Active subfragment of the inhibitory component of troponin.

Abstract

A protein factor, which inhibited the Mg2+activated ATPase [EC 3. 6. 1. 3] activity of actomyosin irrespective of the Ca2+ concentration, was isolated from “ room-temperature” actin, which had been polymerized with 0.1 M KC1 and treated with 1/200 (w/w) of trypsin [EC 3. 4. 4. 4] at 25°C for 3–4min. The minimum amount of the most effective preparation of the inhibitory factor required for reduction of the actomyosin ATPase to that of myosin was half the amount of actin present in the reaction mixture. The ATPase activity of myosin was not affected by the factor. Purified actin, tropomyosin, and troponin were treated with trypsin and then their inhibitory effects on the ATPase activity of actomyosin were tested. After trypsin-treatment only troponin was strongly inhibitory and was effective both in the presence and absence of Ca2+. An inhibitory component with a molecular weight of 23,000 was isolated from purified troponin. On treatment with 1/300 (w/w) of trypsin at 25°C for 3–5min, it was degraded to a subfragment with a molecular weight of 11,000 which still had biological activity. In the presence of tropomyosin, Ca2+-sensitivity of the Mg2+activated ATPase of actomyosin was reversed by combination of the two components of troponin with molecular weights of 23,000 and 19,000 at a weight ratio of 1:4. The reversed Ca2+sensitivity of the ATPase of actomyosin could also be conferred occasionally by a combination of the inhibitory factor isolated from “room-temperature” actin after trypsin-treatment and the two components of troponin with molecular weights of 37,000 and 19,000 at an equal weight ratio.