Myosin subfragment 1 inhibits dissociation of nucleotide and calcium from G-actin.
The dissociation rates of 1,N6-ethenoadenosine 5'-triphosphate (epsilon ATP) and of Ca2+ from G-actin and its complex with myosin subfragment 1 (S1) were measured by recording a large decrease in the fluorescence intensity of the dissociating nucleotide. Under the experimental conditions employed, the binary G-acto-S1A2 complex does not polymerize (Chaussepied, P., and Kasprzak, A. A. (1989) Nature 342, 950-953). The released nucleotide was hydrolyzed either by alkaline phosphatase or by apyrase; to trap Ca2+, EDTA was used. From the anisotropy of N-iodoacetyl-N'-(5-sulfo-1- naphthyl)ethylenediamine (1,5-IAEDANS)-actin, it was established that during the dissociation of epsilon ATP, the G-acto-S1 complex remained stable and the equilibrium of the system was unaltered. The reactions followed first order kinetics. The dissociation rate constant, kd for epsilon ATP decreased from 5.5 x 10(-4) s-1 for free G-actin to 1 x 10(-4) s-1 for G-acto-S1A2; for Ca2+, kd was also similarly reduced from 2.8 x 10(-2) s-1 to 4 x 10(-3) s-1. Two proteolytically derived actin variants were also examined. For free subtilisin-cleaved actin, kd for epsilon ATP was elevated 2-fold but was almost unchanged for Ca2+. In the complex of the cleaved G-actin with S1A2, kd for both epsilon ATP and for Ca2+ were reduced. The removal of the last 3 amino acids from actin produced a derivative whose behavior in binding to S1, as well as in the kinetics of epsilon ATP and Ca2+ dissociation, was undistinguishable from the unmodified protein.
Centre de Recherches de Biochimie Macromoléculaire, Université de Montpellier I, France.
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Kasprzak
D000199:Actins / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D004985:Ethenoadenosine Triphosphate / Q000378:metabolism*; D005456:Fluorescent Dyes; D006868:Hydrolysis; D007700:Kinetics; D015879:Myosin Subfragments / Q000378:metabolism*; D009282:Naphthalenesulfonates; D011189:Potassium Chloride; D011817:Rabbits
D000199:Actins; D004985:Ethenoadenosine Triphosphate; D005456:Fluorescent Dyes; D015879:Myosin Subfragments; D009282:Naphthalenesulfonates; D011189:Potassium Chloride; C006421:1,5-I-AEDANS; D002118:Calcium