Abstract
In buffer containing 50 mM KCl, 1 mM MgCl2, 1 mM EGTA, 5 mM imidazole, pH 7.5, 0.1 mM CaCl2, 0.2 mM dithiothreitol, 0.01% NaN3, and 0.2 mM ATP, the KD for the formation of the 1:1 complex between Acanthamoeba actin and Acanthamoeba profilin was about 5 microM. When the actin was modified by addition of a pyrenyl group to cysteine 374, the KD increased to about 40 microM but the critical concentration (0.16 microM) was unchanged. The very much lower affinity of profilin for modified actin explains the anomalous critical concentrations curves obtained for 5-10% pyrenyl-labeled actin in the presence of profilin and the apparently weak inhibition by profilin of the rate of filament elongation when polymerization is quantified by the increase in fluorescence of pyrenyl-labeled actin. Light-scattering assays of the polymerization of unmodified actin in the absence and presence of profilin gave a similar value for the KD (about 5-10 microM) when determined by the increase in the apparent critical concentration of F-actin at steady state at all concentrations of actin up to 20 microM and by the inhibition of the initial rates of polymerization of actin nucleated by either F-actin or covalently cross-linked actin dimer. In the same buffer, but with ADP instead of ATP, the critical concentration of actin was higher (4.9 microM) and the KD of the profilin-actin complex was lower for both unmodified (1-2 microM) and 100% pyrenyl-labeled actin (4.9 microM).
Highlights
In buffer containing 5 0 mM KCl, 1 mM MgC12, 1 mM EGTA, 5 mM imidazole, pH 7.5,O.l mM CaC12,0.2 mM dithiothreitol, 0.01%NaNS, and 0.2 mM ATP, the K D for the formation of the 1:l complex between Acanthamoeba actin and Acanthamoeba profilin was about 5 p ~ W. hen the actin was modified by addition of a pyrenyl group to cysteine 374, the KD increased to about 40 p~ but the critical concentration
We report in this paper that the apparent actin dependence of the KDfor the profilin-actin complex determined by measurements at steady state and the high calculated from the containing both and MgC12*
Polymerization was quantified by the increase in fluorescence intensity of pyrenyl-labeled actin with excitation a t 368 nm and emission a t 388 nm [4] or by right-angle light scattering with both wavelengths at 400 nm [3].For steady-state measurements, generally actin was first polymerized to steady state in the absence of profilin (25 "C, 3 h) and thendiluted to various concentrations inthe absence or presence of profilin
Summary
The very much lower affinity of profilin for modified actin explains the anomalous critical concentrations curves obtained for 5-10% pyrenyllabeled actin in the presence of profilin and the apparently weak inhibition by profilin of the rate of filament elongation when polymerization is quantified by the increase in fluorescence of pyrenyl-labeled actin. The properties Of Acanthamoeba profilin have been studied in our laboratory [1,2,3,4] and by Pollard and his collaborators essentially the same resultsas for Mg2“free buffers; the decrease in the rate of spontaneous polymerization of actin in the presence of profilin (as monitored by the increase in fluorescence of 5% pyrenyl-labeled actin) and theincrease in the concentration of unpolymerized actin at steady state were both said to be compatible with the simple assumption of the formation of a profilin-actin complex, K D = 2-10 phi, that )could not participate in the nucleation and elongation processes. Fluorescence assay (profilin interacts much more weaklywith pyrenyl-labeled actin than with unmodified actin) and are not observed when polymerization is monitored by light scattering.'
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