Kinetic evidence for a readily exchangeable nucleotide at the terminal subunit of the barbed ends of actin filaments.
The time course of actin depolymerization was quantitatively analyzed to obtain insight into the reactions occurring during actin disassembly. Polymeric actin was diluted, and subsequently the time course of depolymerization was measured. In the presence of 0.5 mM ATP, 100 mM KCl, and 1 mM MgCl2, continuous depolymerization was observed both when the filaments were carefully diluted and when the filaments were fragmented to produce short filaments. The rates of the reactions that are known to occur during depolymerization, such as dissociation and association of ADP- and ATP-actin molecules and exchange of nucleotides bound to monomeric actin, were determined by independent experiments. When the determined rate parameters were used to calculate the time course of depolymerization, consistently in the simulations fast depolymerization of ADP-actin was followed by slower polymerization of ATP-actin that was formed from ADP-actin by nucleotide exchange. The lack of fast depolymerization and subsequent slower polymerization in the experiments suggests that our present conception about actin disassembly requires modification. Good agreement of calculated time courses with the experimentally determined continuous depolymerization was achieved if ADP bound to the terminal subunit of barbed filament ends was assumed to be readily exchangeable for ATP. Fast nucleotide exchange at terminal subunits may contribute to the stability of barbed filament ends and to their role as polymerizing ends in living cells.
Teubner
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Institute of Physiological Chemistry, Ruhr-University Bochum, Germany.
Wegner
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D051339:Actin Depolymerizing Factors; D000199:Actins / Q000378:metabolism*; D000244:Adenosine Diphosphate / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D051345:Destrin; D006868:Hydrolysis; D007700:Kinetics; D008840:Microfilament Proteins / Q000378:metabolism; D011108:Polymers / Q000378:metabolism; D011817:Rabbits; D013997:Time Factors
D051339:Actin Depolymerizing Factors; D000199:Actins; D051345:Destrin; D008840:Microfilament Proteins; D011108:Polymers; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate