Myosin and actin were isolated from bovine thyroid and characterized. New methodology was used to purify myosin since classical methods did not remove thyroglobulin contamination. Thyroid extracts were prepared using 0.3 M ammonium acetate with 2 mM CaCl2. After blue dextran/Sepharose affinity chromatography, the myosin was purified using gel filtration with a discontinuous buffer system as described by Pollard et al. (Pollard, T.D., Thomas, S. M., and Niederman, R. (1974) Anal. Biochem. 60, 258-266). The final product was more than 95% pure as indicated by electron microscopy and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Thyroid myosin resembles myosins from other tissues in regard to subunit size, morphology, ATPase activity, and the activation of Mg2+ATPase by actin. It contains a heavy chain of 200,000 daltons, two light chains of 20,500 and 16,500 daltons, and forms bipolar thick filaments in 0.1 M KCl and Mg2+. Skeletal muscle and thyroid F-actin stimulate thyroid myosin ATPase 2- to 3-fold in the presence of KCl and Mg2+. Ouabain and sodium azide have little or no effect on the ATPase activity indicating the absence of plasma membrane (Na+ + K+) ATPase and minimal contamination by mitochondrial ATPase. Thyroid myosin ATPase activity differed qualitatively from myosins obtained from other tissues. In contrast to the Ca2+ and EDTA activation and Mg2+ inhibition of other vertebrate myosins, the ATPase activity of thyroid myosin is not affected by EDTA and inhibited by Ca2+ and Mg2+. The EDTA/K+ATPase pH curves of thyroid myosin are very different from skeletal muscle myosin. The optimal salt concentration is somewhat lower than that of other myosins. Thyroid actin was purified by repolymerization after extraction from an acetone powder of actomyosin. The electron microscopic appearance of the actin is similar to that of skeletal F-actin.
|Original language||English (US)|
|Number of pages||7|
|Journal||Journal of Biological Chemistry|
|State||Published - 1977|
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology