Formation of the stable myosin-ADP-aluminum fluoride and myosin-ADP-beryllium fluoride complexes and their analysis using 19F NMR.
The effects of aluminum fluoride and beryllium fluoride on smooth muscle myosin and its subfragments were studied. Mg(2+)-ATPase activity was inhibited in the presence of aluminum fluoride (beryllium fluoride). [3H]ADP bound to heavy meromyosin (HMM) in the presence of aluminum fluoride (beryllium fluoride) and was not dissociated after 3 days of dialysis demonstrating that [3H]ADP was trapped in HMM. These results suggest the formation of a stable HMM-ADP-fluoroaluminate (fluoroberyllate) complex. The intrinsic tryptophane fluorescence intensity was increased in the presence of ADP and aluminum fluoride (beryllium fluoride). Acto-S1 was dissociated upon the formation of S1-ADP-fluoroberyllate and actin destabilized S1-ADP-fluoroberyllate complex, while S1-ADP-fluoroaluminate failed to bind to actin. Furthermore, when S1 formed the complex with actin, nucleotide trapping did not occur in the presence of fluoraluminate. These results indicated that the myosin-ADP-fluoroberyllate complex resembles a weak binding state while myosin-ADP-fluoroaluminate complex is a distinct conformation although the binding to actin was also weak. The structure of the ternary complex was investigated using 19F NMR. The 19F NMR spectrum of the S1-ADP-fluoroaluminate complex showed a peak at -66.7 ppm which is due to the binding of fluoraluminate to S1. The peak was not observed when 5'-adenylylimidodiphosphate was substituted for ADP suggesting that aluminum fluoride plays a role as a phosphate analogue. The stoichiometry of the bound fluoride was determined to be 3.8 mol/mol S1 suggesting that the bound species is AlF-4.
Maruta
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S
Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio 44106.
Henry
G D
GD
Sykes
B D
BD
Ikebe
M
M
D000244:Adenosine Diphosphate / Q000737:chemistry*; D000535:Aluminum / Q000737:chemistry* / Q000494:pharmacology; D017607:Aluminum Compounds; D000818:Animals; D001608:Beryllium / Q000737:chemistry* / Q000494:pharmacology; D005459:Fluorides / Q000737:chemistry* / Q000494:pharmacology; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D009130:Muscle, Smooth / Q000187:drug effects; D009132:Muscles / Q000187:drug effects; D015879:Myosin Subfragments / Q000737:chemistry* / Q000187:drug effects; D011817:Rabbits; D014422:Turkeys
D017607:Aluminum Compounds; D015879:Myosin Subfragments; C035982:beryllium fluoride; D000244:Adenosine Diphosphate; D000535:Aluminum; D001608:Beryllium; D005459:Fluorides; C032311:aluminum fluoride