Vanadate-induced changes in myosin subfragment-1 from cardiac muscle.
The interaction between myosin subfragment-1 from bovine cardiac muscle (CS1) and 1,N6-ethenoadenosine diphosphate (epsilon ADP) was studied using steady-state and time-resolved fluorescence methods. The binding constant was found to be 1.52 x 10(6) M-1 at pH 7.5 and 5 degrees C. The intensity decay of epsilon ADP bound to CS1 was resolved into two components over a narrow range of temperatures. The long component was about 22 ns and the short component was between 5 and 7 ns, with fractional amplitudes of about 0.6-0.7 for the long component and 0.3-0.4 for the short component. These data suggest a two-state temperature-sensitive transition of the CS1.epsilon ADP complex. In the presence of orthovanadate (Vi) at 5 degrees C, the decay time of the long component was little affected, whereas the short decay time increased by over 3 ns and the fractional amplitude of the long component decreasing by a factor of 2 to about 0.3 and that of the short component increasing to 0.7. The anisotropy decay of bound epsilon ADP was monoexponential regardless of whether vanadate was present. The recovered single rotational correlation time was 110 ns in the absence of vanadate and 79 ns in the presence of vanadate. The decrease in correlation time suggests an increase in molecular symmetry of the CS1.epsilon ADP.Vi complex. The results are in agreement with previous results obtained from skeletal S1 and indicate that S1 from both isoforms of myosin experiences similar vanadate-induced changes in its hydrodynamic shape. Since the ternary vanadate complex is a stable analogue of the S1.ADP.Pi state, the ligand-induced change in hydrodynamic shape of S1 may be related to the conformational change which myosin head experiences during the ATPase cycle and this change in myosin could be a structural basis for force generation in striated muscle.
Xing
J
J
Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham 35294-2041.
Cheung
H C
HC
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives; D000818:Animals; D002417:Cattle; D005454:Fluorescence Polarization / Q000379:methods; D005456:Fluorescent Dyes; D007700:Kinetics; D009206:Myocardium / Q000378:metabolism*; D015879:Myosin Subfragments / Q000737:chemistry / Q000187:drug effects*; D011487:Protein Conformation / Q000187:drug effects*; D013050:Spectrometry, Fluorescence / Q000379:methods; D014638:Vanadates / Q000494:pharmacology*
D005456:Fluorescent Dyes; D015879:Myosin Subfragments; C015195:1,N(6)-ethenoadenosine diphosphate; D014638:Vanadates; D000244:Adenosine Diphosphate