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Formation of the stable myosin-ADP-aluminum fluoride and myosin-ADP-beryllium fluoride complexes and their analysis using 19F NMR.

1993; Elsevier BV; Volume: 268; Issue: 10 Linguagem: Inglês

10.1016/s0021-9258(18)53150-1

1083-351X

Shinsaku Maruta, Gillian D. Henry, Brian D. Sykes, Mitsuo Ikebe,

Peptidase Inhibition and Analysis

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.