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 (Pi). In the presence of Pi (20 mM), a filament of F-actin had a single high-affinity CB binding site (Kd = 1.4 nM), just like in the case of an ATP-solution [Kd = 5.0 nM: Suzuki, N. & Mihashi, K. (1991) J. Biochem. 109, 19-23]. But in the absence of Pi, there were two low-affinity (Kd = 200 nM) CB binding sites as well as one high-affinity site (Kd = 1.6 nM). We determined the concentration of CB necessary for half-maximal inhibition of growth or shortening of F-actin (Ki) using of pyrene-labeled actin. We obtained Ki = 80 nM for growth and Ki = 800 nM for shortening in the presence of ATP. The addition of Pi to the ATP-solution reduced Ki 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.Pi. When the terminal subunits bind ADP, additional low-affinity CB bindings to the terminal subunits are needed to inhibit the subunit exchange.
Suzuki
N
N
Department of Physics, Faculty of Science, Nagoya University, Aichi.
Mihashi
K
K
D000199:Actins / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D000244:Adenosine Diphosphate / Q000378:metabolism* / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D003571:Cytochalasin B / Q000378:metabolism* / Q000494:pharmacology; D007700:Kinetics; D046911:Macromolecular Substances; D008954:Models, Biological; D009132:Muscles / Q000378:metabolism; D010710:Phosphates / Q000494:pharmacology; D011485:Protein Binding; D011817:Rabbits
D000199:Actins; D046911:Macromolecular Substances; D010710:Phosphates; D003571:Cytochalasin B; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate