Troponin-tropomyosin complex. Column chromatographic separation and activity of the three, active troponin components with and without tropomyosin present.

By means of new or modified procedures troponin-tropomyosin complex was fractionated into three components plus tropomyosin, and then reconstituted in the absence of urea. Tropomyosin was separated from troponin by hydroxyapatite column chromatography, a method giving sharper separation than the previously used technique of isoelectric precipitation. The chromatography also separated the tropomyosin into two fractions. One gave a single band on sodium dodecyl sulfate gel electrophoresis, and the other a double band; and the complete absence of proline suggested that each was a different form of pure tropomyosin. Troponins I, T, and C were separated by DEAE-Sephadex chromatography in 6 M urea as previously, but the separation was found to be much improved if the troponin was treated with a CaZ+ chelator before being applied to the column. The activity of the troponin fractions was assayed at varying ratios to actin both with and without tropomyosin present. It was found that near physiologic ratios to actin, either the combination of troponin I plus tropomyosin or troponin I plus troponin T inhibited the acto-heavy meromyosin ATPase. However, in both cases when troponin C, which is necessary for Ca2+ sensitivity, was added, the inhibition was reversed not only in the presence but also in the absence of Ca2+. Only when both troponin T and tropomyosin were present in addition to troponin I and C did inhibition occur in the absence but not in the presence of Ca2+. We therefore conclude first, that if troponin T is present tropomyosin may not be required for inhibition by troponin I at physiologic ratios to actin, second, that all three components plus tropomyosin are necessary to restore full Ca*+ sensitivity, and third, that this reconstitution can be accomplished by combining the individual components in the absence of urea.