Skeletal muscle myosin II subfragment 1 (S1) ATPase

BTS (2–12 μM) suppresses Ca2+-stimulated myosin S1 ATPase activity in the absence of actin, with an IC50 of about 5 μM [1]. The heavy myosin (HMM) sliding rate is reversibly inhibited by BTS (2–20 μM) [1]. In the presence of ADP, BTS (100 μM) releases myosin from actin [1]. With an IC50 of roughly 3 μM and 1 μM, respectively, BTS (0-20 μM) reversibly reduces Ca2+-activated tension in medium-length skinned skeletal muscle fibers of rabbit and frog [1].

High-throughput screening of chemical libraries[1]
Five microlitres of a mixture of 0.1 mg ml−1 myosin S1, 0.4 mg/ml F-actin, 1 mM DTT in assay buffer (25 mM HEPES pH 7.6, 50 mM KCl, 2 mM MgCl2) was dispensed into a 384-well white cliniplate. Chemical compounds stored in DMSO at a concentration of 5 mg ml−1 were transferred into 384-well plates using a steel pin array mounted on an X,Y,Z robot to give a final concentration of ∼100 μM. The reaction was initiated by adding 5 μl per well of 200 μM ATP then incubated for 1 h at ambient temperature (20–24 °C). To measure the remaining ATP, 40 μl of development solution (0.5 mM luciferin, 1.25 μg ml−1 luciferase, 25 mM Tricine pH 7.8, 5 mM MgSO4, 100 μM EDTA and 1 mM DTT) was added to each well. The luminescence signal in each assay well was then measured using a plate reader within 15 min.

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Steady-State ATPases. [2]
ATPase assays were performed using an NADH-coupled assay monitoring absorbance at 340 nm. These experiments were performed using either an Applied Photophysics stopped-flow apparatus or a standard UV/vis spectrophotometer at 25 °C. ATPase reactions contained 50 nM S1 in buffer A [10 mM imidazole (pH 7.0), 2 mM MgCl2, 1 mM EGTA, and 1 mM DTT] containing 100 U/mL pyruvate kinase, 20 U/mL lactic dehydrogenase, 200 μM NADH, and 500 μM phospho(enol) pyruvate. Actin concentrations varied from 0 to 150 μM. Reactions were initiated by the addition of 2 mM MgATP.[2]

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Internal Tryptophan Fluorescence. [2]
Experiments were performed using an Applied Photophysics stopped-flow apparatus. Protein fluorescence was excited at 295 nm and collected through a 320-nm long-pass emission filter. Experiments were performed in either buffer A (see above) or buffer B [20 mM Tris-HCl (pH 7.0), 200 mM KCl, 2 mM MgCl2, and 1 mM DTT] at 25 °C.[2]

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Pyrene-Actin Fluorescence. [2]
The extent of strong S1 binding to actin under various conditions was measured by fluorescence quenching of pyrene-labeled actin in buffer B at 25 °C. Steady-state measurements were performed on a standard fluorometer using an excitation wavelength of 365 nm. Emission was measured through a 400-nm cutoff filter and a monochromator set to 412 nm. Transient experiments were performed on an Applied Photophysics stopped-flow apparatus, exciting at 365 nm and monitoring emission through a 400-nm cutoff filter.

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Phosphate Release. [2]
Transient phosphate (Pi) release was measured using a fluorescently labeled mutant of phosphate-binding protein (PiBP; 18, 19) with the stopped flow in sequential mixing mode and using a 440-nm long-pass filter (λex = 425 nm). The dead time of the instrument in this configuration was ∼2 ms. Contaminating phosphate was removed from syringes, plastic ware, and the stopped-flow apparatus by incubating overnight with 0.5 mM 7-methylguanosine and 1 U/mL nucleoside phosphorylase in buffer A. Background phosphate was removed from experimental solutions by incubating with 0.1 mM 7-methylguanosine and 0.02 U/mL nucleoside phosphorylase.[2]

In vitro motility[1]
Actin filament sliding on myosin S1 and on HMM was assayed according to Sellers et al.24 with the following modifications. HMM was adsorbed to nitrocellulose-coated coverslips in flow-cell chambers at a concentration of 0.1 mg ml−1. Chambers were blocked with 0.5 mg ml−1 casein, and then F-actin that had been stabilized with TRITC-phalloidin was added at a concentration of 20 nM. Motility was initiated by adding 1 mM ATP in assay buffer (20 mM KCl, 10 mM MOPS pH 7.2, 5 mM MgCl2, 0.1 mM EGTA, 10 mM DTT, 2.5 mg ml−1 glucose, 0.1 mg ml−1 glucose oxidase, 0.02 mg ml−1 catalase) and filament sliding was recorded at 10 s intervals using a 1.4 NA 60× lens (Nikon) and a cooled charge-coupled display (CCD) camera (Princeton Instruments). For control, 5C, 20 μM BTS and 2 μM BTS the number of filaments tracked were 63, 27, 51 and 54 respectively in 8, 3, 6 and 5 individual experiments. Rates are expressed as velocity in μm sec−1 ± standard error.[1]

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Measurement of Ca2+-stimulated S1 ATPase[1]
Myosin S1 was diluted to 0.025 mg ml−1 in reaction buffer (200 mM KCl, 50 mM Tris pH 7.5, 2.5 mM CaCl2, 1 mM MgCl2, 1 mM DTT, 0.2 mM 2-amino-6-mercapto-7methyl-purine riboside (MESG) and 10 U ml−1 purine nucleoside phosphorylase (PNP). The reaction was initiated by adding ATP to 100 μM and absorbance at 360 nm was read every 5 s. Inhibition was assayed at 0, 0.625, 1.25, 2.5, 5 and 10 μM BTS.[1]

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Mant-ADP binding assay[1]
0.5 μM myosin S1 was mixed with 5 μM Mant-ADP (Molecular Probes) in assay buffer (25 mM HEPES pH 7.6, 50 mM KCl, 2 mM MgCl2). Solutions of ADP, BTS and 5% DMSO, or 5% DMSO were added to the mixture of myosin S1 and Mant-ADP to give final ADP or BTS concentrations of 5, 10, 50, 100, 200 or 400 μM. Mant-ADP fluorescence was excited at 290 nm and assayed at 448 nm. Fluorescence values in the presence of ADP or BTS were divided by the fluorescence of the buffer control to correct for dilution effects.[1]

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Cosedimentation of F-actin and HMM[1]
G-actin (35 μM) was polymerized in F-buffer (100 mM KCl, 25 mM Tris pH 7.0, 1 mM MgCl2, 0.2 mM CaCl2, 0.5 mM DTT, 1 mM ATP) for 45 min at 37 °C. F-actin was stabilized by adding phalloidin to 45 μM and was dialysed against spindown buffer (200 mM KCl, 50 mM Tris pH 7.0, 2 mM MgCl2, 1 mM DTT, 50 mM glucose) to remove residual ATP. HMM (0.5 μM) was incubated with or without F-actin (1.5 μM) in binding buffer for 15 min at room temperature, then 10 μl of the mixture was distributed into separate tubes. ADP (50 mM) was preincubated with 50 U ml−1 hexokinase (Sigma) in spindown buffer for 20 min at 37 °C to hydrolyse contaminant ATP. To the actin–myosin mixture, an equal volume of binding buffer alone or binding buffer containing either 2 mM ATP or 2 mM ADP and 0.2 U hexokinase was added. BTS dissolved in DMSO or DMSO alone was added at the time of nucleotide addition to yield a final concentration of 100 μM BTS or 0.1% DMSO. The reaction mixture was incubated at 22 °C for 10 min then centrifuged at 217000 g (75000 rpm) for 10 min in a TLA-100 rotor. The supernatants and pellets were solubilized in SDS–PAGE sample buffer, separated on a 10% SDS–PAGE gel and stained with Coomassie brilliant blue.

Relaxed bundles of rabbit psoas or soleus muscle were chemically skinned in a 50% glycerol-extraction solution (5 mM MgATP, <10−8 M Ca2+, −20 °C)25 to remove the sarcolemma, leaving the contractile apparatus intact. Single fibres were teased from the bundles under silicone oil (12 °C) and rinsed with glycerol-free relaxing solution. Aluminum foil clips (T-clips) were fastened around the ends of the fibres to secure the fibre between hooks on a force transducer and a servomotor (200 μs length changes)[1]

[1]. A small-molecule inhibitor of skeletal muscle myosin II. Nat Cell Biol. 2002 Jan;4(1):83-8.

[2]. Mechanism of inhibition of skeletal muscle actomyosin by N-benzyl-p-toluenesulfonamide. Biochemistry. 2003 May 27;42(20):6128-35.

4-methyl-N-(phenylmethyl)benzenesulfonamide is a sulfonamide.

C14H15NO2S

261.33

261.082

C, 64.34; H, 5.79; N, 5.36; O, 12.24; S, 12.27

1576-37-0

95801

White to off-white solid powder

1.204 g/cm3

419.1ºC at 760 mmHg

115 °C

207.3ºC

3.12E-07mmHg at 25°C

1.589

3.945

1

3

4

18

333

0

S(C1C([H])=C([H])C(C([H])([H])[H])=C([H])C=1[H])(N([H])C([H])([H])C1C([H])=C([H])C([H])=C([H])C=1[H])(=O)=O

WTHKAJZQYNKTCJ-UHFFFAOYSA-N

InChI=1S/C14H15NO2S/c1-12-7-9-14(10-8-12)18(16,17)15-11-13-5-3-2-4-6-13/h2-10,15H,11H2,1H3

N-benzyl-4-methylbenzenesulfonamide

BnNHTs; BTS; N-Benzyl-p-toluenesulfonamide; 1576-37-0; N-Benzyl-4-methylbenzenesulfonamide; N-Tosylbenzylamine; N-Benzyl-4-methyl-benzenesulfonamide; N-Benzyl-p-toluenesulphonamide; Benzenesulfonamide, 4-methyl-N-(phenylmethyl)-; p-Toluenesulfonamide, N-benzyl-;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ≥ 33.33 mg/mL (~127.54 mM)
H2O : ~0.1 mg/mL (~0.38 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (9.57 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (9.57 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (9.57 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)