category:
    Products Biopharma Hplc Analysis Glycan Analysis Glycan Analysis Columns Advancebio Glycan Mapping
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    Structured Review

    Agilent technologies hilic column
    Phosphoenolpyruvate Accumulates in Cells with Low PYK Activity and Inhibits Triosephosphate Isomerase (A) <t>HILIC-MRM</t> quantification of <t>PEP</t> (left panel) Standard addition of PEP to whole-cell methanol/water yeast extracts; and quantification of PEP separated by HILIC using multiple reaction monitoring (MRM). Quantification is demonstrated by linear regression, R 2 vales of > 0.97 were obtained. (Right panel) HILIC-MRM quantification of PEP in TEF pr -PYK1 , CYC pr -PYK1 , and CYC pr -PYK2 yeast; PEP is strongly accumulated in yeast with low PYK activity. Error bars, ±SD, n = 3. (B) PEP inactivates yeast and mammalian TPI. Michaelis-Menten kinetics were determined for yeast TPI (left panel), human TPI (middle panel), and rabbit muscle TPI (right panel). To determine v max and K m , the TPI substrate gly3p was added in incremental doses (left y and lower x axis, blue; y values are normalized to total cellular protein [left and middle panel] or to the purified protein [right panel]). For all TPI isozymes, v max is highlighted with a green dot in the saturation curve; K m values are given in μM (right y and upper x axis, black). Inhibition of TPI activity demonstrated by addition of PEP in incremental concentrations; IC 50 values and the inhibitory constant K i are given in μM, the Kcat in Mol ∗ s −1 . (C) Inactivation of pathogenic TPI alleles by PEP. TPI activity was assayed in transgenic yeast expressing human TPI (WT) or indicated pathogenic TPI alleles without or in the presence of 900 μmol PEP. Error bars, ±SD. Values within bars indicate the absolute enzyme activity in μmol/(min ∗ μg protein). (D) PYK activity does not change oxidant resistance in yeast expressing TPI Ile170Val . The wild-type control, transgenic yeast expressing either PYK1 or PYK2 , and either human TPI or human TPI Ile170Val were spotted as serial dilutions onto SC media without or with diamide (left panel). Spot growth on different concentrations was analyzed with CellProfiler and normalized to the strain with highest PYK activity (right panel). Low PYK activity increased diamide tolerance if expressed in combination with human TPI, but there were no differences in TPI Ile170Val -expressing yeast.
    AdvanceBio Glycan Mapping columns are designed and manufactured to deliver fast high resolution reproducible glycan identification using HILIC chromatography Two UHPLC configurations are available 2 7 µm superficially porous for high resolution and lower backpressure and 1 8 µm for highest resolution AdvanceBio Glycan Mapping columns leverage technology that optimizes results for MS and fluorescence detection They can deliver glycan maps in less than 10 minutes 40 less time than competing products and can be ordered with a selection of standards for performance testing and retention mapping of labeled and unlabeled glycans
    https://www.bioz.com/result/hilic column/product/Agilent technologies
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    hilic column - by Bioz Stars, 2021-09
    86/100 stars

    Images

    1) Product Images from "Pyruvate Kinase Triggers a Metabolic Feedback Loop that Controls Redox Metabolism in Respiring Cells"

    Article Title: Pyruvate Kinase Triggers a Metabolic Feedback Loop that Controls Redox Metabolism in Respiring Cells

    Journal: Cell Metabolism

    doi: 10.1016/j.cmet.2011.06.017

    Phosphoenolpyruvate Accumulates in Cells with Low PYK Activity and Inhibits Triosephosphate Isomerase (A) HILIC-MRM quantification of PEP (left panel) Standard addition of PEP to whole-cell methanol/water yeast extracts; and quantification of PEP separated by HILIC using multiple reaction monitoring (MRM). Quantification is demonstrated by linear regression, R 2 vales of > 0.97 were obtained. (Right panel) HILIC-MRM quantification of PEP in TEF pr -PYK1 , CYC pr -PYK1 , and CYC pr -PYK2 yeast; PEP is strongly accumulated in yeast with low PYK activity. Error bars, ±SD, n = 3. (B) PEP inactivates yeast and mammalian TPI. Michaelis-Menten kinetics were determined for yeast TPI (left panel), human TPI (middle panel), and rabbit muscle TPI (right panel). To determine v max and K m , the TPI substrate gly3p was added in incremental doses (left y and lower x axis, blue; y values are normalized to total cellular protein [left and middle panel] or to the purified protein [right panel]). For all TPI isozymes, v max is highlighted with a green dot in the saturation curve; K m values are given in μM (right y and upper x axis, black). Inhibition of TPI activity demonstrated by addition of PEP in incremental concentrations; IC 50 values and the inhibitory constant K i are given in μM, the Kcat in Mol ∗ s −1 . (C) Inactivation of pathogenic TPI alleles by PEP. TPI activity was assayed in transgenic yeast expressing human TPI (WT) or indicated pathogenic TPI alleles without or in the presence of 900 μmol PEP. Error bars, ±SD. Values within bars indicate the absolute enzyme activity in μmol/(min ∗ μg protein). (D) PYK activity does not change oxidant resistance in yeast expressing TPI Ile170Val . The wild-type control, transgenic yeast expressing either PYK1 or PYK2 , and either human TPI or human TPI Ile170Val were spotted as serial dilutions onto SC media without or with diamide (left panel). Spot growth on different concentrations was analyzed with CellProfiler and normalized to the strain with highest PYK activity (right panel). Low PYK activity increased diamide tolerance if expressed in combination with human TPI, but there were no differences in TPI Ile170Val -expressing yeast.
    Figure Legend Snippet: Phosphoenolpyruvate Accumulates in Cells with Low PYK Activity and Inhibits Triosephosphate Isomerase (A) HILIC-MRM quantification of PEP (left panel) Standard addition of PEP to whole-cell methanol/water yeast extracts; and quantification of PEP separated by HILIC using multiple reaction monitoring (MRM). Quantification is demonstrated by linear regression, R 2 vales of > 0.97 were obtained. (Right panel) HILIC-MRM quantification of PEP in TEF pr -PYK1 , CYC pr -PYK1 , and CYC pr -PYK2 yeast; PEP is strongly accumulated in yeast with low PYK activity. Error bars, ±SD, n = 3. (B) PEP inactivates yeast and mammalian TPI. Michaelis-Menten kinetics were determined for yeast TPI (left panel), human TPI (middle panel), and rabbit muscle TPI (right panel). To determine v max and K m , the TPI substrate gly3p was added in incremental doses (left y and lower x axis, blue; y values are normalized to total cellular protein [left and middle panel] or to the purified protein [right panel]). For all TPI isozymes, v max is highlighted with a green dot in the saturation curve; K m values are given in μM (right y and upper x axis, black). Inhibition of TPI activity demonstrated by addition of PEP in incremental concentrations; IC 50 values and the inhibitory constant K i are given in μM, the Kcat in Mol ∗ s −1 . (C) Inactivation of pathogenic TPI alleles by PEP. TPI activity was assayed in transgenic yeast expressing human TPI (WT) or indicated pathogenic TPI alleles without or in the presence of 900 μmol PEP. Error bars, ±SD. Values within bars indicate the absolute enzyme activity in μmol/(min ∗ μg protein). (D) PYK activity does not change oxidant resistance in yeast expressing TPI Ile170Val . The wild-type control, transgenic yeast expressing either PYK1 or PYK2 , and either human TPI or human TPI Ile170Val were spotted as serial dilutions onto SC media without or with diamide (left panel). Spot growth on different concentrations was analyzed with CellProfiler and normalized to the strain with highest PYK activity (right panel). Low PYK activity increased diamide tolerance if expressed in combination with human TPI, but there were no differences in TPI Ile170Val -expressing yeast.

    Techniques Used: Activity Assay, Hydrophilic Interaction Liquid Chromatography, Standard Addition, Purification, Inhibition, Transgenic Assay, Expressing

    Related Articles

    High Performance Liquid Chromatography:

    Article Title: Crystal structures of human ENPP1 in apo and bound forms
    Article Snippet: .. RapiFluor-MS-labelled N-glycans were separated on an AdvanceBio Glycan Mapping column (Agilent Technologies; 120 Å, 2.1 × 250 mm, 2.7 µm) using an UltiMate 3000 HPLC system (Thermo Fisher, USA) connected to a MicrOTOF-Q II mass spectrometer (Bruker-Daltonics, Germany). ..

    Article Title: Moss-Derived Human Recombinant GAA Provides an Optimized Enzyme Uptake in Differentiated Human Muscle Cells of Pompe Disease
    Article Snippet: .. Labeled glycans were separated on an Agilent AdvanceBio Glycan Mapping column (2.1 × 150 mm, 1.8 μm) with a Security Guard Ultra precolumn (Phenomenex; Aschaffenburg, Germany) on a Nexera X2 HPLC system with a RF-20Axs Fluorescence Detector, equipped with a semimicro flow cell (Shimadzu, Korneuburg, Austria). ..

    Article Title: Microfluidic ion stripper for removal of trifluoroacetic acid from mobile phases used in HILIC-MS of intact proteins
    Article Snippet: .. LC separationsHILIC protein separations were performed on an Agilent HPLC 1290 Infinity II (Waldbronn, Germany), composed of a binary pump, autosampler, column thermostat, and variable wavelength detector, using an Agilent AdvanceBio Glycan Mapping column (150 × 2.1 mm, 1.8 μm particle diameter with 300 Å pore size) with a Phenomenex SecurityGuard Ultra Cartridge (Widepore C4; 2 × 4.6 mm i.d.) (Utrecht, The Netherlands). ..

    Article Title: Reduced myocyte complex N-glycosylation causes dilated cardiomyopathy
    Article Snippet: .. Dried glycans were resuspended in 80% acetonitrile and separated by hydrophilic-interaction liquid chromatography (HILIC) ( ) with an amide glycan-mapping column (683775-913; Agilent Technologies, Santa Clara, CA, USA) attached to an Agilent 1100 HPLC system. ..

    Article Title: Developing a medium combination to attain similar glycosylation profile to originator by DoE and cluster analysis method
    Article Snippet: .. The N-glycans were released from 300 μg antibody by digestion with 0.5 μL PNGaseF (PO704L, Biolabs NEB) followed by labeling with 6 μL 2-AB (76,884, Sigma) at 65 °C for 3 h. Glycosylation patterns were analyzed by HPLC (1260, Agilent) with AdvanceBio glycan mapping column (4.6 mm × 150 mm, Agilent) and fluorescence detector (Ex: 260 nm, Em: 430 nm). ..

    Mass Spectrometry:

    Article Title: Crystal structures of human ENPP1 in apo and bound forms
    Article Snippet: .. RapiFluor-MS-labelled N-glycans were separated on an AdvanceBio Glycan Mapping column (Agilent Technologies; 120 Å, 2.1 × 250 mm, 2.7 µm) using an UltiMate 3000 HPLC system (Thermo Fisher, USA) connected to a MicrOTOF-Q II mass spectrometer (Bruker-Daltonics, Germany). ..

    Hydrophilic Interaction Liquid Chromatography:

    Article Title: The structure of an archaeal oligosaccharyltransferase provides insight into the strict exclusion of proline from the N-glycosylation sequon
    Article Snippet: .. The pyridylaminated oligosaccharides were separated by HILIC (hydrophilic interaction liquid chromatography) chromatography with an AdvanceBio Glycan Mapping column (Agilent Technologies), using an Infinity 1290 UPLC system (Agilent Technologies) equipped with an in-line fluorescence detector. ..

    Article Title: Identification and characterization of a residual host cell protein hexosaminidase B associated with N‐glycan degradation during the stability study of a therapeutic recombinant monoclonal antibody product, et al. Identification and characterization of a residual host cell protein hexosaminidase B associated with N‐glycan degradation during the stability study of a therapeutic recombinant monoclonal antibody product
    Article Snippet: .. N ‐glycan was analyzed by HILIC‐UPLC with AdvanceBio Glycan column (2.1 × 150 mm, 1.8 μm, Agilent). ..

    Article Title: MS-Based Allotype-Specific Analysis of Polyclonal IgG-Fc N-Glycosylation
    Article Snippet: .. The capillary amideHILIC column was packed using the stationary phase obtained from unpacking a HILIC column (Agilent AdvanceBio glycan mapping column, 125 Å, 2.7 μm particles). ..

    Article Title: Reduced myocyte complex N-glycosylation causes dilated cardiomyopathy
    Article Snippet: .. Dried glycans were resuspended in 80% acetonitrile and separated by hydrophilic-interaction liquid chromatography (HILIC) ( ) with an amide glycan-mapping column (683775-913; Agilent Technologies, Santa Clara, CA, USA) attached to an Agilent 1100 HPLC system. ..

    Chromatography:

    Article Title: The structure of an archaeal oligosaccharyltransferase provides insight into the strict exclusion of proline from the N-glycosylation sequon
    Article Snippet: .. The pyridylaminated oligosaccharides were separated by HILIC (hydrophilic interaction liquid chromatography) chromatography with an AdvanceBio Glycan Mapping column (Agilent Technologies), using an Infinity 1290 UPLC system (Agilent Technologies) equipped with an in-line fluorescence detector. ..

    Fluorescence:

    Article Title: The structure of an archaeal oligosaccharyltransferase provides insight into the strict exclusion of proline from the N-glycosylation sequon
    Article Snippet: .. The pyridylaminated oligosaccharides were separated by HILIC (hydrophilic interaction liquid chromatography) chromatography with an AdvanceBio Glycan Mapping column (Agilent Technologies), using an Infinity 1290 UPLC system (Agilent Technologies) equipped with an in-line fluorescence detector. ..

    Article Title: Moss-Derived Human Recombinant GAA Provides an Optimized Enzyme Uptake in Differentiated Human Muscle Cells of Pompe Disease
    Article Snippet: .. Labeled glycans were separated on an Agilent AdvanceBio Glycan Mapping column (2.1 × 150 mm, 1.8 μm) with a Security Guard Ultra precolumn (Phenomenex; Aschaffenburg, Germany) on a Nexera X2 HPLC system with a RF-20Axs Fluorescence Detector, equipped with a semimicro flow cell (Shimadzu, Korneuburg, Austria). ..

    Article Title: Developing a medium combination to attain similar glycosylation profile to originator by DoE and cluster analysis method
    Article Snippet: .. The N-glycans were released from 300 μg antibody by digestion with 0.5 μL PNGaseF (PO704L, Biolabs NEB) followed by labeling with 6 μL 2-AB (76,884, Sigma) at 65 °C for 3 h. Glycosylation patterns were analyzed by HPLC (1260, Agilent) with AdvanceBio glycan mapping column (4.6 mm × 150 mm, Agilent) and fluorescence detector (Ex: 260 nm, Em: 430 nm). ..

    Labeling:

    Article Title: Moss-Derived Human Recombinant GAA Provides an Optimized Enzyme Uptake in Differentiated Human Muscle Cells of Pompe Disease
    Article Snippet: .. Labeled glycans were separated on an Agilent AdvanceBio Glycan Mapping column (2.1 × 150 mm, 1.8 μm) with a Security Guard Ultra precolumn (Phenomenex; Aschaffenburg, Germany) on a Nexera X2 HPLC system with a RF-20Axs Fluorescence Detector, equipped with a semimicro flow cell (Shimadzu, Korneuburg, Austria). ..

    Article Title: Developing a medium combination to attain similar glycosylation profile to originator by DoE and cluster analysis method
    Article Snippet: .. The N-glycans were released from 300 μg antibody by digestion with 0.5 μL PNGaseF (PO704L, Biolabs NEB) followed by labeling with 6 μL 2-AB (76,884, Sigma) at 65 °C for 3 h. Glycosylation patterns were analyzed by HPLC (1260, Agilent) with AdvanceBio glycan mapping column (4.6 mm × 150 mm, Agilent) and fluorescence detector (Ex: 260 nm, Em: 430 nm). ..

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  • 86
    Agilent technologies hilic column
    Phosphoenolpyruvate Accumulates in Cells with Low PYK Activity and Inhibits Triosephosphate Isomerase (A) <t>HILIC-MRM</t> quantification of <t>PEP</t> (left panel) Standard addition of PEP to whole-cell methanol/water yeast extracts; and quantification of PEP separated by HILIC using multiple reaction monitoring (MRM). Quantification is demonstrated by linear regression, R 2 vales of > 0.97 were obtained. (Right panel) HILIC-MRM quantification of PEP in TEF pr -PYK1 , CYC pr -PYK1 , and CYC pr -PYK2 yeast; PEP is strongly accumulated in yeast with low PYK activity. Error bars, ±SD, n = 3. (B) PEP inactivates yeast and mammalian TPI. Michaelis-Menten kinetics were determined for yeast TPI (left panel), human TPI (middle panel), and rabbit muscle TPI (right panel). To determine v max and K m , the TPI substrate gly3p was added in incremental doses (left y and lower x axis, blue; y values are normalized to total cellular protein [left and middle panel] or to the purified protein [right panel]). For all TPI isozymes, v max is highlighted with a green dot in the saturation curve; K m values are given in μM (right y and upper x axis, black). Inhibition of TPI activity demonstrated by addition of PEP in incremental concentrations; IC 50 values and the inhibitory constant K i are given in μM, the Kcat in Mol ∗ s −1 . (C) Inactivation of pathogenic TPI alleles by PEP. TPI activity was assayed in transgenic yeast expressing human TPI (WT) or indicated pathogenic TPI alleles without or in the presence of 900 μmol PEP. Error bars, ±SD. Values within bars indicate the absolute enzyme activity in μmol/(min ∗ μg protein). (D) PYK activity does not change oxidant resistance in yeast expressing TPI Ile170Val . The wild-type control, transgenic yeast expressing either PYK1 or PYK2 , and either human TPI or human TPI Ile170Val were spotted as serial dilutions onto SC media without or with diamide (left panel). Spot growth on different concentrations was analyzed with CellProfiler and normalized to the strain with highest PYK activity (right panel). Low PYK activity increased diamide tolerance if expressed in combination with human TPI, but there were no differences in TPI Ile170Val -expressing yeast.
    Hilic Column, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/hilic column/product/Agilent technologies
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    hilic column - by Bioz Stars, 2021-09
    86/100 stars
      Buy from Supplier

    99
    Agilent technologies poroshell 120 hilic z column
    Phosphoenolpyruvate Accumulates in Cells with Low PYK Activity and Inhibits Triosephosphate Isomerase (A) <t>HILIC-MRM</t> quantification of <t>PEP</t> (left panel) Standard addition of PEP to whole-cell methanol/water yeast extracts; and quantification of PEP separated by HILIC using multiple reaction monitoring (MRM). Quantification is demonstrated by linear regression, R 2 vales of > 0.97 were obtained. (Right panel) HILIC-MRM quantification of PEP in TEF pr -PYK1 , CYC pr -PYK1 , and CYC pr -PYK2 yeast; PEP is strongly accumulated in yeast with low PYK activity. Error bars, ±SD, n = 3. (B) PEP inactivates yeast and mammalian TPI. Michaelis-Menten kinetics were determined for yeast TPI (left panel), human TPI (middle panel), and rabbit muscle TPI (right panel). To determine v max and K m , the TPI substrate gly3p was added in incremental doses (left y and lower x axis, blue; y values are normalized to total cellular protein [left and middle panel] or to the purified protein [right panel]). For all TPI isozymes, v max is highlighted with a green dot in the saturation curve; K m values are given in μM (right y and upper x axis, black). Inhibition of TPI activity demonstrated by addition of PEP in incremental concentrations; IC 50 values and the inhibitory constant K i are given in μM, the Kcat in Mol ∗ s −1 . (C) Inactivation of pathogenic TPI alleles by PEP. TPI activity was assayed in transgenic yeast expressing human TPI (WT) or indicated pathogenic TPI alleles without or in the presence of 900 μmol PEP. Error bars, ±SD. Values within bars indicate the absolute enzyme activity in μmol/(min ∗ μg protein). (D) PYK activity does not change oxidant resistance in yeast expressing TPI Ile170Val . The wild-type control, transgenic yeast expressing either PYK1 or PYK2 , and either human TPI or human TPI Ile170Val were spotted as serial dilutions onto SC media without or with diamide (left panel). Spot growth on different concentrations was analyzed with CellProfiler and normalized to the strain with highest PYK activity (right panel). Low PYK activity increased diamide tolerance if expressed in combination with human TPI, but there were no differences in TPI Ile170Val -expressing yeast.
    Poroshell 120 Hilic Z Column, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/poroshell 120 hilic z column/product/Agilent technologies
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    poroshell 120 hilic z column - by Bioz Stars, 2021-09
    99/100 stars
      Buy from Supplier

    99
    Agilent technologies zorbax hilic plus column
    Phosphoenolpyruvate Accumulates in Cells with Low PYK Activity and Inhibits Triosephosphate Isomerase (A) <t>HILIC-MRM</t> quantification of <t>PEP</t> (left panel) Standard addition of PEP to whole-cell methanol/water yeast extracts; and quantification of PEP separated by HILIC using multiple reaction monitoring (MRM). Quantification is demonstrated by linear regression, R 2 vales of > 0.97 were obtained. (Right panel) HILIC-MRM quantification of PEP in TEF pr -PYK1 , CYC pr -PYK1 , and CYC pr -PYK2 yeast; PEP is strongly accumulated in yeast with low PYK activity. Error bars, ±SD, n = 3. (B) PEP inactivates yeast and mammalian TPI. Michaelis-Menten kinetics were determined for yeast TPI (left panel), human TPI (middle panel), and rabbit muscle TPI (right panel). To determine v max and K m , the TPI substrate gly3p was added in incremental doses (left y and lower x axis, blue; y values are normalized to total cellular protein [left and middle panel] or to the purified protein [right panel]). For all TPI isozymes, v max is highlighted with a green dot in the saturation curve; K m values are given in μM (right y and upper x axis, black). Inhibition of TPI activity demonstrated by addition of PEP in incremental concentrations; IC 50 values and the inhibitory constant K i are given in μM, the Kcat in Mol ∗ s −1 . (C) Inactivation of pathogenic TPI alleles by PEP. TPI activity was assayed in transgenic yeast expressing human TPI (WT) or indicated pathogenic TPI alleles without or in the presence of 900 μmol PEP. Error bars, ±SD. Values within bars indicate the absolute enzyme activity in μmol/(min ∗ μg protein). (D) PYK activity does not change oxidant resistance in yeast expressing TPI Ile170Val . The wild-type control, transgenic yeast expressing either PYK1 or PYK2 , and either human TPI or human TPI Ile170Val were spotted as serial dilutions onto SC media without or with diamide (left panel). Spot growth on different concentrations was analyzed with CellProfiler and normalized to the strain with highest PYK activity (right panel). Low PYK activity increased diamide tolerance if expressed in combination with human TPI, but there were no differences in TPI Ile170Val -expressing yeast.
    Zorbax Hilic Plus Column, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/zorbax hilic plus column/product/Agilent technologies
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    zorbax hilic plus column - by Bioz Stars, 2021-09
    99/100 stars
      Buy from Supplier

    Image Search Results


    Phosphoenolpyruvate Accumulates in Cells with Low PYK Activity and Inhibits Triosephosphate Isomerase (A) HILIC-MRM quantification of PEP (left panel) Standard addition of PEP to whole-cell methanol/water yeast extracts; and quantification of PEP separated by HILIC using multiple reaction monitoring (MRM). Quantification is demonstrated by linear regression, R 2 vales of > 0.97 were obtained. (Right panel) HILIC-MRM quantification of PEP in TEF pr -PYK1 , CYC pr -PYK1 , and CYC pr -PYK2 yeast; PEP is strongly accumulated in yeast with low PYK activity. Error bars, ±SD, n = 3. (B) PEP inactivates yeast and mammalian TPI. Michaelis-Menten kinetics were determined for yeast TPI (left panel), human TPI (middle panel), and rabbit muscle TPI (right panel). To determine v max and K m , the TPI substrate gly3p was added in incremental doses (left y and lower x axis, blue; y values are normalized to total cellular protein [left and middle panel] or to the purified protein [right panel]). For all TPI isozymes, v max is highlighted with a green dot in the saturation curve; K m values are given in μM (right y and upper x axis, black). Inhibition of TPI activity demonstrated by addition of PEP in incremental concentrations; IC 50 values and the inhibitory constant K i are given in μM, the Kcat in Mol ∗ s −1 . (C) Inactivation of pathogenic TPI alleles by PEP. TPI activity was assayed in transgenic yeast expressing human TPI (WT) or indicated pathogenic TPI alleles without or in the presence of 900 μmol PEP. Error bars, ±SD. Values within bars indicate the absolute enzyme activity in μmol/(min ∗ μg protein). (D) PYK activity does not change oxidant resistance in yeast expressing TPI Ile170Val . The wild-type control, transgenic yeast expressing either PYK1 or PYK2 , and either human TPI or human TPI Ile170Val were spotted as serial dilutions onto SC media without or with diamide (left panel). Spot growth on different concentrations was analyzed with CellProfiler and normalized to the strain with highest PYK activity (right panel). Low PYK activity increased diamide tolerance if expressed in combination with human TPI, but there were no differences in TPI Ile170Val -expressing yeast.

    Journal: Cell Metabolism

    Article Title: Pyruvate Kinase Triggers a Metabolic Feedback Loop that Controls Redox Metabolism in Respiring Cells

    doi: 10.1016/j.cmet.2011.06.017

    Figure Lengend Snippet: Phosphoenolpyruvate Accumulates in Cells with Low PYK Activity and Inhibits Triosephosphate Isomerase (A) HILIC-MRM quantification of PEP (left panel) Standard addition of PEP to whole-cell methanol/water yeast extracts; and quantification of PEP separated by HILIC using multiple reaction monitoring (MRM). Quantification is demonstrated by linear regression, R 2 vales of > 0.97 were obtained. (Right panel) HILIC-MRM quantification of PEP in TEF pr -PYK1 , CYC pr -PYK1 , and CYC pr -PYK2 yeast; PEP is strongly accumulated in yeast with low PYK activity. Error bars, ±SD, n = 3. (B) PEP inactivates yeast and mammalian TPI. Michaelis-Menten kinetics were determined for yeast TPI (left panel), human TPI (middle panel), and rabbit muscle TPI (right panel). To determine v max and K m , the TPI substrate gly3p was added in incremental doses (left y and lower x axis, blue; y values are normalized to total cellular protein [left and middle panel] or to the purified protein [right panel]). For all TPI isozymes, v max is highlighted with a green dot in the saturation curve; K m values are given in μM (right y and upper x axis, black). Inhibition of TPI activity demonstrated by addition of PEP in incremental concentrations; IC 50 values and the inhibitory constant K i are given in μM, the Kcat in Mol ∗ s −1 . (C) Inactivation of pathogenic TPI alleles by PEP. TPI activity was assayed in transgenic yeast expressing human TPI (WT) or indicated pathogenic TPI alleles without or in the presence of 900 μmol PEP. Error bars, ±SD. Values within bars indicate the absolute enzyme activity in μmol/(min ∗ μg protein). (D) PYK activity does not change oxidant resistance in yeast expressing TPI Ile170Val . The wild-type control, transgenic yeast expressing either PYK1 or PYK2 , and either human TPI or human TPI Ile170Val were spotted as serial dilutions onto SC media without or with diamide (left panel). Spot growth on different concentrations was analyzed with CellProfiler and normalized to the strain with highest PYK activity (right panel). Low PYK activity increased diamide tolerance if expressed in combination with human TPI, but there were no differences in TPI Ile170Val -expressing yeast.

    Article Snippet: PEP was extracted with methanol/water and separated on a HILIC column (1.7 μm particle size, 2.1 × 100 mm) using ultra-high-pressure binary pump (Agilent 1290) at 800–1000 bar and a flow rate of 1 ml/min.

    Techniques: Activity Assay, Hydrophilic Interaction Liquid Chromatography, Standard Addition, Purification, Inhibition, Transgenic Assay, Expressing