Binding of Divalent Cation and Nucleotide to G-actin in the Presence of Profilin (*)
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 Ca
and Mg
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
to 2 s
, the rate of MgADP dissociation is increased from 0.2 s
to 24 s
. The dependences of the exchange rates on profilin concentration are consistent with a high affinity (5 × 106 to 107M
) of profilin for ATP-G-actin, and a 20-fold lower affinity for ADP-Gactin. 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.
Footnotes
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↵* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
-
↵1 The abbreviations used are:
- BAPTA
-
1,2-bis(O-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid
- 8OH-Q
-
8-hydroxyquinoline (8-quinolinol)
ATP-
1-N6-etheno-ATP
- ADF
-
actin depolymerizing factor
- DTT
-
dithiothreitol
- Ap5A
-
P1,P5-di(adenosine 5′)-pentaphosphate
- PA
-
profilin-actin
- MOPS
-
4-morpholineethanesulfonic acid.
-
↵2 KINSIM software versions were kindly supplied to us by C. Frieden and T. Pollard.
-
- Received July 8, 1994.
- Revision received October 10, 1994.
- © 1995 by The American Society for Biochemistry and Molecular Biology, Inc.
