Kinetics of Smooth Muscle Acrylodan-Tropomyosin Transitions on Actin

Abstract

Actin-tropomyosin generally has the potential to exist in states that are active, inactive or intermediate with respect to activation of myosin S1-ATPase activity. S1 stabilizes the active state while proteins that inhibit ATPase activity may function by stabilizing the inactive state. The transition among states can be initiated by rapidly dissociating S1 from actin-tropomyosin with ATP. We tested the ability of acrylodan labeled smooth muscle tropomyosin to monitor changes in the presence of skeletal muscle troponin. With saturating calcium there was a rapid decrease in acrylodan fluorescence as the actin-tropomyosin made the transition from the active state to the intermediate state. In the absence of calcium the rapid decrease in fluorescence was followed by a slower fluorescence increase as the inactive state became populated. Acrylodan labeled smooth muscle tropomyosin behaved much like labeled skeletal muscle tropomyosin (Borrego-Diaz & Chalovich, 2010). When the same experiment was done with acrylodan labeled tropomyosin-actin-caldesmon a similar pattern of fluorescence decrease and increase occurred. The rapid decrease corresponded to movement to the intermediate state and could not be distinguished from the rate of S1-ATP detachment. The slower redevelopment of fluorescence differed from the case with troponin in that it occurred as caldesmon binding increased to occupy sites vacated by S1. Changes in caldesmon binding were monitored by NBD probes on caldesmon. We sometimes observed an increase in acrylodan fluorescence when caldesmon was mixed with acrylodan-tropomyosin alone. Thus in the case of caldesmon, S1-ATP detachment occurs with a rapid fluorescence decrease that was independent of caldesmon. The slow fluorescence redevelopment occurred as caldesmon bound to actin-tropomyosin. We have not determined if in that case the fluorescence change coincided with tropomyosin movement.