Binding mode of cytochalasin B to F-actin is altered by lateral binding of regulatory proteins.
The binding of cytochalasin B (CB) to F-actin was studied using a trace amount of [3H]-cytochalasin B. F-Actin-bound CB was separated from free CB by ultracentrifugation and the amount of F-actin-bound CB was determined by comparing the radioactivity both in the supernatant and in the precipitate. A filament of pure F-actin possessed one high-affinity binding site for CB (Kd = 5.0 nM) at the B-end. When the filament was bound to native tropomyosin (complex of tropomyosin and troponin), two low-affinity binding sites for CB (Kd = 230 nM) were created, while the high-affinity binding site was reserved (Kd = 3.4 nM). It was concluded that the creation of low-affinity binding sites was primarily due to binding of tropomyosin to F-actin, as judged from the following two observations: (1) a filament of F-actin/tropomyosin complex possessed one high-affinity binding site (Kd = 3.9 nM) plus two low-affinity binding sites (Kd = 550 nM); (2) the Ca2(+)-receptive state of troponin C in F-actin/native tropomyosin complex did not affect CB binding.
Suzuki
N
N
Department of Physics, Faculty of Science, Nagoya University, Aichi.
Mihashi
K
K
D000199:Actins / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D003571:Cytochalasin B / Q000378:metabolism*; D066298:In Vitro Techniques; D007700:Kinetics; D009132:Muscles / Q000378:metabolism; D011817:Rabbits; D014335:Tropomyosin / Q000378:metabolism; D014336:Troponin / Q000378:metabolism
D000199:Actins; D014335:Tropomyosin; D014336:Troponin; D003571:Cytochalasin B