Abstract
Our earlier fluorescence measurements using N-(1-pyrenyl)iodoacetamide-labeled actin revealed that caldesmon interacts with G-actin accelerating its nucleation at low salt concentration and causing polymerization in the absence of sale [Gałazkiewicz, B., Mossakowska, M., Osińska, H. & Dabrowska, R. (1985) FEBS Lett. 184, 144-149]. In this work the caldesmon-induced process of actin polymerization as well as the dynamic properties of the polymers formed have been investigated with the use of fluorescence, electron paramagnetic resonance (EPR) and electron microscopy techniques. Fluorescence titration of N-(1-pyrenyl)iodoacetamide-labeled actin with caldesmon showed saturation of the polymerization at a 1:3 molar ratio of caldesmon/actin monomer. Parallel pelleting experiments revealed, however, that the process of polymer formation is biphasic and only at higher concentrations of caldesmon does the copolymer contain around one caldesmon/three actin monomers. At low concentration of caldesmon a complex of one caldesmon/nine actin monomers is formed. EPR spectroscopy, using maleimide spin label bound at Cys374 of actin, also indicated that one caldesmon molecule polymerizes nine actin monomers. Taken together, these results might suggest the existence of weak and strong forms of actin binding to caldesmon and detection of only the latter by the fluorescence method. Copolymers of actin and caldesmon are indistinguishable from actin polymerized by salt with respect to their appearance in the electron microscope and their ability to interact with heavy meromyosin, although they are characterized by lower torsional flexibility as indicated by immobilization of spin labels attached to actin.
Published Version
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