Considerably more information has been acquired about the three myosin isoenzymes of Acanthamoeba. A 16-amino acid sequence has been identified which contains the ATP-binding site of myosin II: Glu-Ser-Gly-Ala-Gly-Lys-Thr-Gln-Asn-Thr-Me2Lys-Lys-Val-Ile-Gln-Tyr. Radioactive nucleotide is photoaffinity crosslinked to the Gln at position 8 in this sequence. The sequence is highly homologous to a sequence in nematode and rabbit striated myosins that lies in the previously identified """"""""ATP-binding"""""""" region of those myosins but differs from the two sequences that are photoaffinity labeled in rabbit myosin by two different analogues of ATP. A 68-kDa head fragment has been purified from tryptic digests of myosin II and shown to have complete Ca-ATPase activity but no actin-activated MgATPase activity. It did not bind detectably to F-actin. When covalently crosslinked to F-actin by EDC, the 68-kDa showed very high actin-activated ATPase activity; suggesting that the low activity of the non-crosslinked head was due to its poor binding. It is likely that phosphorylation regulates the activity of intact, native myosin II in the same way, i.e. by affecting its binding to F-actin. Kinetic studies of the ATPase activity of myosin IA and IB as a function of myosin concentration at fixed actin concentration have led to the hypothesis that myosin I heavy chain contains two actin-binding sites. This hypothesis was supported by evidence that single molecules of myosin I crosslink F-actin (as measured by the increase in low-shear viscosity) and, in the presence of ATP and when the myosin I is phosphorylated, superprecipitate F-actin. Thus, monomolecular, non-filamentous myosin I can support contractile activity which means that the heavy chain of 125-130 kDa contains all that is required for contractile activity and that myosin filaments are not needed.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Intramural Research (Z01)
Project #
1Z01HL000506-10
Application #
4694483
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
10
Fiscal Year
1985
Total Cost
Indirect Cost
Name
U.S. National Heart Lung and Blood Inst
Department
Type
DUNS #
City
State
Country
United States
Zip Code
Baek, Kyuwon; Liu, Xiong; Ferron, Francois et al. (2008) Modulation of actin structure and function by phosphorylation of Tyr-53 and profilin binding. Proc Natl Acad Sci U S A 105:11748-53
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Liu, Xiong; Shu, Shi; Kovacs, Mihaly et al. (2005) Biological, biochemical, and kinetic effects of mutations of the cardiomyopathy loop of Dictyostelium myosin II: importance of ALA400. J Biol Chem 280:26974-83
Ishikawa, Takashi; Cheng, Naiqian; Liu, Xiong et al. (2004) Subdomain organization of the Acanthamoeba myosin IC tail from cryo-electron microscopy. Proc Natl Acad Sci U S A 101:12189-94
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Brzeska, Hanna; Szczepanowska, Joanna; Matsumura, Fumio et al. (2004) Rac-induced increase of phosphorylation of myosin regulatory light chain in HeLa cells. Cell Motil Cytoskeleton 58:186-99

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