Abstract
Chymotrypsin cleaves Dictyostelium myosin in half, splitting the heavy chain (210,000 daltons) into two fragments of 105,000 daltons each. One of the two major fragments is soluble at low ionic strength and has a native molecular weight of 130,000. As judged by SDS polyacrylamide gel electrophoresis, this soluble fragment consists of the two intact myosin light chains of 18,000 and 16,000 daltons and a 105,000-dalton polypeptide derived from the myosin heavy chain. The soluble fragment retains actin-activated ATPase activity and the ability to bind to actin in an ATP-dissociable fashion. The maximal velocity of the actin- activated ATPase activity of the soluble fragment is 80% of that of uncleaved myosin, although its apparent Km for actin is 12-fold greater than that of myosin. In addition to the major soluble 105,000-dalton fragment discussed above, chymotryptic cleavage of the Dictyostelium myosin also generates fragments that are insoluble at low ionic strength. The major insoluble fragment is 105,000 daltons on an SDS polyacrylamide gel and forms thick filaments that are devoid of myosin heads. A less prevalent insoluble fragment has a molecular weight of 83,000 and is probably a subfragment of the insoluble 105,000-dalton fragment. The heavy chain of myosin is phosphorylated in vivo and the phosphorylation site has been localized to the insoluble fragments, which derive from the tail portion of the myosin molecule.
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- Adelstein R. S., Conti M. A. Phosphorylation of platelet myosin increases actin-activated myosin ATPase activity. Nature. 1975 Aug 14;256(5518):597–598. doi: 10.1038/256597a0. [DOI] [PubMed] [Google Scholar]
- Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
- Clarke M., Spudich J. A. Biochemical and structural studies of actomyosin-like proteins from non-muscle cells. Isolation and characterization of myosin from amoebae of Dictyostelium discoideum. J Mol Biol. 1974 Jun 25;86(2):209–222. doi: 10.1016/0022-2836(74)90013-8. [DOI] [PubMed] [Google Scholar]
- Franke J., Kessin R. A defined minimal medium for axenic strains of Dictyostelium discoideum. Proc Natl Acad Sci U S A. 1977 May;74(5):2157–2161. doi: 10.1073/pnas.74.5.2157. [DOI] [PMC free article] [PubMed] [Google Scholar]
- HUXLEY H. E. ELECTRON MICROSCOPE STUDIES ON THE STRUCTURE OF NATURAL AND SYNTHETIC PROTEIN FILAMENTS FROM STRIATED MUSCLE. J Mol Biol. 1963 Sep;7:281–308. doi: 10.1016/s0022-2836(63)80008-x. [DOI] [PubMed] [Google Scholar]
- Kuczmarski E. R., Spudich J. A. Regulation of myosin self-assembly: phosphorylation of Dictyostelium heavy chain inhibits formation of thick filaments. Proc Natl Acad Sci U S A. 1980 Dec;77(12):7292–7296. doi: 10.1073/pnas.77.12.7292. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lowey S., Slayter H. S., Weeds A. G., Baker H. Substructure of the myosin molecule. I. Subfragments of myosin by enzymic degradation. J Mol Biol. 1969 May 28;42(1):1–29. doi: 10.1016/0022-2836(69)90483-5. [DOI] [PubMed] [Google Scholar]
- Maruta H., Korn E. D. Acanthamoeba cofactor protein is a heavy chain kinase required for actin activation of the Mg2+-ATPase activity of Acanthamoeba myosin I. J Biol Chem. 1977 Dec 10;252(23):8329–8332. [PubMed] [Google Scholar]
- Mockrin S. C., Spudich J. A. Calcium control of actin-activated myosin adenosine triphosphatase from Dictyostelium discoideum. Proc Natl Acad Sci U S A. 1976 Jul;73(7):2321–2325. doi: 10.1073/pnas.73.7.2321. [DOI] [PMC free article] [PubMed] [Google Scholar]
- PARRISH R. G., MOMMAERTS W. F. Studies on myosin. II. Some molecular-kinetic data. J Biol Chem. 1954 Aug;209(2):901–913. [PubMed] [Google Scholar]
- Scordilis S. P., Adelstein R. S. A comparative study of the myosin light chain kinases from myoblast and muscle sources. Studies on the kinases from proliferative rat myoblasts in culture, rat thigh muscle, and rabbit skeletal muscle. J Biol Chem. 1978 Dec 25;253(24):9041–9048. [PubMed] [Google Scholar]
- Spudich J. A., Huxley H. E., Finch J. T. Regulation of skeletal muscle contraction. II. Structural studies of the interaction of the tropomyosin-troponin complex with actin. J Mol Biol. 1972 Dec 30;72(3):619–632. doi: 10.1016/0022-2836(72)90180-5. [DOI] [PubMed] [Google Scholar]
- Trotter J. A., Adelstein R. S. Macrophage myosin. Regulation of actin-activated ATPase, activity by phosphorylation of the 20,000-dalton light chain. J Biol Chem. 1979 Sep 25;254(18):8781–8785. [PubMed] [Google Scholar]
- Uyemura D. G., Brown S. S., Spudich J. A. Biochemical and structural characterization of actin from Dictyostelium discoideum. J Biol Chem. 1978 Dec 25;253(24):9088–9096. [PubMed] [Google Scholar]
- Weeds A. G., Pope B. Studies on the chymotryptic digestion of myosin. Effects of divalent cations on proteolytic susceptibility. J Mol Biol. 1977 Apr;111(2):129–157. doi: 10.1016/s0022-2836(77)80119-8. [DOI] [PubMed] [Google Scholar]