Methods

Displacement of endogenous TN in skinned fibers with TN mutants: A very sensitive method for mapping the functional domains of TN components is the replacement of native TN in the fibers with modified TN and then assaying force development or Ca2+-sensitivity of the modified fibers. In principle, the fibers are briefly treated with 1% Triton X-100 to remove the sarcolemma and subsequently washed to remove the Triton. The fibers are exposed to 10-20-fold excess of exogenous Tn, over the endogenous TN, in a medium containing 250 mM KCl. Finally, unbound TN is removed by washings of the fibers. In the past, most of the replacement studies were performed with skeletal muscle fibers, recently a method for exchanging the TN subunits in cardiac fibers was published (Chandra et al., 1999). The skinned rat cardiac fiber bundles remained relaxed through much of the extraction/reconstitution procedure. The fibers were treated with a mixture of cTN-T-cTN-I to displace the endogenous TN; 70-80% of the endogenous TN subunits were removed. After reconstitution with cTN-C the Ca2+-dependence of the force development by the fibers was restored to an extent of 80-85%.

Reconstitution of a modified TN can also be carried out at the level of myofibrils and the Mg2 -ATPase activity of the modified fibrils can be measured as a function of pCa (Rarick et al., 1997).

Purification of cardiac myofibrils is important for studying the interaction of the troponin components with each other, with tropomyosin and actin. Myofibrils purified by conventional methods are contaminated with mitochondrial, sarcolemmal and sarcoplasmic reticulum membranes. Treatment of these myofibrils by 1% Triton X-100 readily removes the contaminants (Solaro et al., 1971). The MgATPase activity of the purified myofibrils corresponds to that of cardiac actomyosin and they exhibit the normal high Ca2+-sensitivity.

Gel elctrophoresis of the regulatory proteins: In our laboratory, 10% polyacrylamide gels containing 1% SDS, 0.1 M Na-phosphate, pH 7.0 and 8.0 M urea are used for separation of TM, TN-T and TN-I (Fig. H5). Under these conditions TM (zone A, 48-kDa) migrates behind actin, TNT (zone B, 38-kDa) migrates in front of actin, TN-I (zones C and D, 30- and 29 kDa) splits into two components. Zones E and F, correspond to the 27- and 19-kDa myosin LCs. TN-C migrates in front of the 19-kDa LC, it is not visible because it does not stain with Coomassie blue.

I I III

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