The effects of cytochalasins on actin polymerization and actin ATPase provide insights into the mechanism of polymerization.

S.L Brenner,E.D Korn

doi:10.1016/s0021-9258(19)86107-0

Abstract

Substoichiometric concentrations of cytochalasin D inhibited the rate of polymerization of actin in 0.5 mM MgCl2, increased its critical concentration and lowered its steady state viscosity. Stoichiometric concentrations of cytochalasin D in 0.5 mM MgCl2 and even substoichiometric concentrations of cytochalasin D in 30 mM KCl, however, accelerated the rate of actin polymerization, although still lowering the final steady state viscosity. Cytochalasin B, at all concentrations in 0.5 mM MgCl2 or in 30 mM KCl, accelerated the rate of polymerization and lowered the final steady state viscosity. In 0.5 mM MgCl2, cytochalasin D uncoupled the actin ATPase activity from actin polymerization, increasing the ATPase rate by at least 20 times while inhibiting polymerization. Cytochalasin B had a very much lower stimulating effect. Neither cytochalasin D nor B affected the actin ATPase activity in 30 mM KCl. The properties of cytochalasin E were intermediate between those of cytochalasin D and B. Cytochalasin D also stimulated the ATPase activity of monomeric actin in the absence of MgCl2 and KCl and, to a much greater extent, stimulated the ATPase activity of monomeric actin below its critical concentration in 0.5 mM MgCl2. Both above and below its critical concentration and in the presence and absence of cytochalasin D, the initial rate of actin ATPase activity, when little or no polymerization had occurred, was directly proportional to the actin concentration and, therefore, apparently was independent of actin-actin interactions. To rationalize all these data, a working model has been proposed in which the first step of actin polymerization is the conversion of monomeric actin-bound ATP, A . ATP, to monomeric actin-bound ADP and Pi, A* . ADP . Pi, which, like the preferred growing end of an actin filament, can bind cytochalasins.

Highlights

MM KCl, accelerated the rateof actin polym- at steady state andin the presence of excess ATP, there will erization, still lowering the final steady state be a net polymerizing filament end anda net depolymerizing viscosity
We have recentlyshown that substoichiometric concentrations of cytochalasin D decrease the rate of actin polymerization, reduce itsfinal viscosity, and increase the G-actinpool at steady state ( 3 ) .In the context of the treadmilling model, we proposed that these are tehxepected results if cytochalasin
In 0.5 ~ lMglClz~(Fig. lA), substoichiometric concentrations of cytochalasin D (0.02 to 2 p~ uersus 25 p~ actin) inhibited the initial rate and lowered the final viscosity of actin polymerization, as previously reported [3].On the other hand, an approximately stoichiometric concentratoiof ncytochalasin D (20 p ~ac)celerated the initial rateof polymerization but still lowered the steady state viscosity. Under these conditions of polymerization, actin ATPase activity was totally uncoupled from actin polymerization by all concentrations of cytochalasin D that were tested (Fig. 1B)

Summary

Introduction

MM KCl, accelerated the rateof actin polym- at steady state andin the presence of excess ATP, there will erization, still lowering the final steady state be a net polymerizing filament end anda net depolymerizing viscosity. The rate of actin ATPase activity, when little or no polym- increase in steady state ATPase activity caused by stoichioerization had occurred, was directly proportional to metricconcentrations of cytochalasin B [9] and chemical the actin concentration and, apparently was independent of actin-actin interactions. To rationalize all these data, a working model has been proposed in which the first stepof actin polymerization is theconversion of monomeric actin-bound ATP, A-ATP, to monomeric actin-bound ADP and Pi, A* ADP-Pi, which, like the preferred growing end of an actin filament, can bind cytochalasins. Recenbtrief reports [11, 12] of approximately one high affinity binding site for cytochalasins per actin filament are compatiwbliteh this interpretation of cytochalasin action

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Conclusion