Binding of Divalent Cation and Nucleotide to G-actin in the Presence of Profilin

Irina Perelroizen,Marie-France Carlier,Dominique Pantaloni

doi:10.1074/jbc.270.4.1501

Abstract

The effect of profilin, a G-actin binding protein, on the mechanism of exchange of the tightly bound metal ion and nucleotide on G-actin, has been investigated. 1) In low ionic strength buffer, profilin increases the rates of Ca2+ and Mg2+ dissociation from G-actin 250- and 50-fold, respectively. On the profilin-actin complex as well as on G-actin alone, nucleotide exchange is dependent on the concentration of divalent metal ion and is kinetically limited, at low concentration of metal ion, by the dissociation of the metal ion. 2) Under physiological ionic conditions, nucleotide exchange on G-actin is 1 order of magnitude faster than at low ionic strength. The rate of MgATP dissociation is increased by profilin from 0.05 s-1 to 2 s-1, the rate of MgADP dissociation is increased from 0.2 s-1 to 24 s-1. The dependences of the exchange rates on profilin concentration are consistent with a high affinity (5 x 10(6) to 10(7) M-1) of profilin for ATP-G-actin, and a 20-fold lower affinity for ADP-G-actin. Profilin binding to actin lowers the affinity of metal-nucleotide by about 1 order of magnitude. These results restrain the possible roles of profilin in actin assembly in vivo.

Highlights

The effect ofprofilin, a G-actin binding protein, on the mechanism of exchange of the tightly bound metal ion and nucleotide on G-actin, has been investigated. 1) In low ionic strength buffer, profilin increases the rates of Ca2+ and Mg2+ dissociation from G-actin 250- and 50fold, respectively
Analysis of the Profilin Concentration Dependence of the Kinetics of Ca2 + Dissociation from G-actin-The time course of Ca 2+ dissociation from G-actin was monitored by the change in BAPTA absorbance using the stopped-flow
The results described above, showing that profilin binding weakens the binding of the metal ion, let us anticipate that profilin causes a decrease in the overall affinity of ATP or ADP for G-actin, especially at low concentrations of divalent metal ion

Summary

Introduction

The effect ofprofilin, a G-actin binding protein, on the mechanism of exchange of the tightly bound metal ion and nucleotide on G-actin, has been investigated. 1) In low ionic strength buffer, profilin increases the rates of Ca2+ and Mg2+ dissociation from G-actin 250- and 50fold, respectively. The dependences of the exchange rates on profilin concentration are consistent with a high affinity (5 x 106 to 107 M-1) of profilin for ATP-G-actin, and a 20-fold lower affinity for ADP-Gactin. Kinetic data of metal ion and nucleotide exchange on G-actin are consistent with Scheme I, according to which dissociation of the tightly bound divalent cation (M) is kinetically limiting for nucleotide (N) dissociation and binding of the divalent metal ion to actin (A) increases the affinity of nucleotide by 4-6 orders of magnitude. Recent extensive kinetic analysis of Ca 2+ or Mg2+ dependences of the rates of nucleotide exchange in low ionic strength buffer (6) have brought full confirmation for the validity of this scheme with either ATP or ADP as bound nucleotide. Profilin might bind less well to an F-ADP than to an F-ADP-Pi end, or profilin could accelerate Pi release from an F-ADP-Pi end

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Results

Conclusion