The regulation of vertebrate striated muscle contraction by Ca++ has been a topic of considerable interest in recent years. The initial structural biochemical and structural information concerning the thin filament led to the development of the steric blocking theory for the physical mode of action of tropomyosin. The evidence for the movement of tropomyosin relative to actin on Ca++ activation of muscle is well grounded in x-ray diffraction, electron microscopic reconstructions, and in some biochemical and biophysical probe studies. However beginning with Bremel and Weber's discovery of potentiation of the regulated thin filament, a number of other studies indicate that the actual effects of Ca++ involve the kinetics of the activated ATPase reaction as well as the properties of myosin, probably through the phosporylatable light chain of the enzymatically active regions of the molecule. This increasing complexity of the regulation mechanism mandates that the role of tropomyosin in the regulatory process be more intensively studied. It is proposed to investigate the use of a number of relatively newly developed heterobifunctional crosslinking reagents to study their effects on the regulatory ability of reconstituted thin filaments. Attempts will be made to crosslink tropomyosin and actin using these reagents to try to immobilize actin in a less stringent way than has been accomplished before with general crosslinking reagents such as gluteraldehyde. The results of intramolecular crosslinking of tropomyosin and actin before reconstitution of the thin filament will also be studied, both as a control and for its own interest. In this preliminary study the alterations in the activated myosin ATPase and superpercipitation will be examined. The results of one set of preliminary investigations is presented.
