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Product Uses
- Measurement of calcium activated myosin ATPase activity when bound to thin filaments.
- Identification/characterization of proteins or small moleculesthat affect the CTFC regulation and myosin ATPase activity
- Identification/characterization of proteins or small molecules that affect myosin / F-actin interaction
Materials
The cardiac thin filament complex (CTFC) has been assembled from purified F-actin (Cat. # AD99) and Tropomyosin / Troponin protein (TT) complex (Cat. # TT05). Thus, CTFC is composed of six proteins: Actin : Tropomyosin α: Tropomyosin β : Troponin C :Troponin I : Troponin T in a stoichiometric ratio of 7:1:1:1:1:1; see Figure 1. After assembly, the thin filaments are centrifuged at 100,000 x g and the pellets resuspended in reaction buffer which purifies the calcium sensitive complex. The complex has been determined to be biologically active in a calcium activated myosin ATPase assay (see biological activity assay). The complex is supplied as a white lyophilized powder.
Legend: Myosin is a hexameric protein consisting of two heavy chains and two light chains. Myosin can be proteolytically cleaved into heavy meromyosin (HMM) and light meromyosin (LMM) by α-chymotrypsin in the presence of magnesium. In the presence of EDTA, however, α-chymotrypsin produces the soluble myosin S1 fragment (3).
Storage and Resonstitution
Briefly centrifuge to collect the product at the bottom of the tube. Reconstituting a tube of CS-MYS05 with 180 ul of Milli-Q water will generate a 1.4 mg/ml stock of gizzard S1 myosin in the following buffer: 8 mM PIPES pH 7.0, 8 mM MgCl2, 5% (w/v) sucrose and1% (w/v) dextran. The protein should not be exposed to repeated freeze-thaw cycles. The lyophilized protein is stable at 4°C desiccated (<10% humidity) for 1 year.
Purity
Protein purity is determined by scanning densitometry of Coomassie Blue stained protein on a 4-20% gradient polyacrylamide gel. The myosin and its light chains used to produce the myosin S1 fragment was determined to be 90% pure (see Figure2).
Figure 2. Full length and S1 myosin
Legend: A 20 μg sample of CTFC proteins was separated by electrophoresis in a 4-20% SDS-PAGE system (Lane 2) and stained with Coomassie Blue. Molecular weights are 43 kDa actin,38 kDa Troponin T, 32 kDa Tropomyosin (α+β), 24 kDa Troponin I, and 18 kDa Troponin C. Protein concentration was measured with the Precision Red™ Protein Assay Reagent (Cat.# ADV02). See Blue molecular weight markers (Lane 1) are from Life Technologies Inc.
Biological Activity Assay
The biological activity of the CTFC can be determined from its ability to regulate myosin ATPase activity. The CTFC re-creates coated filaments which are analogous to the thin filaments of muscle fibers. Myosin is added in substoichiometric amounts and the reaction is initiated with ATP and calcium. Stringent quality control ensures that in the absence of exogenous calcium, the CTFC completely inhibits myosin ATPase. On addition of 10 µM calcium, myosin ATPase will be restored. Calcium binds to Troponin C which dissociates from F-actin, allowing myosin to bind.
Reagents
1. Cardiac Thin Filament Complex (1 x 1 mg, # TFC01)2. Cardiac Myosin S1 (0.25 mg, # MYS03)3. ATPase Assay Biochem Kit (Cat. # BK051)4. 100 mM ATP in 50 mM Tris-HCl pH 7.5 (100 µl)5. PM12 Reaction buffer (12 mM Pipes-NaOH, pH 6.8, 2 mM MgCl2).
Equipment
1. Spectrophotometer capable of measuring absorbance at 360 nm (+/- 5 nm bandwidth). We recommend a Spectra-Max M2 (Molecular Devices). Filter based machines are not suitable.2. Half area 96 well microtiter plate (Corning Cat.# 3696 or 3697)3. Multi-channel pipette
Method
The following major steps are recognized:
Step 1. Assemble required reagents and compounds (30 min).
Step 2. Prepare Thin Filament stock (15 min).
Step 3. Prepare Motor Mix and plate reader (15 min).
Step 4. Pipette Motor Mix into wells and start reaction/plate reader (10 min).
Thin Filament stock
1. Gently resuspend 1 x 1 mg TFC01 with room temp PM12 buffer to 2 mg/ml; it will be a white solution (500 ul per vial for 1 mg vial).2. Incubate at RT for 10 min.3. Centrifuge at 500 x g for 30s; now it is a clear solution.4. Store at room temperature for up to 20 min.
Myosin reaction stock
1. Dilute S1 myosin to 1.0 mg/ml with ice cold PM12 buffer.2. Mix the following in the stated order at RT, to make 4.0 ml of Myosin/Thin Filament control mixture:
2610 μl of PM12
800 μl 5x MSEG (this is a BK051 component)
500 μl of TFC01
30 μl of Myosin S1 solution
20 μl of 100 mM ATP
40 μl of 100x PNP (this is a BK051 component)
3. Using the pre-warmed half area 96-well plate, pipette the following:4. Pipette 10 μl of 100 μM calcium chloride into “activated” wells.5. Pipette 10 μl of Milli-Q water into “non-activated” wells.6. Pipette 10 μl of 10X [test compound] into appropriate wells.7. Incubate at 37°C for 2 min to warm the mixture.8. Pipette 100 μl of Myosin/Thin Filament mixture into all wells.9. Start protocol, 41 readings, 30 seconds apart, 37°C , OD 360+/- 5 nm.10. Calculate Vmax and compare non-activated to calcium activated samples.
Figure 3: Calcium Dose Response Curve
Legend: The sarcomere assay was set up as described in the protocol above. Calcium was titrated between 2 and 200 μM andthe results plotted on this dose response graph. pCa = 6.1 μM is similar to published pCa values for reconstituted cardiac sarcomeres (Holroyde et al. 1980, Fig.6).
References
1. J.M Murray. 1982. Hybridization and reconstitution of the thin filament. Methods Enzymol. 85: 15-17.2. M.J. Holroyde et al. 1980. The calcium and magnesium binding sites on cardiac troponin their role in the regulation of myofibrillar adenosine triphosphatase. J. Biol. Chem. 255: 11688-11693.
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