Nucleotide-free actin: stabilization by sucrose and nucleotide binding kinetics.
We prepared nucleotide-free actin in buffer containing 48% (w/v) sucrose. Sucrose inhibits the irreversible denaturation of actin that follows nucleotide dissociation [Kasai et al. (1965) Biochim. Biophys. Acta 94, 494-503]. Our conditions removed nucleotide from approximately 80% of the actin. Stabilization of nucleotide-free actin depends on the sucrose concentration. The CD ellipticity (x 10(3) deg cm2 dmol-1) at 222 nm of nucleotide-free actin in 48% sucrose is -3.54. The ellipticity of denatured nucleotide-free actin in dilute buffer is -2.01 and that of native actin is -4.19. In 48% sucrose nucleotide-free actin has 1.12 and native actin has 0.5 solvent-exposed thiol residues. The conformation of native actin is recovered when ATP and Mg2+ are added. Our ability to generate stable nucleotide-free actin permitted us to study the kinetics of nucleotide binding to actin. The observed rate constant of the reaction is linearly dependent on the concentration of epsilon ATP, a fluorescent analog of ATP. The inverse of the association rate constant is proportional to the viscosity of the solvent with an intercept near the origin as expected for a diffusion-limited reaction. The second-order association rate constant for Mg(2+)-ATP and Ca(2+)-ATP binding to nucleotide-free actin in water at 22 degrees C is 5 x 10(6) M-1 s-1. The Smoluchowski collision rate constant for actin and ATP is calculated to be 6.5 x 10(9) M-1 s-1, which makes the "orientation factor" 7.7 x 10(-4). From the ratio of the dissociation and association rate constants, we calculate dissociation equilibrium constants of 1.2 x 10(-9) M for Mg(2+)-ATP-actin, 4.4 x 10(-9) M for Mg(2+)-epsilon ATP-actin, and 1.2 x 10(-10) M for Ca(2+)-ATP-actin.
De La Cruz
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EM
Department of Cell Biology and Anatomy, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
Pollard
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D000199:Actins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D002122:Calcium Chloride / Q000378:metabolism; D002942:Circular Dichroism; D004228:Dithionitrobenzoic Acid; D004355:Drug Stability; D004492:Edetic Acid; D004985:Ethenoadenosine Triphosphate / Q000378:metabolism*; D007202:Indicators and Reagents; D007700:Kinetics; D015636:Magnesium Chloride / Q000378:metabolism; D018482:Muscle, Skeletal / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D011817:Rabbits; D013395:Sucrose; D014783:Viscosity
D000199:Actins; D004985:Ethenoadenosine Triphosphate; D007202:Indicators and Reagents; D015636:Magnesium Chloride; D013395:Sucrose; D000255:Adenosine Triphosphate; D004228:Dithionitrobenzoic Acid; D004492:Edetic Acid; D002122:Calcium Chloride