Kinetics of nucleotide and metal ion interaction with G-actin

The kinetics of interaction of Ca2+ ions and nucleotides with G-actin have been investigated by making use of the enhancement of 1,N6-ethenoadenosine 5'-triphosphate (epsilon ATP) fluorescence on binding to actin, the enhancement of 2-[[2-[bis(carboxymethyl)amino]-5-methylphenoxy] methyl]-6-methoxy-8-[bis(carboxymethyl)amino]quinoline (Quin-2) fluorescence on binding to Ca2+, and the sensitivity of the fluorescence of an N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine (1,5-AEDANS) group on Cys-374 to metal ion binding. It is concluded that metal ion dissociation is the rate-limiting step in nucleotide dissociation (0.016 s-1 for Ca2+ at pH 7.2 and 21 degrees C) and that earlier conclusions that metal ion release is relatively fast and subsequent nucleotide release slow are incorrect. Results presented here and obtained by others on the metal ion concentration dependence of the effective rate of nucleotide exchange can be interpreted in the light of this conclusion in terms of a limiting rate which corresponds to that of metal ion release and an "apparent" dissociation constant for Ca2+ which is without direct physical significance. This apparent dissociation constant is more than 2 orders of magnitude greater than the real dissociation constant of Ca2+ from the Ca-actin-ATP complex, which was estimated to be 2 X 10(-9) M from a titration with Quin-2. Confirmation that the rate of Ca2+ release is rate limiting both in nucleotide dissociation reactions and in replacement of Ca2+ by Mg2+ was obtained with 1,5-AEDANS-actin, since both the replacement of Ca2+ by Mg2+ and the removal of Ca2+ to give the actin-ATP complex occurred at the same (slow) rate.(ABSTRACT TRUNCATED AT 250 WORDS

Divalent cation-, nucleotide-, and polymerization-dependent changes in the conformation of subdomain 2 of actin.

Conformational changes in subdomain 2 of actin were investigated using fluorescence probes dansyl cadaverine (DC) or dansyl ethylenediamine (DED) covalently attached to Gln41. Examination of changes in the fluorescence emission spectra as a function of time during Ca2+/Mg2+ and ATP/ADP exchange at the high-affinity site for divalent cation-nucleotide complex in G-actin confirmed a profound influence of the type of nucleotide but failed to detect a significant cation-dependent difference in the environment of Gln41. No significant difference between Ca- and Mg-actin was also seen in the magnitude of the fluorescence changes resulting from the polymerization of these two actin forms. Evidence is presented that earlier reported cation-dependent differences in the conformation of the loop 38-52 may be related to time-dependent changes in the conformation of subdomain 2 in DED- or DC-labeled G-actin, accelerated by substitution of Mg2+ for Ca2+ in CaATP-G-actin and, in particular, by conversion of MgATP- into MgADP-G-actin. These spontaneous changes are associated with a denaturation-driven release of the bound nucleotide that is promoted by two effects of DED or DC labeling: lowered affinity of actin for nucleotide and acceleration of ATP hydrolysis on MgATP-G-actin that converts it into a less stable MgADP form. Evidence is presented that the changes in the environment of Gln41 accompanying actin polymerization result in part from the release of Pi after the hydrolysis of ATP on the polymer. A similarity of this change to that accompanying replacement of the bound ATP with ADP in G-actin is discussed