Exchange of ADP, ATP and 1:N6‐Ethenoadenosine 5′‐Triphosphate at G‐Actin: Equilibrium and Kinetics

Abstract

Nucleotide exchange was monitored by the strong increase of fluorescence accompanying binding of 1:N6‐ethanoadenosine 5′‐triphosphate (ɛATP) to G‐actin. ɛATP · G‐actin was titrated by ADP and a relative binding constant KADP/ɛATP= 0.03 was obtained. With KɛATP/ATP= 0.19, the physiologically important relative binding constant KADP/ATP= 0.0057 was calculated. From kinetic measurements of the exchange reaction a rate constant of ADP dissociation k−= 8 × 10−4 s−1 was derived which was compared with the corresponding values for ATP (8 × 10−4 S−1) and eATP (4 × 10−3 S−1) (all values in 20 mM Tris · HC1 buffer, pH 8.2 containing 0.8 mM CaCl2 at21°C). The exchange of ADP versus ATP is an important step in the steady‐state equilibrium of actin polymerization. Since polymerization of ATP · G‐actin to ADP · F‐actin is irreversible, depolymerization can take place only by dissociation of ADP · G‐actin followed by nucleotide exchange. It was found from a comparison of k‐ with the rate constants of the polymerization steps that the ratio of ADP · G‐actin to ATP · G‐actin is much higher in the steady state than at equilibrium. Copyright © 1979, Wiley Blackwell. All rights reserved