Dictyostelium discoideum myosin II: characterization of functional myosin motor fragments.
The transient kinetic properties of the recombinant myosin head fragments M761 and M781, which both lack the light chain binding domain (LCBD) and correspond to the first 761 and 781 residues of Dictyostelium discoideum myosin II, were compared with those of the subfragment 1-like fragment M864 and a shorter catalytic domain fragment M754. The properties of M761, M781, and M864 are almost identical in regard to nucleotide binding, nucleotide hydrolysis, actin binding, and the interactions between actin and nucleotide binding sites. Only the rate of the hydrolysis step was significantly faster for M761 and the affinity of M781 for actin significantly weaker than for M864. This indicates that the LCBD plays no major role in the biochemical behavior of the myosin head. In contrast, loss of the peptide between 754 and 761 produced several major changes in the property of M754 as documented previously [Woodward, S. K. A., Geeves, M. A., & Manstein, D. J. (1995) Biochemistry 34, 16056-16064]. We further show that C-terminal extension of M761 with one or two alpha-actinin repeats has very little effect on the behavior of the protein. The recombinant nature of M761 and the fact that it can be produced and purified in large amounts make it an ideal construct for systematic studies of the structure, kinetics, and function of the myosin motor.
Kurzawa
S E
SE
Max-Planck-Institut für Molekulare Physiologie, Dortmund, Germany.
Manstein
D J
DJ
Geeves
M A
MA
D000199:Actins / Q000378:metabolism; D000244:Adenosine Diphosphate / Q000378:metabolism / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002645:Chickens; D004023:Dictyostelium / Q000502:physiology*; D007700:Kinetics; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000502:physiology; D015879:Myosin Subfragments / Q000737:chemistry* / Q000378:metabolism*; D009218:Myosins / Q000737:chemistry* / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D000199:Actins; D015879:Myosin Subfragments; D010446:Peptide Fragments; D011994:Recombinant Proteins; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D009218:Myosins