Interaction and polymerization of the G-actin-myosin head complex: effect of DNase I.
The properties of polymerization and interaction of the G-actin-myosin S1 complexes (formed with either the S1(A1) or the S1(A2) isoform) have been studied by light-scattering and fluorescence measurements in the absence and in the presence of DNase I. In the absence of DNase I, the G-actin-S1(A1) and G-actin-S1(A2) complexes were found to be characterized by different limiting concentrations (l.c.), defined as the complex concentrations above which the polymerization occurs spontaneously within 20 h at 20 degrees C in a "no salt" buffer (l.c. = 0.42 and 8.8 microM for G-actin-S1(A1) and G-actin-S1(A2), respectively). The occurrence of a limiting concentration for either complex together with the kinetic properties of the polymerization led us to conclude that the G-actin-S1 polymerization occurs via a nucleation-elongation process. Fluorescence titrations and proteolysis experiments revealed that G-actin interacts with S1 with a 1:1 stoichiometry (independently of the presence of ATP) with dissociation constants, in the absence of nucleotide, of 20 and 50 nM for the G-actin-S1(A1) and G-actin-S1(A2) complexes, respectively. In the presence of at least a 1.5-fold excess of DNase I, the polymerization of the G-actin-S1 complexes was blocked even at high protein concentration or in the presence of salts. In addition, the affinity of either S1 isoform to actin was reduced 4-5-fold by DNase I, while the stoichiometry of the G-actin-S1 complexes was not changed. However, since the dissociation constants remain in the submicromolar range, we could demonstrate the existence of ternary DNase I-G-actin-S1 complexes stable under polymerizing conditions. Finally, the study of the effect of nucleotides and of various salts on the G-actin-S1 interaction further showed significant differences between the G-actin-S1 and F-actin-S1 interactions.
Lheureux
K
K
Centre de Recherches de Biochimie Macromoléculaire, CNRS-UPR9008, INSERM-U249, Montpellier, France.
Forné
T
T
Chaussepied
P
P
D000199:Actins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D003850:Deoxyribonuclease I / Q000378:metabolism / Q000494:pharmacology*; D007700:Kinetics; D008027:Light; D046911:Macromolecular Substances; D009132:Muscles / Q000378:metabolism; D015879:Myosin Subfragments / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding; D011817:Rabbits; D012542:Scattering, Radiation; D013050:Spectrometry, Fluorescence; D013997:Time Factors
D000199:Actins; D046911:Macromolecular Substances; D015879:Myosin Subfragments; D003850:Deoxyribonuclease I