A novel method of extraction of TnC from skeletal muscle myofibrils.

Incubation of mechanically skinned barnacle myofibrillar bundles in 10 mM orthovanadate (pH 6.6) results in the loss of Ca2+-dependent force generation, which reduces to 0.98+/-0.006% (mean +/-SEM, n=25) of control levels. Analysis of myofibrillar bundles by gel electrophoresis showed that tension loss is primarily due to the extraction of troponin C (TnC) (65.4+/-5.04% mean +/-SEM, n=5). This is a novel finding, since treating cardiac fibres with orthovanadate results in the removal of both TnC and troponin I (TnI) (28). Ca2+ dependence was restored to the myofibrillar bundles following reconstitution with either native isoform of barnacle TnC (BTnC1: 78. 72+/-12.8%, n=9, BTnC2: 82.73+/-20.3%, n=3). The reversible loss of Ca2+-dependent tension generation following the removal and replacement of TnC indicates that the regulation of contraction in the barnacle is controlled by thin-filament regulatory proteins.