Activity of Pks13-TE was assessed using 4-methylumbelliferyl heptanoate (4-MUH, Sigma) as a fluorogenic substrate in a 96-well plate format (Richardson and Smith, 2007 (link)). To make initial velocity measurements, Pks13-TE (1 μM) in 0.1 M Tris-HCl, pH 7 buffer was incubated with different concentrations of 4-MUH (2-150 μM in DMSO) in a 100 μL reaction volume, and the fluorescence of the hydrolyzed product 4-methylumbelliferone was read (excitation at 355 nm and emission at 460 nm) (PolarStar Omega plate reader BMG Labtech) at 5-10 min intervals over 80-120 min. The reaction rate was observed to be linear in the measured range. 4-MUH in buffer alone was included as a control to quantify its background hydrolysis. Data points were plotted as an average of triplicates and each experiment was repeated twice independently. The initial velocity data was curve fit to Michaelis-Menten equation by nonlinear regression using Prism software (GraphPad) to determine the kinetic parameters Km and Vmax. The assay and data analysis for Pks13-TE mutants was done the same way as that for the wild-type protein with the 4-MUH concentration varying from 2 to 300 μM.
4 muh
Manufactured by Merck Group
Sourced in United States
4-MUH is a laboratory reagent used as an analytical tool in various scientific applications. It is a fluorogenic substrate that can be used to detect and measure the activity of specific enzymes. The core function of 4-MUH is to provide a quantitative assessment of enzymatic activity through fluorescent signal detection.
Automatically generated - may contain errors
Lab products found in correlation
4 muh, by BMG Labtech (2 mentions)
Prism software, by GraphPad (2 mentions)
Polarstar omega, by BMG Labtech (2 mentions)
Synergy h4 plate reader, by Agilent Technologies (1 mentions)
Zardaverine, by Merck Group (1 mentions)
Butylated hydroxytoluene, by Merck Group (1 mentions)
Wwl70, by Cayman Chemical (1 mentions)
Kt 182, by Cayman Chemical (1 mentions)
5 protocols using 4 muh
1
Kinetic Analysis of Pks13-TE Activity
2
Fluorescence-based Enzymatic Hydrolysis Assay
3
Analytical Reagents for Biochemical Assays
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AG, KT‐182, N‐oleylethanolamine‐d2 (OEA‐d2) and WWL70 were supplied by Cayman Chemicals (Ann Arbor, MI, USA) and 4‐MUH, 4‐MU, ACEA, butylated hydroxytoluene and zardaverine by Sigma‐Aldrich (Madrid, Spain). TOFA was supplied by Abcam (Cambridge, UK).
4
Kinetic Characterization of Pks13-TE Enzyme
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Enzyme activity of Pks13-TE
was assessed using 4-methylumbelliferyl heptanoate (4-MUH, Sigma)
as a fluorogenic substrate in a 96-well plate format. To make initial
velocity measurements, Pks13-TE (0.5 μM) in 0.1 M Tris-HCl,
pH 7 buffer was incubated with different concentrations of 4-MUH (0.2–200
μM in DMSO, 1% DMSO final) in a 100 μL reaction volume,
and the fluorescence of the hydrolyzed product 4-methylumbelliferone
was read (excitation at 355 nm and emission at 460 nm) using a PolarStar
Omega plate reader (BMG Labtech) at 5–10 min intervals over
120–140 min. The reaction rate was observed to be linear in
the measured range. 4-MUH in buffer alone was included as a control
to quantify its background hydrolysis. Data points were plotted as
an average of duplicates and analyzed using Prism software (GraphPad)
to determine the kinetic parameters Km, Vmax, and kcat. The experiment was repeated in 20 mM HEPES, 134 mM potassium acetate,
8 mM sodium acetate, 4 mM sodium chloride, and 0.8 mM magnesium acetate,
pH 7.2.
was assessed using 4-methylumbelliferyl heptanoate (4-MUH, Sigma)
as a fluorogenic substrate in a 96-well plate format. To make initial
velocity measurements, Pks13-TE (0.5 μM) in 0.1 M Tris-HCl,
pH 7 buffer was incubated with different concentrations of 4-MUH (0.2–200
μM in DMSO, 1% DMSO final) in a 100 μL reaction volume,
and the fluorescence of the hydrolyzed product 4-methylumbelliferone
was read (excitation at 355 nm and emission at 460 nm) using a PolarStar
Omega plate reader (BMG Labtech) at 5–10 min intervals over
120–140 min. The reaction rate was observed to be linear in
the measured range. 4-MUH in buffer alone was included as a control
to quantify its background hydrolysis. Data points were plotted as
an average of duplicates and analyzed using Prism software (GraphPad)
to determine the kinetic parameters Km, Vmax, and kcat. The experiment was repeated in 20 mM HEPES, 134 mM potassium acetate,
8 mM sodium acetate, 4 mM sodium chloride, and 0.8 mM magnesium acetate,
pH 7.2.
5
Fluorogenic TE Activity Assay for PPI Inhibition
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The fluorogenic TE activity
assay was performed as previously described.22 (link) Briefly, each assay was performed in opaque black, flat-bottom 96-well
plates (Corning), with each well containing 500 nM purified TE in
buffer A (100 mM Tris-HCl, 50 mM NaCl, 0.05% Brij35, pH 7.5). For
PPI inhibition, recombinant TE was preincubated with PPIs at 37 °C
for 30 min. The hydrolysis reaction was started by addition of 300
μM 4-MUH (Sigma) and incubation at 37 °C for 1 h. Fluorescence
due to liberated 4-MU was measured at 355/460 nm. The Ki value for each inhibitor candidate was calculated using
the Cheng and Prusoff equation.37 (link)
assay was performed as previously described.22 (link) Briefly, each assay was performed in opaque black, flat-bottom 96-well
plates (Corning), with each well containing 500 nM purified TE in
buffer A (100 mM Tris-HCl, 50 mM NaCl, 0.05% Brij35, pH 7.5). For
PPI inhibition, recombinant TE was preincubated with PPIs at 37 °C
for 30 min. The hydrolysis reaction was started by addition of 300
μM 4-MUH (Sigma) and incubation at 37 °C for 1 h. Fluorescence
due to liberated 4-MU was measured at 355/460 nm. The Ki value for each inhibitor candidate was calculated using
the Cheng and Prusoff equation.37 (link)
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