Related Experiment Videos
Temperature-dependent transitions of the myosin-product intermediate at 10 degrees C in the Mn(II)-ATP hydrolysis
Abstract:
1. While below 10 degrees C, the initial burst of Pi liberation in the hydrolysis of Mn(II)-ATP by heavy meromyosin or myosin subfragment 1 was inhibited by the pre-addition of ADP without any change in the steady-state activity, it was not inhibited above 10 degrees C. The burst size was about one mole per two moles of myosin active sites. 2. Above 10 degrees C, the ultraviolet absorption spectrum of heavy meromyosin induced by ATP in MnCl2 was similar to that induced in MgCl2 and the spectral decay to the ADP-induced level occurred only after all the ATP in the solution was depleted. In contrast, below 10 degrees C the spectrum induced by ATP in MnCl2 decayed to the ADP-induced level within a few seconds after the addition of ATP, although ATP was present in the solution. 3. These two results indicate that in Mn-ATP above 10 degrees C at the burst site there is a myosin*-ADP-Pi complex generated by ATP hydrolysis while below 10 degrees C there is a myosin-product complex identical with the one generated by adding ADP (and Pi) to myosin. 4. At tempertures both above and below 10 degrees C, the Mn-ATP hydrolysis of heavy meromyosin was activated by actin and superprecipitation of actomyosin occurred. Characteristics of these phenomena showed a transition at around 10 degrees C.
Insights
Temperature significantly impacts myosin
Area of Science:
- Biochemistry
- Muscle Physiology
Background:
- Myosin's interaction with ATP is crucial for muscle contraction.
- Understanding the kinetics of ATP hydrolysis by myosin is key to elucidating muscle function.
Purpose of the Study:
- To investigate the temperature-dependent mechanisms of ATP hydrolysis by heavy meromyosin (HMM) in the presence of manganese.
- To characterize the intermediates formed during Mn-ATP hydrolysis by HMM and the effect of actin.
Main Methods:
- Enzyme kinetics studies measuring inorganic phosphate (Pi) liberation.
- Spectroscopic analysis of ultraviolet absorption changes.
- Observation of actomyosin superprecipitation.
Main Results:
- Below 10°C, ADP pre-addition inhibited the initial Pi burst, suggesting a distinct myosin-product complex.
- Above 10°C, the myosin*-ADP-Pi complex persisted until ATP depletion, unlike the rapid decay below 10°C.
- Actin activation of Mn-ATP hydrolysis and actomyosin superprecipitation exhibited a transition around 10°C.
Conclusions:
- A temperature-dependent transition in the mechanism of ATP hydrolysis by HMM occurs around 10°C.
- The nature of the myosin-product complex differs significantly below and above this critical temperature.
- Actin's regulatory role in myosin ATPase activity is also modulated by temperature.