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Biochemistry. 1993 Sep 28;32(38):9994-10004.

Analysis of the interactions of actin depolymerizing factor with G- and F-actin.

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  • 1Department of Biochemistry, Colorado State University, Fort Collins 80523.

Abstract

Chick actin depolymerizing factor (ADF) is an actin binding protein previously shown to rapidly depolymerize actin filaments in vitro, yielding a 1:1 complex of ADF and actin monomer. Here we show that ADF protects actin monomer from denaturation by EDTA by inhibiting the exchange of actin-bound nucleotide. Under low ionic strength conditions, the approximate dissociation constant (KD) for the ADF-actin complex determined from exchange of nucleotide (1,N6-etheno-ATP) is about 150 and is calcium-independent. Addition of ADF to monomeric actin inhibits actin assembly as well as the ATP hydrolysis that normally accompanies assembly. Complex formation is demonstrated between ADF and actin containing either ATP, ADP, or AMPPNP as the bound nucleotide. A KD of 0.1-0.2 microM was calculated for both the ADF-ATP-actin and ADF-AMPPNP-actin complexes, whereas the KD for the ADF-ADP-actin complex is about 1.3 microM. ADF can either depolymerize or cosediment with F-actin in a stoichiometric fashion, but these reciprocal activities are pH-dependent. At pHs between 6.5 and 7.1, ADF cosediments with F-actin and demonstrates only weak depolymerizing activity. ADF binding is cooperative and saturates at a 1:1 ADF:actin molar ratio. At pHs between 7.1 and 7.7, ADF shows increasing depolymerizing activity and less F-actin binding. At pH 8.0, ADF depolymerizes F-actin in a stoichiometric manner. Both the F-actin binding and the depolymerizing activities of ADF are inhibited by phalloidin.(ABSTRACT TRUNCATED AT 250 WORDS)

PMID:
8399168
[PubMed - indexed for MEDLINE]
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