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J. Biochem, 1976, Vol. 80, No. 1 111-120
© 1976 Japanese Biochemical Society

research-article

Ca2+-induced Conformational Changes of Spin-labeled g2 Chain Bound to Myosin and the Effect of Phosphorylation1

Yoh OKAMOTO and Koichi YAGI

Department of Chemistry, Faculty of Science, Hokkaido University Kita-ku, Sapporo, Hokkaido 060

One of the low molecular weight components of myosin, g2, was isolated by alkali treatment of myosin and was chemically modified with a spin label reagent, 4-maleimido-2, 2, 6, 6-tetramethylpiperidinooxyl. The label on g2 showed a rather weakly immobilized ESR spectrum and it was clearly affected by Ca2+; the half-maximal change was at around pCa 4. The spin-labeled g2 was incorporated into myosin by exchange with the intrinsic g2 of myosin in 0.6 M KSCN or 4 M LiCl. The label on g2 became strongly immobilized on association with myosin. Under the conditions used, ESR spectral change due to Ca2+ occurred at two different concentration ranges, which were as low as pCa 8 and at around pCa 4.

Phosphorylated g2 was isolated from myosin after the protein kinase (EC 2.7.1.37 [EC] )-catalyzed phosphorylation of myosin and it was also modified with the maleimide label. Dephosphorylation of the phosphorylated g2 was performed using E. coli alkaline phosphatase (EC 3.1.3.1 [EC] ). The effects of Ca2+ on the ESR spectra of phosphorylated and dephosphorylated g2 were investigated in the state associated with myosin. A change in the ESR spectrum from strongly immobilized to weakly immobilized states was observed with both g2 chains on the addition of Ca2+. However, the effective concentration ranges of Ca2+ were quite different; around pCa 4 for the phosphorylated g2 and around pCa 8 for the dephosphorylated g2. The results indicate that g2 undergoes a conformational change at physiological levels of Ca2+ sufficient to saturate troponin, but it does not do so after phosphorylation.

1This study was supported by grants from the Muscular Dystrophy Associations of America, Inc. and from the Ministry of Education, Science and Culture, of Japan.


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