THE FUNCTIONAL-ROLE OF THE DOMAINS OF TROPONIN-C INVESTIGATED WITH THROMBIN FRAGMENTS OF TROPONIN-C RECONSTITUTED INTO SKINNED MUSCLE-FIBERS

Proteolysis of rabbit fast skeletal troponin-C (RSTnC) with thrombin p roduces four separate fragments containing the following Ca2+-binding site(s): TH1 (residues 1-120) sites I-III; TH2 (121-159) site IV; TH3 (1-100) sites I and II; and TH4 (101-120) site III. We studied the abi lity of these fragments 60 restore the steady state isometric force in TnC-depleted skinned skeletal muscle fibers, Interestingly, we found that all investigated fragments of RSTnC possessed some of the propert ies of native RSTnC, but none of them could fully regulate contraction in the fibers like intact RSTnC, TH1 was the most effective in the fo rce restoration (65%) whereas the smaller fragments developed about 50 % (TH3 and TH4) or 20% (TH2) of the initial force of unextracted fiber s. Additionally, much higher concentrations of TH2, TH3, and TH4 compa red to RSTnC or TH1 were necessary for force development suggesting a decreased affinity of these fragments to their binding site(s) in the fibers, Like intact RSTnC, TH1 was able to interact with the fibers in a Ca2+-independent (Mg2+-dependent) manner, indicating that at a mini mum, Ca2+-binding site III is required for this type of binding, The i nitial binding of the other fragments to the TnC-depleted fibers occur red only in the presence of Ca2+, TH2 and TH4 appeared to bind to two different binding sites in the fibers, The binding to one of the sites caused partial force restoration, This binding of TH2 and TH4 was abo lished when Ca2+ was removed, TH2 and TH4 binding to the second site r equired Ca2+ initially but was maintained in the presence of Mg2+. Thi s interaction of TH2 and TH4 partially blocked the rebinding of RSTnC to the fibers, The latter results suggest that site III or IV in these small fragments, when removed from the constraints of the parent prot ein, may assume conformations that allow them to function, to a certai n extent, like both the regulatory sites (I and II) and the Ca2+-Mg2sites (III and IV) of TnC.