mant atp  (Jena Bioscience)


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  • 95
    Name:
    Mant ATP
    Description:

    Catalog Number:
    nu-202l
    Price:
    343.57
    Applications:
    Conformational dynamic: DnaB/C-protein[1], Csk[2] Inhibition of AC-isoforms[3] Fluorescence stop-flow kinetics: PKA[4] FRET: AC[5], myosin V[6]
    Purity:
    ≥ 95 % (HPLC)
    Category:
    Nucleotides Nucleosides
    Buy from Supplier


    Structured Review

    Jena Bioscience mant atp
    The effect of ionomycin on <t>ATP</t> release and vesicular fusion in MDCK cells (A) total internal reflection fluorescence (TIRF) microscopy of MDCK cells loaded with <t>MANT-ATP</t> (25 μM, 5 h, 37°C) showing vesicular fusion after the addition of ionomycin (1 μM) . Arrows indicate vesicles that abruptly disappeared from the evanescence field. (B) Ionomycin (1 μM)-induced vesicular fusion as indicated by wide-field fluorescence microscopy of MDCK cells loaded with quinacrine (5 μM, 30 min, 37°C). Arrows indicate vesicles that display an abruptly drop in fluorescence. (C) Right: Original trace of the effect of ionomycin (1 μM, 37°C) on the relative fluorescence intensity of quinacrine-loaded MDCK cells. The left panel present the mean ± s.e.m. from 6 experiments. (D) ATP release from MDCK cells induced by apical application of ionomycin (IONO, 1 μM). The arrow indicates the addition of ionomycin or the control solution; the mean ± s.e.m. values are presented ( n = 10). The asterisk indicates statistical significance.

    https://www.bioz.com/result/mant atp/product/Jena Bioscience
    Average 95 stars, based on 24 article reviews
    Price from $9.99 to $1999.99
    mant atp - by Bioz Stars, 2020-08
    95/100 stars

    Images

    1) Product Images from "Renal epithelial cells can release ATP by vesicular fusion"

    Article Title: Renal epithelial cells can release ATP by vesicular fusion

    Journal: Frontiers in Physiology

    doi: 10.3389/fphys.2013.00238

    The effect of ionomycin on ATP release and vesicular fusion in MDCK cells (A) total internal reflection fluorescence (TIRF) microscopy of MDCK cells loaded with MANT-ATP (25 μM, 5 h, 37°C) showing vesicular fusion after the addition of ionomycin (1 μM) . Arrows indicate vesicles that abruptly disappeared from the evanescence field. (B) Ionomycin (1 μM)-induced vesicular fusion as indicated by wide-field fluorescence microscopy of MDCK cells loaded with quinacrine (5 μM, 30 min, 37°C). Arrows indicate vesicles that display an abruptly drop in fluorescence. (C) Right: Original trace of the effect of ionomycin (1 μM, 37°C) on the relative fluorescence intensity of quinacrine-loaded MDCK cells. The left panel present the mean ± s.e.m. from 6 experiments. (D) ATP release from MDCK cells induced by apical application of ionomycin (IONO, 1 μM). The arrow indicates the addition of ionomycin or the control solution; the mean ± s.e.m. values are presented ( n = 10). The asterisk indicates statistical significance.
    Figure Legend Snippet: The effect of ionomycin on ATP release and vesicular fusion in MDCK cells (A) total internal reflection fluorescence (TIRF) microscopy of MDCK cells loaded with MANT-ATP (25 μM, 5 h, 37°C) showing vesicular fusion after the addition of ionomycin (1 μM) . Arrows indicate vesicles that abruptly disappeared from the evanescence field. (B) Ionomycin (1 μM)-induced vesicular fusion as indicated by wide-field fluorescence microscopy of MDCK cells loaded with quinacrine (5 μM, 30 min, 37°C). Arrows indicate vesicles that display an abruptly drop in fluorescence. (C) Right: Original trace of the effect of ionomycin (1 μM, 37°C) on the relative fluorescence intensity of quinacrine-loaded MDCK cells. The left panel present the mean ± s.e.m. from 6 experiments. (D) ATP release from MDCK cells induced by apical application of ionomycin (IONO, 1 μM). The arrow indicates the addition of ionomycin or the control solution; the mean ± s.e.m. values are presented ( n = 10). The asterisk indicates statistical significance.

    Techniques Used: Fluorescence, Microscopy

    Spontaneous vesicular fusion in MDCK cells . Total internal reflection fluorescence (TIRF) microscopy of MDCK cells loaded with MANT-ATP (25 μM, 5 h, 37°C). Arrows indicate vesicles that abruptly disappeared from the evanescence field.
    Figure Legend Snippet: Spontaneous vesicular fusion in MDCK cells . Total internal reflection fluorescence (TIRF) microscopy of MDCK cells loaded with MANT-ATP (25 μM, 5 h, 37°C). Arrows indicate vesicles that abruptly disappeared from the evanescence field.

    Techniques Used: Fluorescence, Microscopy

    2) Product Images from "Structure-Activity Relationships for the Interactions of 2'- and 3'-(O)-(N-Methyl)anthraniloyl-Substituted Purine and Pyrimidine Nucleotides with Mammalian Adenylyl Cyclases"

    Article Title: Structure-Activity Relationships for the Interactions of 2'- and 3'-(O)-(N-Methyl)anthraniloyl-Substituted Purine and Pyrimidine Nucleotides with Mammalian Adenylyl Cyclases

    Journal: Biochemical pharmacology

    doi: 10.1016/j.bcp.2011.05.010

    Fluorescence emission spectra of 2’-MANT- and 3’-MANT-nucleotides bound to VC1:VC1 homodimer: Comparison with MANT-GTP and MANT-ATP
    Figure Legend Snippet: Fluorescence emission spectra of 2’-MANT- and 3’-MANT-nucleotides bound to VC1:VC1 homodimer: Comparison with MANT-GTP and MANT-ATP

    Techniques Used: Fluorescence

    Fluorescence emission spectra of 2’-MANT- and 3’-MANT-nucleotides bound to VC1:IIC2 heterodimer: Comparison with MANT-GTP and MANT-ATP
    Figure Legend Snippet: Fluorescence emission spectra of 2’-MANT- and 3’-MANT-nucleotides bound to VC1:IIC2 heterodimer: Comparison with MANT-GTP and MANT-ATP

    Techniques Used: Fluorescence

    3) Product Images from "Renal epithelial cells can release ATP by vesicular fusion"

    Article Title: Renal epithelial cells can release ATP by vesicular fusion

    Journal: Frontiers in Physiology

    doi: 10.3389/fphys.2013.00238

    The effect of ionomycin on ATP release and vesicular fusion in MDCK cells (A) total internal reflection fluorescence (TIRF) microscopy of MDCK cells loaded with MANT-ATP (25 μM, 5 h, 37°C) showing vesicular fusion after the addition of ionomycin (1 μM) . Arrows indicate vesicles that abruptly disappeared from the evanescence field. (B) Ionomycin (1 μM)-induced vesicular fusion as indicated by wide-field fluorescence microscopy of MDCK cells loaded with quinacrine (5 μM, 30 min, 37°C). Arrows indicate vesicles that display an abruptly drop in fluorescence. (C) Right: Original trace of the effect of ionomycin (1 μM, 37°C) on the relative fluorescence intensity of quinacrine-loaded MDCK cells. The left panel present the mean ± s.e.m. from 6 experiments. (D) ATP release from MDCK cells induced by apical application of ionomycin (IONO, 1 μM). The arrow indicates the addition of ionomycin or the control solution; the mean ± s.e.m. values are presented ( n = 10). The asterisk indicates statistical significance.
    Figure Legend Snippet: The effect of ionomycin on ATP release and vesicular fusion in MDCK cells (A) total internal reflection fluorescence (TIRF) microscopy of MDCK cells loaded with MANT-ATP (25 μM, 5 h, 37°C) showing vesicular fusion after the addition of ionomycin (1 μM) . Arrows indicate vesicles that abruptly disappeared from the evanescence field. (B) Ionomycin (1 μM)-induced vesicular fusion as indicated by wide-field fluorescence microscopy of MDCK cells loaded with quinacrine (5 μM, 30 min, 37°C). Arrows indicate vesicles that display an abruptly drop in fluorescence. (C) Right: Original trace of the effect of ionomycin (1 μM, 37°C) on the relative fluorescence intensity of quinacrine-loaded MDCK cells. The left panel present the mean ± s.e.m. from 6 experiments. (D) ATP release from MDCK cells induced by apical application of ionomycin (IONO, 1 μM). The arrow indicates the addition of ionomycin or the control solution; the mean ± s.e.m. values are presented ( n = 10). The asterisk indicates statistical significance.

    Techniques Used: Fluorescence, Microscopy

    Spontaneous vesicular fusion in MDCK cells . Total internal reflection fluorescence (TIRF) microscopy of MDCK cells loaded with MANT-ATP (25 μM, 5 h, 37°C). Arrows indicate vesicles that abruptly disappeared from the evanescence field.
    Figure Legend Snippet: Spontaneous vesicular fusion in MDCK cells . Total internal reflection fluorescence (TIRF) microscopy of MDCK cells loaded with MANT-ATP (25 μM, 5 h, 37°C). Arrows indicate vesicles that abruptly disappeared from the evanescence field.

    Techniques Used: Fluorescence, Microscopy

    4) Product Images from "Renal epithelial cells can release ATP by vesicular fusion"

    Article Title: Renal epithelial cells can release ATP by vesicular fusion

    Journal: Frontiers in Physiology

    doi: 10.3389/fphys.2013.00238

    The effect of ionomycin on ATP release and vesicular fusion in MDCK cells (A) total internal reflection fluorescence (TIRF) microscopy of MDCK cells loaded with MANT-ATP (25 μM, 5 h, 37°C) showing vesicular fusion after the addition of ionomycin (1 μM) . Arrows indicate vesicles that abruptly disappeared from the evanescence field. (B) Ionomycin (1 μM)-induced vesicular fusion as indicated by wide-field fluorescence microscopy of MDCK cells loaded with quinacrine (5 μM, 30 min, 37°C). Arrows indicate vesicles that display an abruptly drop in fluorescence. (C) Right: Original trace of the effect of ionomycin (1 μM, 37°C) on the relative fluorescence intensity of quinacrine-loaded MDCK cells. The left panel present the mean ± s.e.m. from 6 experiments. (D) ATP release from MDCK cells induced by apical application of ionomycin (IONO, 1 μM). The arrow indicates the addition of ionomycin or the control solution; the mean ± s.e.m. values are presented ( n = 10). The asterisk indicates statistical significance.
    Figure Legend Snippet: The effect of ionomycin on ATP release and vesicular fusion in MDCK cells (A) total internal reflection fluorescence (TIRF) microscopy of MDCK cells loaded with MANT-ATP (25 μM, 5 h, 37°C) showing vesicular fusion after the addition of ionomycin (1 μM) . Arrows indicate vesicles that abruptly disappeared from the evanescence field. (B) Ionomycin (1 μM)-induced vesicular fusion as indicated by wide-field fluorescence microscopy of MDCK cells loaded with quinacrine (5 μM, 30 min, 37°C). Arrows indicate vesicles that display an abruptly drop in fluorescence. (C) Right: Original trace of the effect of ionomycin (1 μM, 37°C) on the relative fluorescence intensity of quinacrine-loaded MDCK cells. The left panel present the mean ± s.e.m. from 6 experiments. (D) ATP release from MDCK cells induced by apical application of ionomycin (IONO, 1 μM). The arrow indicates the addition of ionomycin or the control solution; the mean ± s.e.m. values are presented ( n = 10). The asterisk indicates statistical significance.

    Techniques Used: Fluorescence, Microscopy

    Spontaneous vesicular fusion in MDCK cells . Total internal reflection fluorescence (TIRF) microscopy of MDCK cells loaded with MANT-ATP (25 μM, 5 h, 37°C). Arrows indicate vesicles that abruptly disappeared from the evanescence field.
    Figure Legend Snippet: Spontaneous vesicular fusion in MDCK cells . Total internal reflection fluorescence (TIRF) microscopy of MDCK cells loaded with MANT-ATP (25 μM, 5 h, 37°C). Arrows indicate vesicles that abruptly disappeared from the evanescence field.

    Techniques Used: Fluorescence, Microscopy

    5) Product Images from "Renal epithelial cells can release ATP by vesicular fusion"

    Article Title: Renal epithelial cells can release ATP by vesicular fusion

    Journal: Frontiers in Physiology

    doi: 10.3389/fphys.2013.00238

    The effect of ionomycin on ATP release and vesicular fusion in MDCK cells (A) total internal reflection fluorescence (TIRF) microscopy of MDCK cells loaded with MANT-ATP (25 μM, 5 h, 37°C) showing vesicular fusion after the addition of ionomycin (1 μM) . Arrows indicate vesicles that abruptly disappeared from the evanescence field. (B) Ionomycin (1 μM)-induced vesicular fusion as indicated by wide-field fluorescence microscopy of MDCK cells loaded with quinacrine (5 μM, 30 min, 37°C). Arrows indicate vesicles that display an abruptly drop in fluorescence. (C) Right: Original trace of the effect of ionomycin (1 μM, 37°C) on the relative fluorescence intensity of quinacrine-loaded MDCK cells. The left panel present the mean ± s.e.m. from 6 experiments. (D) ATP release from MDCK cells induced by apical application of ionomycin (IONO, 1 μM). The arrow indicates the addition of ionomycin or the control solution; the mean ± s.e.m. values are presented ( n = 10). The asterisk indicates statistical significance.
    Figure Legend Snippet: The effect of ionomycin on ATP release and vesicular fusion in MDCK cells (A) total internal reflection fluorescence (TIRF) microscopy of MDCK cells loaded with MANT-ATP (25 μM, 5 h, 37°C) showing vesicular fusion after the addition of ionomycin (1 μM) . Arrows indicate vesicles that abruptly disappeared from the evanescence field. (B) Ionomycin (1 μM)-induced vesicular fusion as indicated by wide-field fluorescence microscopy of MDCK cells loaded with quinacrine (5 μM, 30 min, 37°C). Arrows indicate vesicles that display an abruptly drop in fluorescence. (C) Right: Original trace of the effect of ionomycin (1 μM, 37°C) on the relative fluorescence intensity of quinacrine-loaded MDCK cells. The left panel present the mean ± s.e.m. from 6 experiments. (D) ATP release from MDCK cells induced by apical application of ionomycin (IONO, 1 μM). The arrow indicates the addition of ionomycin or the control solution; the mean ± s.e.m. values are presented ( n = 10). The asterisk indicates statistical significance.

    Techniques Used: Fluorescence, Microscopy

    Spontaneous vesicular fusion in MDCK cells . Total internal reflection fluorescence (TIRF) microscopy of MDCK cells loaded with MANT-ATP (25 μM, 5 h, 37°C). Arrows indicate vesicles that abruptly disappeared from the evanescence field.
    Figure Legend Snippet: Spontaneous vesicular fusion in MDCK cells . Total internal reflection fluorescence (TIRF) microscopy of MDCK cells loaded with MANT-ATP (25 μM, 5 h, 37°C). Arrows indicate vesicles that abruptly disappeared from the evanescence field.

    Techniques Used: Fluorescence, Microscopy

    6) Product Images from "The crystal structure and biochemical characterization of Kif15: a bifunctional molecular motor involved in bipolar spindle formation and neuronal development"

    Article Title: The crystal structure and biochemical characterization of Kif15: a bifunctional molecular motor involved in bipolar spindle formation and neuronal development

    Journal: Acta Crystallographica Section D: Biological Crystallography

    doi: 10.1107/S1399004713028721

    MT pelleting assays in the presence of Kif15 motor or tail domains. ( a ) MT pelleting assays of the Kif15 motor domain in the presence of various nucleotides. Increasing amounts of Kif15 19–375 (1–12 µ M ) were incubated with MTs (5 µ M ) in the presence of 2 m M Mg 2+ -ATP, 2 m M AMP-PNP or 4 mU apyrase. Sample s of supernatants and pellets were analysed by SDS–PAGE. ( b ) MT binding of Kif15 19–375 in the presence of 2 m M Mg 2+ -ATP, 2 m M AMP-PNP or 4 mU apyrase. The plotted data relate to the amounts (µ M ) of Kif15 19–375 recovered from supernatant and pellet (pelleted with MTs) fractions of reactions run in the presence of various nucleotides. Data were obtained by analysing the SDS–PAGE ( ImageJ 143.u) presented in ( a ). ( c ) MT pelleting assays of the Kif15 tail domain. Increasing amounts of Kif15 1149–1388 (1–10 µ M ) were incubated with MTs (5 µ M ). Samples of supernatants and pellets were analysed by SDS–PAGE. The plotted data relate to the amounts (µ M ) of Kif15 1149–1388 recovered from supernatant and pellet (pelleted with MTs) fractions. Data were obtained by analysing the SDS–PAGE ( ImageJ 143.u) presented on the left side of the figure.
    Figure Legend Snippet: MT pelleting assays in the presence of Kif15 motor or tail domains. ( a ) MT pelleting assays of the Kif15 motor domain in the presence of various nucleotides. Increasing amounts of Kif15 19–375 (1–12 µ M ) were incubated with MTs (5 µ M ) in the presence of 2 m M Mg 2+ -ATP, 2 m M AMP-PNP or 4 mU apyrase. Sample s of supernatants and pellets were analysed by SDS–PAGE. ( b ) MT binding of Kif15 19–375 in the presence of 2 m M Mg 2+ -ATP, 2 m M AMP-PNP or 4 mU apyrase. The plotted data relate to the amounts (µ M ) of Kif15 19–375 recovered from supernatant and pellet (pelleted with MTs) fractions of reactions run in the presence of various nucleotides. Data were obtained by analysing the SDS–PAGE ( ImageJ 143.u) presented in ( a ). ( c ) MT pelleting assays of the Kif15 tail domain. Increasing amounts of Kif15 1149–1388 (1–10 µ M ) were incubated with MTs (5 µ M ). Samples of supernatants and pellets were analysed by SDS–PAGE. The plotted data relate to the amounts (µ M ) of Kif15 1149–1388 recovered from supernatant and pellet (pelleted with MTs) fractions. Data were obtained by analysing the SDS–PAGE ( ImageJ 143.u) presented on the left side of the figure.

    Techniques Used: Incubation, SDS Page, Binding Assay

    Overall structure of the binary Kif15 19–375 –Mg 2+ -ADP complex and comparison of the sequence/secondary structure of the Kif15 and Eg5 motor domains. ( a ) Stereo plots of the front and back view of the human Kif15 motor domain. α-Helices are coloured blue, β-strands green and loops/turns grey. The switch II cluster (α4–L12–α5) is highlighted in claret and the neck linker following the C-terminal helix α6 is shown in cyan. Mg 2+ -ADP is shown as a ball-and-stick model. ( b ) Structural and sequence alignment of the Eg5 (PDB entry 3hqd ; Parke et al. , 2009 ▶ ) and Kif15 motor domains. Residue 16 of Eg5 is aligned with residue 24 of Kif15. Identical residues are coloured white on a red background and similar residues are shaded in red. The position of the ATP-binding pocket (N1–N4), the switch I and II regions and the position of the neck-linker regions are underlined in black.
    Figure Legend Snippet: Overall structure of the binary Kif15 19–375 –Mg 2+ -ADP complex and comparison of the sequence/secondary structure of the Kif15 and Eg5 motor domains. ( a ) Stereo plots of the front and back view of the human Kif15 motor domain. α-Helices are coloured blue, β-strands green and loops/turns grey. The switch II cluster (α4–L12–α5) is highlighted in claret and the neck linker following the C-terminal helix α6 is shown in cyan. Mg 2+ -ADP is shown as a ball-and-stick model. ( b ) Structural and sequence alignment of the Eg5 (PDB entry 3hqd ; Parke et al. , 2009 ▶ ) and Kif15 motor domains. Residue 16 of Eg5 is aligned with residue 24 of Kif15. Identical residues are coloured white on a red background and similar residues are shaded in red. The position of the ATP-binding pocket (N1–N4), the switch I and II regions and the position of the neck-linker regions are underlined in black.

    Techniques Used: Sequencing, Binding Assay

    Characterization of the basal and MT-stimulated ATPase activities of Kif15 19–375 and Kif15 1–375 . ( a ) Influence of the NaCl concentration on the basal ATPase activity of Kif15 19–375 (red) and Kif15 1–375 (blue) in the presence of 1 m M ATP. ( b ) Optimization of the basal ATPase activity in the presence of increasing ATP concentrations measured at 75 m M NaCl for Kif15 19–375 (red) and 50 m M NaCl for Kif15 1–375 (blue). ( c ) Salt dependence of the MT-stimulated Kif15 19–375 ATPase activity (red) in the absence (circles) and in the presence of 50 m M (filled circles), 100 m M (squares) and 150 m M (filled squares) KCl and salt dependence of the MT-stimulated Kif15 1–375 ATPase activity (blue) in the absence (filled diamonds) and the presence (squares) of 50 m M KCl. Data were measured at increasing MT concentrations ranging from 0 to 10 µ M in the presence of 1 m M ATP. ( d ) Optimization of the ATP concentration for the MT-stimulated ATPase activity of Kif15 19–375 (red) and Kif15 1–375 (blue) in the presence of increasing ATP concentrations, measured at 3 µ M MTs, in the absence of salt.
    Figure Legend Snippet: Characterization of the basal and MT-stimulated ATPase activities of Kif15 19–375 and Kif15 1–375 . ( a ) Influence of the NaCl concentration on the basal ATPase activity of Kif15 19–375 (red) and Kif15 1–375 (blue) in the presence of 1 m M ATP. ( b ) Optimization of the basal ATPase activity in the presence of increasing ATP concentrations measured at 75 m M NaCl for Kif15 19–375 (red) and 50 m M NaCl for Kif15 1–375 (blue). ( c ) Salt dependence of the MT-stimulated Kif15 19–375 ATPase activity (red) in the absence (circles) and in the presence of 50 m M (filled circles), 100 m M (squares) and 150 m M (filled squares) KCl and salt dependence of the MT-stimulated Kif15 1–375 ATPase activity (blue) in the absence (filled diamonds) and the presence (squares) of 50 m M KCl. Data were measured at increasing MT concentrations ranging from 0 to 10 µ M in the presence of 1 m M ATP. ( d ) Optimization of the ATP concentration for the MT-stimulated ATPase activity of Kif15 19–375 (red) and Kif15 1–375 (blue) in the presence of increasing ATP concentrations, measured at 3 µ M MTs, in the absence of salt.

    Techniques Used: Concentration Assay, Activity Assay

    7) Product Images from "The crystal structure and biochemical characterization of Kif15: a bifunctional molecular motor involved in bipolar spindle formation and neuronal development"

    Article Title: The crystal structure and biochemical characterization of Kif15: a bifunctional molecular motor involved in bipolar spindle formation and neuronal development

    Journal: Acta Crystallographica Section D: Biological Crystallography

    doi: 10.1107/S1399004713028721

    MT pelleting assays in the presence of Kif15 motor or tail domains. ( a ) MT pelleting assays of the Kif15 motor domain in the presence of various nucleotides. Increasing amounts of Kif15 19–375 (1–12 µ M ) were incubated with MTs (5 µ M ) in the presence of 2 m M Mg 2+ -ATP, 2 m M AMP-PNP or 4 mU apyrase. Sample s of supernatants and pellets were analysed by SDS–PAGE. ( b ) MT binding of Kif15 19–375 in the presence of 2 m M Mg 2+ -ATP, 2 m M AMP-PNP or 4 mU apyrase. The plotted data relate to the amounts (µ M ) of Kif15 19–375 recovered from supernatant and pellet (pelleted with MTs) fractions of reactions run in the presence of various nucleotides. Data were obtained by analysing the SDS–PAGE ( ImageJ 143.u) presented in ( a ). ( c ) MT pelleting assays of the Kif15 tail domain. Increasing amounts of Kif15 1149–1388 (1–10 µ M ) were incubated with MTs (5 µ M ). Samples of supernatants and pellets were analysed by SDS–PAGE. The plotted data relate to the amounts (µ M ) of Kif15 1149–1388 recovered from supernatant and pellet (pelleted with MTs) fractions. Data were obtained by analysing the SDS–PAGE ( ImageJ 143.u) presented on the left side of the figure.
    Figure Legend Snippet: MT pelleting assays in the presence of Kif15 motor or tail domains. ( a ) MT pelleting assays of the Kif15 motor domain in the presence of various nucleotides. Increasing amounts of Kif15 19–375 (1–12 µ M ) were incubated with MTs (5 µ M ) in the presence of 2 m M Mg 2+ -ATP, 2 m M AMP-PNP or 4 mU apyrase. Sample s of supernatants and pellets were analysed by SDS–PAGE. ( b ) MT binding of Kif15 19–375 in the presence of 2 m M Mg 2+ -ATP, 2 m M AMP-PNP or 4 mU apyrase. The plotted data relate to the amounts (µ M ) of Kif15 19–375 recovered from supernatant and pellet (pelleted with MTs) fractions of reactions run in the presence of various nucleotides. Data were obtained by analysing the SDS–PAGE ( ImageJ 143.u) presented in ( a ). ( c ) MT pelleting assays of the Kif15 tail domain. Increasing amounts of Kif15 1149–1388 (1–10 µ M ) were incubated with MTs (5 µ M ). Samples of supernatants and pellets were analysed by SDS–PAGE. The plotted data relate to the amounts (µ M ) of Kif15 1149–1388 recovered from supernatant and pellet (pelleted with MTs) fractions. Data were obtained by analysing the SDS–PAGE ( ImageJ 143.u) presented on the left side of the figure.

    Techniques Used: Incubation, SDS Page, Binding Assay

    Overall structure of the binary Kif15 19–375 –Mg 2+ -ADP complex and comparison of the sequence/secondary structure of the Kif15 and Eg5 motor domains. ( a ) Stereo plots of the front and back view of the human Kif15 motor domain. α-Helices are coloured blue, β-strands green and loops/turns grey. The switch II cluster (α4–L12–α5) is highlighted in claret and the neck linker following the C-terminal helix α6 is shown in cyan. Mg 2+ -ADP is shown as a ball-and-stick model. ( b ) Structural and sequence alignment of the Eg5 (PDB entry 3hqd ; Parke et al. , 2009 ▶ ) and Kif15 motor domains. Residue 16 of Eg5 is aligned with residue 24 of Kif15. Identical residues are coloured white on a red background and similar residues are shaded in red. The position of the ATP-binding pocket (N1–N4), the switch I and II regions and the position of the neck-linker regions are underlined in black.
    Figure Legend Snippet: Overall structure of the binary Kif15 19–375 –Mg 2+ -ADP complex and comparison of the sequence/secondary structure of the Kif15 and Eg5 motor domains. ( a ) Stereo plots of the front and back view of the human Kif15 motor domain. α-Helices are coloured blue, β-strands green and loops/turns grey. The switch II cluster (α4–L12–α5) is highlighted in claret and the neck linker following the C-terminal helix α6 is shown in cyan. Mg 2+ -ADP is shown as a ball-and-stick model. ( b ) Structural and sequence alignment of the Eg5 (PDB entry 3hqd ; Parke et al. , 2009 ▶ ) and Kif15 motor domains. Residue 16 of Eg5 is aligned with residue 24 of Kif15. Identical residues are coloured white on a red background and similar residues are shaded in red. The position of the ATP-binding pocket (N1–N4), the switch I and II regions and the position of the neck-linker regions are underlined in black.

    Techniques Used: Sequencing, Binding Assay

    Characterization of the basal and MT-stimulated ATPase activities of Kif15 19–375 and Kif15 1–375 . ( a ) Influence of the NaCl concentration on the basal ATPase activity of Kif15 19–375 (red) and Kif15 1–375 (blue) in the presence of 1 m M ATP. ( b ) Optimization of the basal ATPase activity in the presence of increasing ATP concentrations measured at 75 m M NaCl for Kif15 19–375 (red) and 50 m M NaCl for Kif15 1–375 (blue). ( c ) Salt dependence of the MT-stimulated Kif15 19–375 ATPase activity (red) in the absence (circles) and in the presence of 50 m M (filled circles), 100 m M (squares) and 150 m M (filled squares) KCl and salt dependence of the MT-stimulated Kif15 1–375 ATPase activity (blue) in the absence (filled diamonds) and the presence (squares) of 50 m M KCl. Data were measured at increasing MT concentrations ranging from 0 to 10 µ M in the presence of 1 m M ATP. ( d ) Optimization of the ATP concentration for the MT-stimulated ATPase activity of Kif15 19–375 (red) and Kif15 1–375 (blue) in the presence of increasing ATP concentrations, measured at 3 µ M MTs, in the absence of salt.
    Figure Legend Snippet: Characterization of the basal and MT-stimulated ATPase activities of Kif15 19–375 and Kif15 1–375 . ( a ) Influence of the NaCl concentration on the basal ATPase activity of Kif15 19–375 (red) and Kif15 1–375 (blue) in the presence of 1 m M ATP. ( b ) Optimization of the basal ATPase activity in the presence of increasing ATP concentrations measured at 75 m M NaCl for Kif15 19–375 (red) and 50 m M NaCl for Kif15 1–375 (blue). ( c ) Salt dependence of the MT-stimulated Kif15 19–375 ATPase activity (red) in the absence (circles) and in the presence of 50 m M (filled circles), 100 m M (squares) and 150 m M (filled squares) KCl and salt dependence of the MT-stimulated Kif15 1–375 ATPase activity (blue) in the absence (filled diamonds) and the presence (squares) of 50 m M KCl. Data were measured at increasing MT concentrations ranging from 0 to 10 µ M in the presence of 1 m M ATP. ( d ) Optimization of the ATP concentration for the MT-stimulated ATPase activity of Kif15 19–375 (red) and Kif15 1–375 (blue) in the presence of increasing ATP concentrations, measured at 3 µ M MTs, in the absence of salt.

    Techniques Used: Concentration Assay, Activity Assay

    8) Product Images from "Dynamics of human protein kinase Aurora A linked to drug selectivity"

    Article Title: Dynamics of human protein kinase Aurora A linked to drug selectivity

    Journal: eLife

    doi: 10.7554/eLife.36656

    Mechanism of ATP binding to Aurora A at 10°C. ( A ) Binding of mant-ATP to Aurora A was followed by an increase in fluorescence with biphasic kinetics. The plot of k o b s versus concentration of mant-ATP of the fast phase ( B ) yields k 2 = 0.8 ± 0.2 μM −1 s −1 and k - 2 = 50 ± 8 s −1 and the slow phase ( C ) reached a plateau around 21 ± 1 s −1 ( k 3 + k - 3 ). ( D ) Dissociation kinetics of 10 μM Aurora A/10 μM mant-ATP complex was measured after a 11-fold dilution into buffer and yields k o f f o b s = 17.2 ± 1 s −1 . ( E, F ) Macroscopic dissociation constant of Aurora A with mant-ATP measured by fluorescence energy transfer. ( E ) Emission spectra (excitation at 290 nm) of 1 μM Aurora A (green), 160 μM mant-ATP (red), and 1 μM Aurora A/160 μM mant-ATP (blue). ( F ) The change in fluorescence at 450 nm ( Δ F 450 ) versus mant-ATP concentrations yields K D = 22 ± 6 μM. ( G ) Global fitting (red) of all kinetics data (black) in KinTek Explorer to the binding scheme shown in ( H ) results in the kinetic constants given in the scheme and an overall K D = 48 ± 8 μM, calculated from all rate constants. Fluorescence traces are the average of at least five replicate measurements (n > 5), and error bars and uncertainties given in B, C, D, F, and H denote the (propagated) standard deviation in the fitted parameter.
    Figure Legend Snippet: Mechanism of ATP binding to Aurora A at 10°C. ( A ) Binding of mant-ATP to Aurora A was followed by an increase in fluorescence with biphasic kinetics. The plot of k o b s versus concentration of mant-ATP of the fast phase ( B ) yields k 2 = 0.8 ± 0.2 μM −1 s −1 and k - 2 = 50 ± 8 s −1 and the slow phase ( C ) reached a plateau around 21 ± 1 s −1 ( k 3 + k - 3 ). ( D ) Dissociation kinetics of 10 μM Aurora A/10 μM mant-ATP complex was measured after a 11-fold dilution into buffer and yields k o f f o b s = 17.2 ± 1 s −1 . ( E, F ) Macroscopic dissociation constant of Aurora A with mant-ATP measured by fluorescence energy transfer. ( E ) Emission spectra (excitation at 290 nm) of 1 μM Aurora A (green), 160 μM mant-ATP (red), and 1 μM Aurora A/160 μM mant-ATP (blue). ( F ) The change in fluorescence at 450 nm ( Δ F 450 ) versus mant-ATP concentrations yields K D = 22 ± 6 μM. ( G ) Global fitting (red) of all kinetics data (black) in KinTek Explorer to the binding scheme shown in ( H ) results in the kinetic constants given in the scheme and an overall K D = 48 ± 8 μM, calculated from all rate constants. Fluorescence traces are the average of at least five replicate measurements (n > 5), and error bars and uncertainties given in B, C, D, F, and H denote the (propagated) standard deviation in the fitted parameter.

    Techniques Used: Binding Assay, Fluorescence, Concentration Assay, Standard Deviation

    9) Product Images from "Kif2C Minimal Functional Domain Has Unusual Nucleotide Binding Properties That Are Adapted to Microtubule Depolymerization *"

    Article Title: Kif2C Minimal Functional Domain Has Unusual Nucleotide Binding Properties That Are Adapted to Microtubule Depolymerization *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.317859

    A model for the MT depolymerization mechanism of Kif2C and other kinesin-13s. A MT is represented as a single protofilament, and Kif2C is represented as a single motor domain with the Neck shown as an attached helix. The functionally important family-specific Loop 2 is highlighted . The nucleotide bound to Kif2C is indicated with the letter: T for ATP, D for ADP, and D-Pi for ADP-Pi. In solution, Kif2C releases ADP autonomously and then binds ATP, so that the Kif2C pool is mostly ATP-loaded. Kif2C-ATP is strongly biased toward binding directly to the end of MT, which is shown by a thick solid line . Binding triggers ATP hydrolysis by Kif2C, which in turn leads to the release of tubulin from MT. The requirement for ATP hydrolysis is shown by an asterisk . Alternatively, when Kif2C-ATP binds to the MT lattice (shown by a dashed line ), ATP hydrolysis is also triggered, which yields Kif2C-ADP-P i that diffuses along MTs. The release of P i tightens MT-binding by Kif2C, where Kif2C readily releases ADP, binds another ATP, and may start the next round of Kif2C diffusion along MT. This only leads to tubulin disassembly from MT if ATP-bound Kif2C reaches the MT end.
    Figure Legend Snippet: A model for the MT depolymerization mechanism of Kif2C and other kinesin-13s. A MT is represented as a single protofilament, and Kif2C is represented as a single motor domain with the Neck shown as an attached helix. The functionally important family-specific Loop 2 is highlighted . The nucleotide bound to Kif2C is indicated with the letter: T for ATP, D for ADP, and D-Pi for ADP-Pi. In solution, Kif2C releases ADP autonomously and then binds ATP, so that the Kif2C pool is mostly ATP-loaded. Kif2C-ATP is strongly biased toward binding directly to the end of MT, which is shown by a thick solid line . Binding triggers ATP hydrolysis by Kif2C, which in turn leads to the release of tubulin from MT. The requirement for ATP hydrolysis is shown by an asterisk . Alternatively, when Kif2C-ATP binds to the MT lattice (shown by a dashed line ), ATP hydrolysis is also triggered, which yields Kif2C-ADP-P i that diffuses along MTs. The release of P i tightens MT-binding by Kif2C, where Kif2C readily releases ADP, binds another ATP, and may start the next round of Kif2C diffusion along MT. This only leads to tubulin disassembly from MT if ATP-bound Kif2C reaches the MT end.

    Techniques Used: Binding Assay, Diffusion-based Assay

    10) Product Images from "Renal epithelial cells can release ATP by vesicular fusion"

    Article Title: Renal epithelial cells can release ATP by vesicular fusion

    Journal: Frontiers in Physiology

    doi: 10.3389/fphys.2013.00238

    The effect of ionomycin on ATP release and vesicular fusion in MDCK cells (A) total internal reflection fluorescence (TIRF) microscopy of MDCK cells loaded with MANT-ATP (25 μM, 5 h, 37°C) showing vesicular fusion after the addition of ionomycin (1 μM) . Arrows indicate vesicles that abruptly disappeared from the evanescence field. (B) Ionomycin (1 μM)-induced vesicular fusion as indicated by wide-field fluorescence microscopy of MDCK cells loaded with quinacrine (5 μM, 30 min, 37°C). Arrows indicate vesicles that display an abruptly drop in fluorescence. (C) Right: Original trace of the effect of ionomycin (1 μM, 37°C) on the relative fluorescence intensity of quinacrine-loaded MDCK cells. The left panel present the mean ± s.e.m. from 6 experiments. (D) ATP release from MDCK cells induced by apical application of ionomycin (IONO, 1 μM). The arrow indicates the addition of ionomycin or the control solution; the mean ± s.e.m. values are presented ( n = 10). The asterisk indicates statistical significance.
    Figure Legend Snippet: The effect of ionomycin on ATP release and vesicular fusion in MDCK cells (A) total internal reflection fluorescence (TIRF) microscopy of MDCK cells loaded with MANT-ATP (25 μM, 5 h, 37°C) showing vesicular fusion after the addition of ionomycin (1 μM) . Arrows indicate vesicles that abruptly disappeared from the evanescence field. (B) Ionomycin (1 μM)-induced vesicular fusion as indicated by wide-field fluorescence microscopy of MDCK cells loaded with quinacrine (5 μM, 30 min, 37°C). Arrows indicate vesicles that display an abruptly drop in fluorescence. (C) Right: Original trace of the effect of ionomycin (1 μM, 37°C) on the relative fluorescence intensity of quinacrine-loaded MDCK cells. The left panel present the mean ± s.e.m. from 6 experiments. (D) ATP release from MDCK cells induced by apical application of ionomycin (IONO, 1 μM). The arrow indicates the addition of ionomycin or the control solution; the mean ± s.e.m. values are presented ( n = 10). The asterisk indicates statistical significance.

    Techniques Used: Fluorescence, Microscopy

    Spontaneous vesicular fusion in MDCK cells . Total internal reflection fluorescence (TIRF) microscopy of MDCK cells loaded with MANT-ATP (25 μM, 5 h, 37°C). Arrows indicate vesicles that abruptly disappeared from the evanescence field.
    Figure Legend Snippet: Spontaneous vesicular fusion in MDCK cells . Total internal reflection fluorescence (TIRF) microscopy of MDCK cells loaded with MANT-ATP (25 μM, 5 h, 37°C). Arrows indicate vesicles that abruptly disappeared from the evanescence field.

    Techniques Used: Fluorescence, Microscopy

    11) Product Images from "Asymmetric nucleotide transactions of the HslUV protease"

    Article Title: Asymmetric nucleotide transactions of the HslUV protease

    Journal: Journal of molecular biology

    doi: 10.1016/j.jmb.2008.05.070

    HslU binding to FL-gt1, a fluorescent substrate-mimic peptide. ( A ) Binding of FL-gt1 (100 nM) by wild-type HslU or E257Q HslU in the presence/absence of HslV 12 . Binding reactions contained 500 μM ATPγS (HslU) or 500 μM ATP (E257Q HslU) and were performed at 25 °C in PD buffer. The fitted lines are for non-cooperative binding with K d ’s of 0.76 μM (HslUV), 0.93 μM (HslU), 3.0 μM (E257Q HslUV), and 5.6 μM (E257Q). ( B ) Binding of E257Q HslU (1.5 μM hexamer) to FL-gt1 (0.1 μM) as a function of ATPγS concentration (top panel) or N6-methyl-ATP concentration (bottom panel). The data were fitted to the Hill equation. Half-maximal binding occurred at 0.8 μM ATPγS (n = 2.3 ± 0.23), 34 μM N6-methyl-ATP without HslV (n = 1.8 ± 0.15), and 2.3 μM N6-methyl-ATP with 3 μM HslV 12 (n = 1.5 ± 0.12).
    Figure Legend Snippet: HslU binding to FL-gt1, a fluorescent substrate-mimic peptide. ( A ) Binding of FL-gt1 (100 nM) by wild-type HslU or E257Q HslU in the presence/absence of HslV 12 . Binding reactions contained 500 μM ATPγS (HslU) or 500 μM ATP (E257Q HslU) and were performed at 25 °C in PD buffer. The fitted lines are for non-cooperative binding with K d ’s of 0.76 μM (HslUV), 0.93 μM (HslU), 3.0 μM (E257Q HslUV), and 5.6 μM (E257Q). ( B ) Binding of E257Q HslU (1.5 μM hexamer) to FL-gt1 (0.1 μM) as a function of ATPγS concentration (top panel) or N6-methyl-ATP concentration (bottom panel). The data were fitted to the Hill equation. Half-maximal binding occurred at 0.8 μM ATPγS (n = 2.3 ± 0.23), 34 μM N6-methyl-ATP without HslV (n = 1.8 ± 0.15), and 2.3 μM N6-methyl-ATP with 3 μM HslV 12 (n = 1.5 ± 0.12).

    Techniques Used: Binding Assay, Concentration Assay

    HslU·HslV binding. ( A ) Binding of HslU (top) or E257Q HslU (bottom) to HslV 12 (15 nM) was assayed by changes in the rate of Z-Gly-Gly-AMC peptide cleavage. Reactions contained 1 mM ATPγS or 1 mM ATP and were performed in PD buffer at 25 °C. Data were fit to a quadratic form of a hyperbolic binding isotherm. Apparent K D values were 12 ± 2 nM (HslU; ATPγS), 78 ± 10 nM (HslU; ATP), 19 ± 5 nM (E257Q HslU; ATP) and 21 ± 2 nM (E257Q HslU; ATPγS). ( B (10 μM Arc-IA37-st11-titin-ssrA) strengthened binding of HslV to HslU (0.3 μM hexamer), as measured by changes in the rate of HslU ATP hydrolysis. HslUV·Arc complexes were preassembled by incubating for 5 min with 10 μM ATPγS at 37 °C. Reactions in PD buffer at 37 °C were initiated by addition of ATP and a regeneration system. ( C ) HslU activation of HslV 12 (100 nM) cleavage of Z-Gly-Gly-Leu-AMC (200 μM) was assayed in the presence of 100, 200, 400 or 800 nM ATPγS. ( D ) Rates of Z-Gly-Gly-Leu-AMC (200 μM) cleavage were assayed in the presence of 1 μM HslU hexamer, 100 nM ATPγS, and increasing HslV 12 . ( E ) N6-methyl-ATP supports hyperbolic activation of HslU 6 (50 nM) stimulation of HslV 12 (200 nM) cleavage of Z-Gly-Gly-Leu-AMC (200 μM). The solid line is a fit (R = 0.997) to the equation activity = 100/(1+([0.85 μM][N6-methyl-ATP])).
    Figure Legend Snippet: HslU·HslV binding. ( A ) Binding of HslU (top) or E257Q HslU (bottom) to HslV 12 (15 nM) was assayed by changes in the rate of Z-Gly-Gly-AMC peptide cleavage. Reactions contained 1 mM ATPγS or 1 mM ATP and were performed in PD buffer at 25 °C. Data were fit to a quadratic form of a hyperbolic binding isotherm. Apparent K D values were 12 ± 2 nM (HslU; ATPγS), 78 ± 10 nM (HslU; ATP), 19 ± 5 nM (E257Q HslU; ATP) and 21 ± 2 nM (E257Q HslU; ATPγS). ( B (10 μM Arc-IA37-st11-titin-ssrA) strengthened binding of HslV to HslU (0.3 μM hexamer), as measured by changes in the rate of HslU ATP hydrolysis. HslUV·Arc complexes were preassembled by incubating for 5 min with 10 μM ATPγS at 37 °C. Reactions in PD buffer at 37 °C were initiated by addition of ATP and a regeneration system. ( C ) HslU activation of HslV 12 (100 nM) cleavage of Z-Gly-Gly-Leu-AMC (200 μM) was assayed in the presence of 100, 200, 400 or 800 nM ATPγS. ( D ) Rates of Z-Gly-Gly-Leu-AMC (200 μM) cleavage were assayed in the presence of 1 μM HslU hexamer, 100 nM ATPγS, and increasing HslV 12 . ( E ) N6-methyl-ATP supports hyperbolic activation of HslU 6 (50 nM) stimulation of HslV 12 (200 nM) cleavage of Z-Gly-Gly-Leu-AMC (200 μM). The solid line is a fit (R = 0.997) to the equation activity = 100/(1+([0.85 μM][N6-methyl-ATP])).

    Techniques Used: Binding Assay, Activation Assay, Activity Assay

    HslU. ( A ) Two HslU 6 ATPases (blue) can assemble with the HslV 12 peptidase (magenta). ( B ) HslU hexamers have six potential nucleotide binding sites, located at domain and subunit interfaces. From 3–6 nucleotides bind HslU 6 in different crystal structures. ( C (10 μM). Reactions components were preincubated with 10 μM ATPγS to promote HslU or HslUV association prior to addition of ATP.
    Figure Legend Snippet: HslU. ( A ) Two HslU 6 ATPases (blue) can assemble with the HslV 12 peptidase (magenta). ( B ) HslU hexamers have six potential nucleotide binding sites, located at domain and subunit interfaces. From 3–6 nucleotides bind HslU 6 in different crystal structures. ( C (10 μM). Reactions components were preincubated with 10 μM ATPγS to promote HslU or HslUV association prior to addition of ATP.

    Techniques Used: Binding Assay

    12) Product Images from "Interactions of Bordetella pertussis adenylyl cyclase toxin CyaA with calmodulin mutants and calmodulin antagonists: Comparison with membranous adenylyl cyclase I"

    Article Title: Interactions of Bordetella pertussis adenylyl cyclase toxin CyaA with calmodulin mutants and calmodulin antagonists: Comparison with membranous adenylyl cyclase I

    Journal: Biochemical pharmacology

    doi: 10.1016/j.bcp.2012.01.005

    Concentration/response curves for the interaction of CyaA with CaM and CaM mutants in FRET assays. FRET was determined as described in Section 2. The cuvette contained 300 nM of 2′-MANT-3′-d-ATP and 300 nM of CyaA-N. For assigning a FRET
    Figure Legend Snippet: Concentration/response curves for the interaction of CyaA with CaM and CaM mutants in FRET assays. FRET was determined as described in Section 2. The cuvette contained 300 nM of 2′-MANT-3′-d-ATP and 300 nM of CyaA-N. For assigning a FRET

    Techniques Used: Concentration Assay, Chick Chorioallantoic Membrane Assay

    13) Product Images from "Renal epithelial cells can release ATP by vesicular fusion"

    Article Title: Renal epithelial cells can release ATP by vesicular fusion

    Journal: Frontiers in Physiology

    doi: 10.3389/fphys.2013.00238

    The effect of hyposmotic stress on vesicular fusion of quinacrine-loaded vesicles by TIRF microscopy of MDCK cells . (A) Original traces of the total fluorescence intensity of 5 cells as determined by TIRF microscopy. The mean ± s.e.m. values are shown on the right as n = 17. (B) Effect of apically-induced hypotonic stress (50%) on the level of extracellular ATP. Data are presented as the mean ± s.e.m. ( n = 10). The asterisk indicates statistical significance.
    Figure Legend Snippet: The effect of hyposmotic stress on vesicular fusion of quinacrine-loaded vesicles by TIRF microscopy of MDCK cells . (A) Original traces of the total fluorescence intensity of 5 cells as determined by TIRF microscopy. The mean ± s.e.m. values are shown on the right as n = 17. (B) Effect of apically-induced hypotonic stress (50%) on the level of extracellular ATP. Data are presented as the mean ± s.e.m. ( n = 10). The asterisk indicates statistical significance.

    Techniques Used: Microscopy, Fluorescence

    The effect of ionomycin on ATP release and vesicular fusion in MDCK cells (A) total internal reflection fluorescence (TIRF) microscopy of MDCK cells loaded with MANT-ATP (25 μM, 5 h, 37°C) showing vesicular fusion after the addition of ionomycin (1 μM) . Arrows indicate vesicles that abruptly disappeared from the evanescence field. (B) Ionomycin (1 μM)-induced vesicular fusion as indicated by wide-field fluorescence microscopy of MDCK cells loaded with quinacrine (5 μM, 30 min, 37°C). Arrows indicate vesicles that display an abruptly drop in fluorescence. (C) Right: Original trace of the effect of ionomycin (1 μM, 37°C) on the relative fluorescence intensity of quinacrine-loaded MDCK cells. The left panel present the mean ± s.e.m. from 6 experiments. (D) ATP release from MDCK cells induced by apical application of ionomycin (IONO, 1 μM). The arrow indicates the addition of ionomycin or the control solution; the mean ± s.e.m. values are presented ( n = 10). The asterisk indicates statistical significance.
    Figure Legend Snippet: The effect of ionomycin on ATP release and vesicular fusion in MDCK cells (A) total internal reflection fluorescence (TIRF) microscopy of MDCK cells loaded with MANT-ATP (25 μM, 5 h, 37°C) showing vesicular fusion after the addition of ionomycin (1 μM) . Arrows indicate vesicles that abruptly disappeared from the evanescence field. (B) Ionomycin (1 μM)-induced vesicular fusion as indicated by wide-field fluorescence microscopy of MDCK cells loaded with quinacrine (5 μM, 30 min, 37°C). Arrows indicate vesicles that display an abruptly drop in fluorescence. (C) Right: Original trace of the effect of ionomycin (1 μM, 37°C) on the relative fluorescence intensity of quinacrine-loaded MDCK cells. The left panel present the mean ± s.e.m. from 6 experiments. (D) ATP release from MDCK cells induced by apical application of ionomycin (IONO, 1 μM). The arrow indicates the addition of ionomycin or the control solution; the mean ± s.e.m. values are presented ( n = 10). The asterisk indicates statistical significance.

    Techniques Used: Fluorescence, Microscopy

    Spontaneous vesicular fusion in MDCK cells . Total internal reflection fluorescence (TIRF) microscopy of MDCK cells loaded with MANT-ATP (25 μM, 5 h, 37°C). Arrows indicate vesicles that abruptly disappeared from the evanescence field.
    Figure Legend Snippet: Spontaneous vesicular fusion in MDCK cells . Total internal reflection fluorescence (TIRF) microscopy of MDCK cells loaded with MANT-ATP (25 μM, 5 h, 37°C). Arrows indicate vesicles that abruptly disappeared from the evanescence field.

    Techniques Used: Fluorescence, Microscopy

    14) Product Images from "Kif2C Minimal Functional Domain Has Unusual Nucleotide Binding Properties That Are Adapted to Microtubule Depolymerization *"

    Article Title: Kif2C Minimal Functional Domain Has Unusual Nucleotide Binding Properties That Are Adapted to Microtubule Depolymerization *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.317859

    A model for the MT depolymerization mechanism of Kif2C and other kinesin-13s. A MT is represented as a single protofilament, and Kif2C is represented as a single motor domain with the Neck shown as an attached helix. The functionally important family-specific Loop 2 is highlighted . The nucleotide bound to Kif2C is indicated with the letter: T for ATP, D for ADP, and D-Pi for ADP-Pi. In solution, Kif2C releases ADP autonomously and then binds ATP, so that the Kif2C pool is mostly ATP-loaded. Kif2C-ATP is strongly biased toward binding directly to the end of MT, which is shown by a thick solid line . Binding triggers ATP hydrolysis by Kif2C, which in turn leads to the release of tubulin from MT. The requirement for ATP hydrolysis is shown by an asterisk . Alternatively, when Kif2C-ATP binds to the MT lattice (shown by a dashed line ), ATP hydrolysis is also triggered, which yields Kif2C-ADP-P i that diffuses along MTs. The release of P i tightens MT-binding by Kif2C, where Kif2C readily releases ADP, binds another ATP, and may start the next round of Kif2C diffusion along MT. This only leads to tubulin disassembly from MT if ATP-bound Kif2C reaches the MT end.
    Figure Legend Snippet: A model for the MT depolymerization mechanism of Kif2C and other kinesin-13s. A MT is represented as a single protofilament, and Kif2C is represented as a single motor domain with the Neck shown as an attached helix. The functionally important family-specific Loop 2 is highlighted . The nucleotide bound to Kif2C is indicated with the letter: T for ATP, D for ADP, and D-Pi for ADP-Pi. In solution, Kif2C releases ADP autonomously and then binds ATP, so that the Kif2C pool is mostly ATP-loaded. Kif2C-ATP is strongly biased toward binding directly to the end of MT, which is shown by a thick solid line . Binding triggers ATP hydrolysis by Kif2C, which in turn leads to the release of tubulin from MT. The requirement for ATP hydrolysis is shown by an asterisk . Alternatively, when Kif2C-ATP binds to the MT lattice (shown by a dashed line ), ATP hydrolysis is also triggered, which yields Kif2C-ADP-P i that diffuses along MTs. The release of P i tightens MT-binding by Kif2C, where Kif2C readily releases ADP, binds another ATP, and may start the next round of Kif2C diffusion along MT. This only leads to tubulin disassembly from MT if ATP-bound Kif2C reaches the MT end.

    Techniques Used: Binding Assay, Diffusion-based Assay

    15) Product Images from "Structural and Functional Characterization of the JH2 Pseudokinase Domain of JAK Family Tyrosine Kinase 2 (TYK2) *"

    Article Title: Structural and Functional Characterization of the JH2 Pseudokinase Domain of JAK Family Tyrosine Kinase 2 (TYK2) *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M115.672048

    ATP binding to TYK2 JH2. A , surface plasmon resonance sensorgram of ATP binding to TYK2 JH2. B , Mant-ATP titration curve. Points represent the average from four individual measurements. Error bar s are standard deviation. C , thermal melting curve of JH2
    Figure Legend Snippet: ATP binding to TYK2 JH2. A , surface plasmon resonance sensorgram of ATP binding to TYK2 JH2. B , Mant-ATP titration curve. Points represent the average from four individual measurements. Error bar s are standard deviation. C , thermal melting curve of JH2

    Techniques Used: Binding Assay, SPR Assay, Titration, Standard Deviation

    A small molecule inhibitor binding to TYK2 JH2. A , chemical structure of the pyrazine inhibitor and surface plasmon resonance sensorgram of the pyrazine inhibitor binding to TYK2 JH2. B , molecular interactions of the pyrazine inhibitor in the ATP-binding
    Figure Legend Snippet: A small molecule inhibitor binding to TYK2 JH2. A , chemical structure of the pyrazine inhibitor and surface plasmon resonance sensorgram of the pyrazine inhibitor binding to TYK2 JH2. B , molecular interactions of the pyrazine inhibitor in the ATP-binding

    Techniques Used: Binding Assay, SPR Assay

    Crystal structure of TYK2 JH2 in complex with ATP-γS. A , overall structure of the TYK2 pseudokinase domain. The protein is shown in graphic representation with the activation loop highlighted in violet , αC helix in green , G-loop in orange
    Figure Legend Snippet: Crystal structure of TYK2 JH2 in complex with ATP-γS. A , overall structure of the TYK2 pseudokinase domain. The protein is shown in graphic representation with the activation loop highlighted in violet , αC helix in green , G-loop in orange

    Techniques Used: Activation Assay

    Functional characterization of TYK2 JH2. A , ATP hydrolysis as measured by ADP-Glo assay. Error bar s are S.D. from triplicate experiments. B , analysis of TYK2 signaling in mammalian cells. TYK2-deficient 11,1 cells ( left ) or Sββ cells (
    Figure Legend Snippet: Functional characterization of TYK2 JH2. A , ATP hydrolysis as measured by ADP-Glo assay. Error bar s are S.D. from triplicate experiments. B , analysis of TYK2 signaling in mammalian cells. TYK2-deficient 11,1 cells ( left ) or Sββ cells (

    Techniques Used: Functional Assay, Glo Assay

    16) Product Images from "Characterization of the ATPase FlaI of the motor complex of the Pyrococcus furiosus archaellum and its interactions between the ATP-binding protein FlaH"

    Article Title: Characterization of the ATPase FlaI of the motor complex of the Pyrococcus furiosus archaellum and its interactions between the ATP-binding protein FlaH

    Journal: PeerJ

    doi: 10.7717/peerj.4984

    ATP binding and hydrolysis of Pf FlaI. (A) Fluorescence increase at increasing concentrations of MANT-ATP upon addition of 20 nM, 100 nM and 5 µM Pf FlaI. Lines depict the linear fits of the two observed phases. The lines cross at a MANT-ATP concentration of 1.7 µM. (B) Total fluorescence after addition of increasing amounts of ATP to a solution containing 20 nM Pf FlaI and 10 nM of MANT-ATP. The data were fitted with the Hill equation: \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}$F=({F}_{\max }+({F}_{\min }-{F}_{\max })\ast [\text{ATP}]^{n})/({\text{IC}}_{50}^{n}+[\text{ATP}]^{n})$\end{document} F = F max + F min − F max ∗ ATP n ∕ IC 50 n + ATP n resulting in a best fit ( R 2 = 0.98) with IC 50 = 260 nM and n = 0.67. (C, D) ATP hydrolysis by 12.5 µg/ml Pf FlaI at different temperatures and at different pHs respectively. (E) ATPase activity of Pf FlaI at different ATP concentrations. The curve was fitted to the Michaelis-Menten equation ( V = V max∗[ATP]∕( Km + [ATP])), resulting in a K m of 580 nM. The inset shows the same data plotted according to the Hill equation (Hill coefficient = 0.9). Experiments were performed with at least two biological and three technical replicates. Error bars depict the standard error obtained from the technical replicates.
    Figure Legend Snippet: ATP binding and hydrolysis of Pf FlaI. (A) Fluorescence increase at increasing concentrations of MANT-ATP upon addition of 20 nM, 100 nM and 5 µM Pf FlaI. Lines depict the linear fits of the two observed phases. The lines cross at a MANT-ATP concentration of 1.7 µM. (B) Total fluorescence after addition of increasing amounts of ATP to a solution containing 20 nM Pf FlaI and 10 nM of MANT-ATP. The data were fitted with the Hill equation: \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}$F=({F}_{\max }+({F}_{\min }-{F}_{\max })\ast [\text{ATP}]^{n})/({\text{IC}}_{50}^{n}+[\text{ATP}]^{n})$\end{document} F = F max + F min − F max ∗ ATP n ∕ IC 50 n + ATP n resulting in a best fit ( R 2 = 0.98) with IC 50 = 260 nM and n = 0.67. (C, D) ATP hydrolysis by 12.5 µg/ml Pf FlaI at different temperatures and at different pHs respectively. (E) ATPase activity of Pf FlaI at different ATP concentrations. The curve was fitted to the Michaelis-Menten equation ( V = V max∗[ATP]∕( Km + [ATP])), resulting in a K m of 580 nM. The inset shows the same data plotted according to the Hill equation (Hill coefficient = 0.9). Experiments were performed with at least two biological and three technical replicates. Error bars depict the standard error obtained from the technical replicates.

    Techniques Used: Binding Assay, Fluorescence, Concentration Assay, AST Assay, Activity Assay

    17) Product Images from "Structure-Activity Relationships for the Interactions of 2'- and 3'-(O)-(N-Methyl)anthraniloyl-Substituted Purine and Pyrimidine Nucleotides with Mammalian Adenylyl Cyclases"

    Article Title: Structure-Activity Relationships for the Interactions of 2'- and 3'-(O)-(N-Methyl)anthraniloyl-Substituted Purine and Pyrimidine Nucleotides with Mammalian Adenylyl Cyclases

    Journal: Biochemical pharmacology

    doi: 10.1016/j.bcp.2011.05.010

    Fluorescence emission spectra of 2’-MANT- and 3’-MANT-nucleotides bound to VC1:VC1 homodimer: Comparison with MANT-GTP and MANT-ATP
    Figure Legend Snippet: Fluorescence emission spectra of 2’-MANT- and 3’-MANT-nucleotides bound to VC1:VC1 homodimer: Comparison with MANT-GTP and MANT-ATP

    Techniques Used: Fluorescence

    Fluorescence emission spectra of 2’-MANT- and 3’-MANT-nucleotides bound to VC1:IIC2 heterodimer: Comparison with MANT-GTP and MANT-ATP
    Figure Legend Snippet: Fluorescence emission spectra of 2’-MANT- and 3’-MANT-nucleotides bound to VC1:IIC2 heterodimer: Comparison with MANT-GTP and MANT-ATP

    Techniques Used: Fluorescence

    18) Product Images from "Structure-Activity Relationships for the Interactions of 2'- and 3'-(O)-(N-Methyl)anthraniloyl-Substituted Purine and Pyrimidine Nucleotides with Mammalian Adenylyl Cyclases"

    Article Title: Structure-Activity Relationships for the Interactions of 2'- and 3'-(O)-(N-Methyl)anthraniloyl-Substituted Purine and Pyrimidine Nucleotides with Mammalian Adenylyl Cyclases

    Journal: Biochemical pharmacology

    doi: 10.1016/j.bcp.2011.05.010

    Fluorescence emission spectra of 2’-MANT- and 3’-MANT-nucleotides bound to VC1:VC1 homodimer: Comparison with MANT-GTP and MANT-ATP
    Figure Legend Snippet: Fluorescence emission spectra of 2’-MANT- and 3’-MANT-nucleotides bound to VC1:VC1 homodimer: Comparison with MANT-GTP and MANT-ATP

    Techniques Used: Fluorescence

    Fluorescence emission spectra of 2’-MANT- and 3’-MANT-nucleotides bound to VC1:IIC2 heterodimer: Comparison with MANT-GTP and MANT-ATP
    Figure Legend Snippet: Fluorescence emission spectra of 2’-MANT- and 3’-MANT-nucleotides bound to VC1:IIC2 heterodimer: Comparison with MANT-GTP and MANT-ATP

    Techniques Used: Fluorescence

    Related Articles

    Staining:

    Article Title: Renal epithelial cells can release ATP by vesicular fusion
    Article Snippet: .. The images obtained the staining patterns of the two dyes seemed very similar, even though quinacrine appeared to stain more vesicles than MANT-ATP. ..

    Incubation:

    Article Title: Renal epithelial cells can release ATP by vesicular fusion
    Article Snippet: .. After prolonged incubation, we observed that MANT-ATP accumulated in vesicles in a similar manner to quinacrine. ..

    other:

    Article Title: The crystal structure and biochemical characterization of Kif15: a bifunctional molecular motor involved in bipolar spindle formation and neuronal development
    Article Snippet: Syringe 1 contained the Kif1519–375 or Kif151–375 single head–Mant-ADP complex, which was formed by adding stock 5 mM Mant-ATP to 2 µM Kif15 in BRB80 buffer with rapid mixing and incubating on ice for 30 min. Syringe 2 contained MTs at various concentrations plus 2 mM Mg2+ -ATP chasing nucleotide.

    Article Title: The crystal structure and biochemical characterization of Kif15: a bifunctional molecular motor involved in bipolar spindle formation and neuronal development
    Article Snippet: Kif15 was pre-incubated on ice to load the active site with Mant-ATP.

    Fluorescence:

    Article Title: Renal epithelial cells can release ATP by vesicular fusion
    Article Snippet: .. Using either quinacrine or MANT-ATP, we were able to observe spontaneous drops in vesicular fluorescence using TIRF microscopy. .. The sudden fall in vesicular fluorescence were taken as account for direct vesicular fusion with the plasma membrane.

    Article Title: Dynamics of human protein kinase Aurora A linked to drug selectivity
    Article Snippet: .. For the mant-ATP experiment, the dissociation constant was measured at 10°C using fluorescence energy transfer from tryptophan residues in Aurora A to mant-ATP by setting the excitation wavelength to 290 nm (5 nm bandwidth) and collecting the emission intensity from 310 to 550 nm (5 nm bandwidth) in increments of 2 nm. ..

    Microscopy:

    Article Title: Renal epithelial cells can release ATP by vesicular fusion
    Article Snippet: .. Using either quinacrine or MANT-ATP, we were able to observe spontaneous drops in vesicular fluorescence using TIRF microscopy. .. The sudden fall in vesicular fluorescence were taken as account for direct vesicular fusion with the plasma membrane.

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    Jena Bioscience mant atp
    The effect of ionomycin on <t>ATP</t> release and vesicular fusion in MDCK cells (A) total internal reflection fluorescence (TIRF) microscopy of MDCK cells loaded with <t>MANT-ATP</t> (25 μM, 5 h, 37°C) showing vesicular fusion after the addition of ionomycin (1 μM) . Arrows indicate vesicles that abruptly disappeared from the evanescence field. (B) Ionomycin (1 μM)-induced vesicular fusion as indicated by wide-field fluorescence microscopy of MDCK cells loaded with quinacrine (5 μM, 30 min, 37°C). Arrows indicate vesicles that display an abruptly drop in fluorescence. (C) Right: Original trace of the effect of ionomycin (1 μM, 37°C) on the relative fluorescence intensity of quinacrine-loaded MDCK cells. The left panel present the mean ± s.e.m. from 6 experiments. (D) ATP release from MDCK cells induced by apical application of ionomycin (IONO, 1 μM). The arrow indicates the addition of ionomycin or the control solution; the mean ± s.e.m. values are presented ( n = 10). The asterisk indicates statistical significance.
    Mant Atp, supplied by Jena Bioscience, used in various techniques. Bioz Stars score: 95/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    The effect of ionomycin on ATP release and vesicular fusion in MDCK cells (A) total internal reflection fluorescence (TIRF) microscopy of MDCK cells loaded with MANT-ATP (25 μM, 5 h, 37°C) showing vesicular fusion after the addition of ionomycin (1 μM) . Arrows indicate vesicles that abruptly disappeared from the evanescence field. (B) Ionomycin (1 μM)-induced vesicular fusion as indicated by wide-field fluorescence microscopy of MDCK cells loaded with quinacrine (5 μM, 30 min, 37°C). Arrows indicate vesicles that display an abruptly drop in fluorescence. (C) Right: Original trace of the effect of ionomycin (1 μM, 37°C) on the relative fluorescence intensity of quinacrine-loaded MDCK cells. The left panel present the mean ± s.e.m. from 6 experiments. (D) ATP release from MDCK cells induced by apical application of ionomycin (IONO, 1 μM). The arrow indicates the addition of ionomycin or the control solution; the mean ± s.e.m. values are presented ( n = 10). The asterisk indicates statistical significance.

    Journal: Frontiers in Physiology

    Article Title: Renal epithelial cells can release ATP by vesicular fusion

    doi: 10.3389/fphys.2013.00238

    Figure Lengend Snippet: The effect of ionomycin on ATP release and vesicular fusion in MDCK cells (A) total internal reflection fluorescence (TIRF) microscopy of MDCK cells loaded with MANT-ATP (25 μM, 5 h, 37°C) showing vesicular fusion after the addition of ionomycin (1 μM) . Arrows indicate vesicles that abruptly disappeared from the evanescence field. (B) Ionomycin (1 μM)-induced vesicular fusion as indicated by wide-field fluorescence microscopy of MDCK cells loaded with quinacrine (5 μM, 30 min, 37°C). Arrows indicate vesicles that display an abruptly drop in fluorescence. (C) Right: Original trace of the effect of ionomycin (1 μM, 37°C) on the relative fluorescence intensity of quinacrine-loaded MDCK cells. The left panel present the mean ± s.e.m. from 6 experiments. (D) ATP release from MDCK cells induced by apical application of ionomycin (IONO, 1 μM). The arrow indicates the addition of ionomycin or the control solution; the mean ± s.e.m. values are presented ( n = 10). The asterisk indicates statistical significance.

    Article Snippet: Using either quinacrine or MANT-ATP, we were able to observe spontaneous drops in vesicular fluorescence using TIRF microscopy.

    Techniques: Fluorescence, Microscopy

    Spontaneous vesicular fusion in MDCK cells . Total internal reflection fluorescence (TIRF) microscopy of MDCK cells loaded with MANT-ATP (25 μM, 5 h, 37°C). Arrows indicate vesicles that abruptly disappeared from the evanescence field.

    Journal: Frontiers in Physiology

    Article Title: Renal epithelial cells can release ATP by vesicular fusion

    doi: 10.3389/fphys.2013.00238

    Figure Lengend Snippet: Spontaneous vesicular fusion in MDCK cells . Total internal reflection fluorescence (TIRF) microscopy of MDCK cells loaded with MANT-ATP (25 μM, 5 h, 37°C). Arrows indicate vesicles that abruptly disappeared from the evanescence field.

    Article Snippet: Using either quinacrine or MANT-ATP, we were able to observe spontaneous drops in vesicular fluorescence using TIRF microscopy.

    Techniques: Fluorescence, Microscopy

    Fluorescence emission spectra of 2’-MANT- and 3’-MANT-nucleotides bound to VC1:VC1 homodimer: Comparison with MANT-GTP and MANT-ATP

    Journal: Biochemical pharmacology

    Article Title: Structure-Activity Relationships for the Interactions of 2'- and 3'-(O)-(N-Methyl)anthraniloyl-Substituted Purine and Pyrimidine Nucleotides with Mammalian Adenylyl Cyclases

    doi: 10.1016/j.bcp.2011.05.010

    Figure Lengend Snippet: Fluorescence emission spectra of 2’-MANT- and 3’-MANT-nucleotides bound to VC1:VC1 homodimer: Comparison with MANT-GTP and MANT-ATP

    Article Snippet: MANT-GTP ( 1 ), 2’-d-3’-MANT-GTP ( 2 ), 3’-d-2’-MANT-GTP ( 3 ), MANT-GTPγS ( 4 ), MANT-ATP ( 5 ), 2’-d-3’-MANT-ATP ( 6 ), 3’-d-2’-MANT-ATP ( 7 ), MANT-ITPγS ( 9 ), MANT-XTP ( 10 ), ANT-GTP ( 14 ) and MANT-ADP ( 16 ) were obtained from Jena Bioscience (Jena, Germany).

    Techniques: Fluorescence

    Fluorescence emission spectra of 2’-MANT- and 3’-MANT-nucleotides bound to VC1:IIC2 heterodimer: Comparison with MANT-GTP and MANT-ATP

    Journal: Biochemical pharmacology

    Article Title: Structure-Activity Relationships for the Interactions of 2'- and 3'-(O)-(N-Methyl)anthraniloyl-Substituted Purine and Pyrimidine Nucleotides with Mammalian Adenylyl Cyclases

    doi: 10.1016/j.bcp.2011.05.010

    Figure Lengend Snippet: Fluorescence emission spectra of 2’-MANT- and 3’-MANT-nucleotides bound to VC1:IIC2 heterodimer: Comparison with MANT-GTP and MANT-ATP

    Article Snippet: MANT-GTP ( 1 ), 2’-d-3’-MANT-GTP ( 2 ), 3’-d-2’-MANT-GTP ( 3 ), MANT-GTPγS ( 4 ), MANT-ATP ( 5 ), 2’-d-3’-MANT-ATP ( 6 ), 3’-d-2’-MANT-ATP ( 7 ), MANT-ITPγS ( 9 ), MANT-XTP ( 10 ), ANT-GTP ( 14 ) and MANT-ADP ( 16 ) were obtained from Jena Bioscience (Jena, Germany).

    Techniques: Fluorescence