FUNCTIONAL-CHARACTERIZATION OF THE 2 ISOFORMS OF TROPONIN-C FROM THE ARTHROPOD BALANUS-NUBILUS

Two isoforms of troponin C (BTnC1 and BTnC2) from the striated muscle of the arthropod Balanus nubilus Darwin (giant barnacle) have been purified (Potter et al., 1987; Collins et al., 1991). Both isoforms were present in all of the white striated muscle fibres studied but not in the red fibres. The ratio of BTnC2 to BTnC1 in different fibre types varied between 3:1 and 1:1. Both forms of TnC could be readily extracted from myofibrillar bundles of barnacle muscle in low ionic strength EDTA solutions, reducing force activation to < 10%. Both forms either separately or together reassociated with the TnC-depleted fibres in a relaxing (LR) solution (pCa > 8.0, [Mg2+] free = 1 mM, I = 0.15 M), and the reconstituted fibres could be subsequently activated in contraction (LA) solution (pCa = < 3.8, [Mg2+] free = 1 mM, I = 0.15 M,). The dissociation of BTnC 1 + 2 is blocked in low ionic strength solutions containing Mg2+ (greater-than-or-equal-to 10 mM). The two isoforms of crayfish TnC (CrTnC1 and CrTnC2) were also found to be equivalent to the barnacle TnCs in their ability to reactivate TnC-depleted barnacle myofibrillar bundles. Similar experiments using rabbit skeletal muscle TnC (STnC) (I = 0.15 M) in BTnC-depleted myofibrillar bundles of barnacle showed considerable variability. STnC could associate, although weakly, with the depleted bundles in either LR or LA, and force could be partially restored. In neither situation was it as effective as either BTnC or CrTnC. Interestingly, bovine cardiac TnC (CTnC), although it did not associate at pCa > 7.0, did associate and effectively activate force at pCa < 3.8, but dissociated on return to pCa > 7.0 (LR). Neither barnacle TnC isoform associated with TnC-depleted skinned fibres from rabbit skeletal muscle at pCa > 7.0, but did associate and activate these fibres at pCa < 3.8. Once these fibres were returned to LR and then placed in LA at pCa 3.8 all BTnC-restored force was lost, indicating a dissociation of BTnC once the Ca2+ is lowered, as observed with CTnC in barnacle myofibrillar bundles. Finally, the inhibitory effect of BTnI on force and the absence of an effect of calmodulin, trifluoperazine or ATP-gamma-S on force were all taken as evidence for a thin filament regulated Ca2+ control system.