Interaction of myosin subfragment 1 with Cibacron Blue F3GA

Cibacron Blue F3GA and its immobilized derivatives have been shown before to bind and inhibit nucleotide-dependent enzymes and, among them, myosin subfragment 1. Experiments have been carried out to examine the mechanism of the subfragment 1--dye interaction. Binding of subfragment 1 to immobilized dye (Affi-Gel Blue) does not involve the ATP binding site on myosin. Subfragment 1 hydrolyzes MgATP and CaATP while bound to the Affi-Gel Blue column. Inactivated subfragment 1, which contains [3H]ADP noncovalently trapped at the active site, binds and elutes from the Affi-Gel Blue column in the same manner as unmodified, active protein. Free Cibacron Blue inhibits the ATPase activity of subfragment 1. The inhibition is pH, salt, and time dependent. Complete inhibition correlates with the noncovalent binding of four to five dye molecules per mole of subfragment 1. Three to four of these dye molecules can be preferentially removed from subfragment 1 in the presence of 1 M KCl without relieving the inhibition. This inhibition, which can be traced to one dye molecule per subfragment 1, is reversible and is facilitated in the presence of MgADP and MgATP, suggesting that the dye does not bind at the active site of subfragment 1. Our observations are explained in terms of hydrophobic and electrostatic protein--dye interactions.