THE EFFECT OF CA(2-FILAMENTS WITH OR WITHOUT BOUND MYOSIN SUBFRAGMENT-1() ON THE CONFORMATION OF TROPOMYOSIN AND ACTIN IN REGULATED ACTIN)

The effects of Ca2+ and myosin subfragment 1 on the conformation of tr opomyosin and actin in regulated actin filaments in ghost fibers were investigated by means of the polarized fluorescence technique. Regulat ed thin filaments were reconstituted in skeletal muscle ghost fibers b y incorporation into the fibers of either skeletal muscle troponin-tro pomyosin or smooth-muscle caldesmon-calmodulin-tropomyosin complexes. Tropomyosin and actin were specifically labeled with fluorescent probe s, 1,5-IAEDANS and phalloidin-rhodamine, respectively. Analysis of the fluorescence parameters indicated that the binding of Ca2+ to regulat ed actin filaments induces conformational changes in tropomyosin and a ctin that lead to the strengthening of the intraction between these tw o proteins and weakening of the binding of actin monomers in the filam ent. These changes become larger when regulated actin forms rigor link s with myosin subfragment 1. No notable alterations in the position of tropomyosin relative to actin in the frontal plane of the fiber were detected either upon binding of Ca2+ or upon the additional binding of myosin subfragment 1 to regulated actin.