Effect of removal and reconstitution of troponins C and I on the Ca2+‐activated tension development of single glycerinated rabbit skeletal muscle fibers

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Abstract
The effect of troponin T treatment on the Ca2+‐activated tension of single glycerinated rabbit skeletal muscle fibers was examined. The tension of the fiber was completely desensitized to Ca2+ by incubation in a solution containing an excessive amount of troponin T and reached a level of about 70% of the maximum tension of the control fiber. SDS/PAGE showed that most of troponins C and I was removed from the fiber by troponin T treatment. During the course of troponin T treatment, the cooperativity of Ca2+ activation (Hill coefficient) was decreased while pCa at half‐maximal Ca2+‐sensitive tension @K) increased. Using the 26‐K fragment of troponin T, the study indicated that the removal of troponins C and I was due to the replacement of the troponin C · I · T complex in the myofibrils of the fiber with the added troponin T. The troponin‐T‐treated fiber was again sensitized to Ca2+ by the addition of troponin C · I. The removal of troponin C by treatment with trans‐1,2‐cyclohexanediamine‐N,N,N′,N′‐tetraacetic acid did not change the minimum tension of the fiber, from which troponin C · I was partially removed by troponin T treatment, but it decreased the height of maximum tension with a concomitant decrease in the Hill coefficient as well as a decrease in pK. The above findings suggested that pK is determined by the balance between two opposite actions through troponins C and I, while the extent of cooperativity of Ca2+ activation seemed to be related mainly to the content of troponin C.