J. Biochem, 1982, Vol. 91, No. 6 1907-1915
© 1982 Japanese Biochemical Society
research-article |
Filament Formation from Gizzard Myosin for Calcium Sensitizing Effect of Skeletal Troponin on the Contractile Activities of Gizzard Myosin1
Department of Chemistry, Faculty of Science, Tokyo Institute of Technology Meguro-ku, Tokyo 152
In our previous report (J. Biochem. 89, 87–101, 1981), it was shown that the contractile (ATPase and superprecipitation) activities of chicken-gizzard acto-phospho-rylated myosin (acto-PM) both with and without tropomyosin (TM) were insensitive to Ca2+ but were made sensitive to Ca2+ by addition of rabbit skeletal troponin (and TM). In the present study, we were able to observe that the other three cases of Ca2+-insensitive activities of acto-unphosphorylated myosin with TM (acto-UM-TM) were also made sensitive to Ca2+ by addition of troponin: (a) the activities in ATP concentrations as low as 1 µM, (b) those in MgCl2 concentrations as high as 30 mM and (c) those in ITP media.
We also studied the turbidity of gizzard myosin suspensions as a function of the ITP concentration in the presence of 10 mM MgCl2. We thus observed that the turbidity of UM suspensions and that of PM suspensions decreased as the ITP concentration increased to higher than approximately 0.3 mM and to higher than approximately 1 mM, respectively. Moreover, we observed that the ITPase activity of UM, that of acto-UM and its superprecipitation activity decreased when the ITP concentration increased to higher than approximately 0.3 mM, and that the ITPase activity of PM, that of acto-PM and its superprecipitation activity decreased when the ITP concentration increased to higher than approximately 1 mM. It appeared, therefore, that the decreases in the activities all resulted from dissociation of myosin filaments, which was indicated by the decrease in the turbidity of myosin suspensions.
On the other hand, as the MgCl2 concentration increased, the ITP concentration required to decrease the turbidity of myosin suspensions increased, but the ITP concentration required for causing a decrease in the superprecipitation activity remained practically unaltered. It appeared therefore that dissociation of actomyo-sin into actin and myosin was responsible for the decrease in the contractile activities that was observed when the ITP concentration was higher than approximately 0.3 mM or 1 mM.
All the results described above were interpreted as indicating that actin-linked regulation of muscle contraction works only when myosin is in the form of filaments.
1Supported by Research Grants from the Ministry of Education, Science and Culture of Japan.
2Present address: Department of Chemistry, Gunma University, Faculty of Technology, Kiryu, Gunma 376.