Kinetics of Regulated Actin Transitions Measured by Probes on Tropomyosin

Abstract

Changes in the muscle regulatory protein complex, troponin, are important for modulation of activity and may occur as a result of disease-causing mutations. Both increases and decreases in the rate of ATP hydrolysis by myosin may occur as dictated by changes in the distribution of actin-tropomyosin-troponin among its different states. It is important to measure the rates of transition among these states to study physiological adaptation and disease processes. We show here that acrylodan or pyrene probes on tropomyosin can be used to monitor the transition from active to intermediate and inactive states of actin-tropomyosin-troponin. Transitions measured in the absence of calcium had two phases, as previously reported for some other probes on troponin and actin. The first step was a rapid equilibrium that favored the formation of the intermediate state and had an apparent rate constant less than that of S1-ATP dissociation. The second fluorescence transition was slower, with an apparent constant that increased from approximately 5 to 80/s over a range of 1-37 degrees C. Only the initial rapid transition was seen in the presence of saturating calcium. The acrylodan probe had the advantage of yielding a larger signal than the pyrene probe. Furthermore, the acrylodan signal decreased in going from the active state to the intermediate state, and then increased upon going to the inactive state.

The Rates of Ca2+ Dissociation and Cross-bridge Detachment from Ventricular Myofibrils as Reported by a Fluorescent Cardiac Troponin C

“…This may suggest that crossbridge detachment observed by TnC IANBD T53C is sensed and limited by movement of another myofilament protein (tropomyosin (Tm), TnI, or TnT) or that the rate of cross-bridge detachment from the nucleotide free myofibrils is slower than that measured from reconstituted actomyosin. The idea that Tm movement may limit the observed rate of cross-bridge detachment is consistent with skeletal thin filament studies where myosin detachment was Ͼ500/s as measured by light scatter but sensed at ϳ200/s by a fluorescent Tm (47). However, by following a change in Trp fluorescence, ATP also hyperbolically accelerated the rate of cross-bridge detachment in skeletal myofibrils (in the absence of ADP) (36).…”

“…In the absence of ADP, ATP-induced detachment of myosin from reconstituted skeletal and cardiac actomyosin is extremely fast (Ͼ500/s) (15,47). This may suggest that crossbridge detachment observed by TnC IANBD T53C is sensed and limited by movement of another myofilament protein (tropomyosin (Tm), TnI, or TnT) or that the rate of cross-bridge detachment from the nucleotide free myofibrils is slower than that measured from reconstituted actomyosin.…”

Acrylodan-Labeled Smooth Muscle Tropomyosin Reports Differences in the Effects of Troponin and Caldesmon in the Transition from the Active State to the Inactive State

“…Acrylodan-labeled and pyrene-labeled smooth muscle tropomyosin were prepared as described for skeletal muscle tropomyosin (24) using a 10:1 molar ratio of acrylodan or N-(1-pyrene)iodoacetamide to tropomyosin. These conditions gave >70% labeling using extinction coefficients of 22,000 M −1 cm −1 at 344 nm for pyrene and 14,400 M −1 cm −1 at 372 nm for acrylodan (33).…”

“…The differential equations used to simulate the ATP dissociation studies (Model A) were shown earlier (24). S1-ATP detachment was simulated by examining the change of ATP-M-A with time.…”

“…However, image reconstructions of actin filaments containing smooth muscle tropomyosin and an actin binding caldesmon fragment show that tropomyosin does not occupy the same inhibitory position that is stabilized by troponin in the absence of calcium (23). We reexamined the question of tropomyosin movement using an acrylodan probe on smooth muscle tropomyosin that has certain advantages over pyrene tropomyosin for measuring transitions of actin-tropomyosin-troponin (24). …”