Slow Myosin ATP Turnover in the Super-Relaxed State in Tarantula Muscle

Abstract

We measured the nucleotide turnover activity of myosin in tarantula leg-muscle fibers by observing single turnovers of the fluorescent nucleotide analog, mantATP, as monitored by the decrease in fluorescence when mantATP is replaced by ATP in a chase experiment. We find a multi-exponential process, with approximately two-thirds of the myosin showing a very slow nucleotide turnover time constant, ∼30 minutes. This slow turnover state is termed the super-relaxed state (SRX) and is a highly novel adaptation for energy conservation in an animal that spends extremely long periods of time in a quiescent state (days) employing a lie-in-wait hunting strategy. If fibers are incubated in mantADP and chased with ADP, the SRX is not seen, indicating that relaxed myosins are responsible for the SRX. Phosphorylation of the myosin regulatory light chain eliminates the fraction of myosin with the very long lifetime. The presence of the SRX measured here correlates well with the binding of myosin to the core of the thick filament in a structure known as the interacting-head motif (or J-motif) observed previously by electron microscopy. Both the structural array and the long-lived SRX require ATP, both are lost upon myosin phosphorylation, and both appear to be more stable in tarantula than in skeletal or cardiac preparations. EPR spectroscopy of a spin-labeled nucleotide bound to the motor domain of myosin in relaxed tarantula fibers likewise shows orientation that is lost when the myosin is phosphorylated. Together, the data support the hypothesis that the SRX myosin and the myosin seen in the EM of the order helical array in tarantula filaments are the same.