Nucleotide and actin binding properties of the isolated motor domain from Dictyostelium discoideum myosin.
Nucleotide and actin binding properties of the truncated myosin head (S1dC) from Dictyostelium myosin II were studied in solution using rabbit skeletal myosin subfragment 1 as a reference material. S1dC and subfragment 1 had similar affinities for ADP analogues, epsilon ADP and TNP-ADP. The complexes of epsilon ADP and BeFx or AIF4- were less stable with S1dC than with subfragment 1. Stern-Volmer constants for acrylamide quenching of S1dC complexes with epsilon ADP, epsilon ADP.AIF4- and epsilon ADP.BeFx were 2.6, 2.9 and 2.2 M-1, respectively. The corresponding values for subfragment 1 were 2.6, 1.5 and 1.1 M-1. The environment of the nucleotide binding site was probed by using a hydrophobic fluorescent probe, PPBA. PPBA was a competitive inhibitor of S1dC Ca(2+)-ATPase (Ki = 1.6 microM). The binding of nucleotides to subfragment 1 enhanced PPBA fluorescence and caused blue shifts in the wavelength of its maximum emission in the order: ATP approximately ADP.AIF4- approximately ADP.BeFx > ATP gamma S > ADP > PPi. In the case of S1dC, the effects of different nucleotides were smaller and indistinguishable from each other. S1dC bound actin tighter than S1 (Kd = 7 nM and 60 nM, respectively). The actin activated MgATPase activity of S1dC varied between preparations, and the Vmax and K(m) values ranged between 3 and 7 s-1 and 60 and 190 microM, respectively. S1dC showed lower structural stability than S1 as revealed by their thermal inactivations at 35 degrees C. These results show that the nucleotide and actin binding of S1dC and subfragment 1 are similar but there are some differences in nucleotide and phosphate analogue-induced changes and the communication between the nucleotide and actin binding sites in these proteins.
Bobkov
A A
AA
Department of Chemistry and Biochemistry, University of California, Los Angeles 90095, USA.
Sutoh
K
K
Reisler
E
E
D020106:Acrylamide; D000178:Acrylamides; D000199:Actins / Q000378:metabolism*; D000244:Adenosine Diphosphate / Q000031:analogs & derivatives / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism; D004023:Dictyostelium / Q000502:physiology*; D007700:Kinetics; D015879:Myosin Subfragments / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D009218:Myosins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*
D000178:Acrylamides; D000199:Actins; D015879:Myosin Subfragments; D020106:Acrylamide; D000244:Adenosine Diphosphate; C036745:2',3'-(O-(2,4,6-trinitrocyclohexadienylidine))adenosine 5'-diphosphate; D017301:Ca(2+) Mg(2+)-ATPase; D009218:Myosins