Abstract
It has been shown previously, using G-actin labeled with N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylene-diamine, that Mg2+ induces a conformational change in monomeric G-actin as a consequence of binding to a tight divalent cation binding site (Frieden, C. (1982) J. Biol. Chem. 257, 2882-2886). Using the same fluorescent probe, we show that, subsequent to the Mg2+-induced conformational change, cytochalasin D induces a fluorescence decrease. The data are consistent with a mechanism which proposes that, after Mg2+ binding, cytochalasin D binds and induces a second conformational change which results in overall tight binding of the cytochalasin. The initial binding of cytochalasin D to monomeric actin labeled with the fluorescent probe was found to be 200 microM, and the forward and reverse rate constants for the subsequent conformational change were 350 s-1 and 8 s-1, respectively, with an overall dissociation constant to the Mg2+-induced form of 4.6 microM. The conformational change does not occur in monomeric actin in the presence of Ca2+ rather than Mg2+, but Ca2+ competes with Mg2+ for the tight binding site on the G-actin molecule. Direct binding studies show that actin which has not been labeled with the fluorophore binds cytochalasin D more tightly. The conformational change induced by Mg2+ and cytochalasin D precedes the formation of an actin dimer.
Highlights
It has been shown previously, using G-actin labeled accomplishes this by binding two actin monomers [10] with with N-iodoacetyl-N’-(5-sulfo-l-naphthyl)ethylene-thesubsequent complex serving as anucleusfor further diamine, that Mg2+induces a conformationalchange in elongation [11].Cytochalasin D (CD’) has been shownto bind monomeric G-actin as a consequence of binding to a to monomeric actin and, in the presencoef M e, induces the tight divalent cation binding site (Frieden, C. (1982)
We have shown elsewhere that the CD-induced dimerization of actin requiresMg2+ [12], presumably aas consequence of theconformationalchange induced by Mg2+ binding to G-actin[14, 15].The Mg2+-inducedconformational change is monitored by the increase in the fluorescence of a fluorotochalasin D to monomeric actin labeled with the flu- phore N-iodoacetyl-N’-(5-sulfo-l-naphthyl)ethylene~amine orescent probe was found to be 200 PM, and the for- (AEDANS) attached to Cys-374, while CD causes a timeward and reverse rate constants for the subsequent dependent decrease in the fluorescence of M p - AEDANS
The change does not occur in monomeric actin in the pres- CD-induced conformational change may be a prerequisite of ence of CaZ+rather than Mg2+,but Ca” competes with CD-induced dimerization and subsequent nucleation[16]
Summary
We have shown elsewhere that the CD-induced dimerization of actin requiresMg2+ [12], presumably aas consequence of theconformationalchange induced by Mg2+ binding to G-actin[14, 15].The Mg2+-inducedconformational change is monitored by the increase in the fluorescence of a fluorotochalasin D to monomeric actin labeled with the flu- phore N-iodoacetyl-N’-(5-sulfo-l-naphthyl)ethylene~amine orescent probe was found to be 200 PM, and the for- (AEDANS) attached to Cys-374, while CD causes a timeward and reverse rate constants for the subsequent dependent decrease in the fluorescence of M p - AEDANS - conformational change were 350 s” and 8 s-’, respec- actin. Like some of these regulatory proteins, the cytochalasins bind tightly to one end (the fast growing end) of actin filaments [1,2,3], effectively inhibiting elongation at this end [4,5,6,7]. We assumed the mechanism shown in Scheme I [15]: A
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