HIGH-AFFINITY BINDING OF CYTOCHALASIN-B TO THE B-END OF F-ACTIN LOSES ITS INHIBITORY EFFECT ON SUBUNIT EXCHANGE WHEN THE BOUND NUCLEOTIDE IS ADP

We investigated the mode of binding of cytochalasin B (CB) to F-actin in an ADP-solution with and without inorganic phosphate (P(i)). In the presence of P(i) (20 mM), a filament of F-actin had a single high-affinity CB binding site (K(d) = 1.4 nM), just like in the case of an ATP-solution [K(d) = 5.0 nM: Suzuki, N. & Mihashi, K. (1991) J. Biochem. 109, 19-23]. But in the absence of P(i), there were two low-affinity (K(d) = 200 nM) CB binding sites as well as one high-affinity site (K(d) = 1.6 nM). We determined the concentration of CB necessary for half-maximal inhibition of growth or shortening of F-actin (K(i)) using of pyrene-labeled actin. We obtained K(i) = 80 nM for growth and K(i) = 800 nM for shortening in the presence of ATP. The addition of P(i) to the ATP-solution reduced K(i) for growth to 9 nM. We propose a model explaining these results. In the model, high-affinity CB binding to the terminal subunit dimer can inhibit subunit exchange at the B-end only when the terminal subunits bind ATP or ADP.P(i). When the terminal subunits bind ADP, additional low-affinity CB bindings to the terminal subunits are needed to inhibit the subunit exchange.