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The Tropomyosin / Troponin protein (TT) complex has been purified from bovine cardiac muscle (1). The TT complex is composed of five proteins Tropomyosin alpha: Tropomyosin beta: TroponinC : Troponin I : Troponin T in a stoichiometric ratio of 1:1:1:1:1, see Figure 1. The complex has been determined to be biologically active in an F-actin / calcium activated myosin ATPase assay (see biological activity assay). The complex is supplied as a white lyophilized powder.
Figure 1—Schematic diagram of muscle thin filament.
Storage and Reconstitution
Briefly centrifuge to collect the product at the bottom of the tube. The protein should be reconstituted to 3 mg/ml by the addition of ice cold Milli-Q water. The protein will be in the following buffer: 20 mM PIPES pH 7.0, 25 mM KCl, 5% (w/v) sucrose and 1% (w/v) dextran. In order to maintain high biological activity of the protein, it is recommended that the protein solution be aliquoted into "experiment sized" amounts, snap frozen in liquid nitrogen and stored at -70°C. The protein is stable for 6 months if stored at -70°C. 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.
Protein purity is determined by scanning densitometry of Coomassie Blue stained protein on a 4-20% gradient polyacrylamide gel. The TT complex was determined to be 85% pure (see Figure 2a). Antibodies against each component were used to verify each one was present (see Figure 2b)
Figure 2. Tromomyosin / Troponin TCI Complex Protein Purity Determination.
Figure 2a):A 20 μg sample of TT complex protein was separated by electrophoresis in a 4-20% SDS-PAGE system and stained with Coomassie Blue. Arrow 1 indicates Troponin T (38 kDal), arrow 2 indicates Tropomyosin(alpha+beta, 32 kDal), arrow 3 indicates Troponin I(24 kDal) and arrow 4 indicates Troponin C (18 kDal).
Figure 2b: A 2 μg sample of TT complex protein was separated by electrophoresis in a 4-20% SDS-PAGE system, blotted onto PVDF membrane and probed with component specific monoclonal antibodies. Lane 3: Tropomyosin(alpha+beta, 32 kDal, MAb clone:TM311), Lane 4: Troponin T (38 kDal, MAb clone JLT-12 from Sigma) (antibody gave 24 kDal signal—cross reacts with Troponin I ?), Lane 5: Troponin I (24 kDal, MAb clone MAB1691), and Lane 6: Troponin C (18 kDal, MAb clone 7B9).
Protein quantitation was performed using the Precision Red™ Protein Assay Reagent (Cat.# ADV02). See Blue molecular weight markers are from Life Technologies Inc.
Biological Activity Assay
The biological activity of the TT complex can be determined from it’s ability to regulate F-actin activated myosin ATPase. The assay is constructed by first polymerizing actin to form F-actin, then theTT complex is mixed with the actin filaments in a stoichiometric amount. This creates coated filaments which are analogous to the thin filaments of muscle fibers. These filaments are centrifuged at 100,000 xg and the pellets resuspended in reaction buffer which purifies the calcium sensitive complex. Myosin is added in substoichiometric amounts and the reaction initiated with ATP and calcium. Stringent quality control ensures that in the absence of calcium the TT complex completely inhibits myosin ATPase. Ona ddition of 10 uM calcium myosin ATPase will be restored. Calcium binds to Troponin C which dissociates from F-actin allowing myosin to bind.
1. Cardiac TT complex (1 x 1 mg, # TT05)
2. Cardiac Myosin S1 (0.25 mg), # MYS03)
3. Cardiac Actin (1 mg, Cat. # AD99-A)
4. ATPase Assay Biochem Kit (Cat. # BK051)
5. 100 mM ATP in 50 mM Tris-HCl pH 7.5 (100ul)
6. 1 M Dithiothreitol in water (100 ul).
7. PM12 Reaction buffer (12 mM Pipes-KOH, pH 7.0, 2 mM MgCl2).
8. 500 mM EGTA-Na pH 8.0.
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
The following major steps are recognized:
Step 1. Assemble required reagents and compounds. (30min).
Step 2. Prepare F-actin polymer stock. (1h).
Step 3. Prepare Thin Filament stock (1.5h)
Step 4. Prepare Motor Mix and plate reader. (15min).
Step 5. Pipette Motor Mix into wells and start reaction/plate reader. (10min).
F-actin polymer stock
1. Resuspend AD99 with 2.5ml of Buffer to 0.4 mg/ml (measure protein concentration for better reproducibility), in buffer 5mM Pipes-KOH pH 7.0, 500uM ATP, 500uM DTT.
2. Place at RT for 10min to solubilize the actin.
3. Then add 2.0 mM MgCl2 and 2.0 mM EGTA and incubate at RT for 20 min to polymerize. (shelf life 1h at RT).
Thin Filament stock
1. Resuspend 1 x 1mg TT05 on ice with ice cold water to 5mg/ml. (200ul per vial for 1mg vial).
2. Mix the following to make 1.2 ml of actin/TT05 (TF):
1000 μl of F-actin stock
200 μl of TT05
3. Incubate at RT for 20min.
4. Centrifuge at 100K xg 4°C for 1h.
5. Resuspend pellet in 1200 μl of RT PM12 buffer.
6. Store at RT for up to 2h, or 4°C up to 3 days.
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 2.08 ml of actin/TT/myosin (ATM) control mixture:
400 μl of PM12
400 μl 5x MSEG (this is a BK051 component)
1200 μl of TF
40 μl of Myosin S1 solution.
40 μl of 100x PNP (this is a BK051 component)
10.4 μl of 100mM ATP
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 10 x [test compound] into appropriate wells.
7. Incubate at 37°C for 2min to warm the mixture.
8. Pipette 100 μl of ATM mixture into all wells.
9. Start protocol, 41 readings, 30 seconds apart, 37°C , OD 360nm.
10. Calculate Vmax and compare non-activated to calcium activated samples.
Figure 3 legend: The sarcomere assay was set up as described in the protocol above. Calcium was titrated between 2 and 200 μM and the 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).
1. J.M Murray. 1982. Hybridization and reconstitution of the thin filament. Methods in Enzymology, 85, p.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.
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