Myosin ATP and

Although this model is very attractive because of its simplicity, further experiments have shown that there are two problems with it (Eisenberg and Hill, 1985). First, myosin.ATP binds as well to actin as does myosin.ADP.Pj such that there is a rapid equilibrium between actomyosin.ATP and myosin.ATP, and between... 

White et aP measured the rates of phosphate release during a single turnover of actomyosin nucleoside triphosphate (NTP) hydrolysis using a double-mixing stopped-flow spectrofluorometer, at very low ionic strength to increase the affinity of myosin-ATP and myo-sin-ADP-Pi to actin. Myosin subfragment 1 and a series of nucleoside triphosphates were mixed and incubated for approximately 1-10 s to allow NTP to bind to myosin and generate a steady-state mixture of myosin-NTP and myosin-NDP-Pi. The steady-state intermediates were then mixed with actin. 

Within each sarcomere the relative sliding of thick and thin filaments is brought about by "cross-bridges," parts of the myosin molecules that stick out from the myosin filaments and interact cyclically with the thin actin filaments, transporting them hy a kind of rowing action. During this process, the hydrolysis of ATP to ADP and phosphate couples the conformational... 

Gopal, D., Pavlov, D. I., Levitsky, D. I., et al., 1996. Chemomechanical trans-dnction in the actomyosin molecnlar motor by 2, 3 -dideoxydidehydro-ATP and characterizadon of its interacdon with myosin snbfragment 1 in die presence and absence of acdn. Biochemistry 35 10149-10157. 

In an effort to understand how actin-actin interactions might be affected by the binding of the myosin head, and in order to gain more insight into the nature of the actin-myosin interface, we have investigated the nature of the kinetic actin-myosin intermediates involved in the process of S)-induced polymerization of G-actin. For this purpose, a variety of fluorescent probes (e.g., pyrene, NBD, AEDANS) have been covalently attached to the C-terminus of G-actin to probe the G-actin-S] interaction under conditions of tightest binding, i.e., in the absence of ATP. 

Adelstein, R.S. Eisenberg, E. (1980). Regulation and kinetics of the actin-myosin-ATP interaction. Ann. Rev. Biochem. 49,921-956. 

Enzymatic activities. The hydrolysis of ATP by actin-activated myosin is the characteristic enzymatic activity of muscle, smooth muscle included. All forms of smooth muscle myosin are slower than those of other muscles. The binding site for ATP and a reduced enzymatic activity are still present in monomeric myosin. The enzymatic activity of monomeric myosin is altered by a conformational change, (the 10S-6S transition) and the species of cations present in the reaction mixture. These differences relate to the possible mechanisms of regulation. 

Myosin-ATP has a low affinity for actin, and actin is thus released. This last step is a key component of relaxation and is dependent upon the binding of ATP to the actin-myosin complex. 

Relaxation occurs when sarcoplasmic Ca falls below 10 mol/L owing to its resequestration into the sarcoplasmic reticulum by Ca ATPase. TpC.dCa thus loses its Ca. Consequently, troponin, via interaction with tropomyosin, inhibits further myosin head and F-actin interaction, and in the presence of ATP the myosin head detaches from the F-actin. 

When smooth muscle myosin is bound to F-actin in the absence of other muscle proteins such as tropomyosin, there is no detectable ATPase activity. This absence of activity is quite unlike the situation described for striated muscle myosin and F-actin, which has abundant ATPase activity. Smooth muscle myosin contains fight chains that prevent the binding of the myosin head to F-actin they must be phosphorylated before they allow F-actin to activate myosin ATPase. The ATPase activity then attained hydrolyzes ATP about tenfold more slowly than the corresponding activity in skeletal muscle. The phosphate on the myosin fight chains may form a chelate with the Ca bound to the tropomyosin-TpC-actin complex, leading to an increased rate of formation of cross-bridges between the myosin heads and actin. The phosphorylation of fight chains initiates the attachment-detachment contraction cycle of smooth muscle. 

Figure 49-14. Regulation of smooth muscle contraction by Ca. pL-myosin is the phosphorylated light chain of myosin L-myosin is the dephosphorylated light chain. (Adapted from Adelstein RS, Eisenberg R Regulation and kinetics of actin-myosin ATP interaction. Annu Rev Biochem 1980 49 921.)...

The hydrolysis of ATP is used to drive movement of the filaments. ATP binds to myosin heads and is hydrolyzed to ADP and P, by the ATPase activity of the actomyosin complex. 

Walker, J. E., Saraste, M., Runswick, M. J. and Gay, N. J. (1982). Distantly related sequences in the alpha- and beta-subunits of ATP synthase, myosin, kinases and other ATP-requiring enzymes and a common nucleotide binding fold, EMBO J., 1, 945-951. 

ATP is used not only to power muscle contraction, but also to re-establish the resting state of the cell. At the end of the contraction cycle, calcium must be transported back into the sarcoplasmic reticulum, a process which is ATP driven by an active pump mechanism. Additionally, an active sodium-potassium ATPase pump is required to reset the membrane potential by extruding sodium from the sarcoplasm after each wave of depolarization. When cytoplasmic Ca2- falls, tropomyosin takes up its original position on the actin and prevents myosin binding and the muscle relaxes. Once back in the sarcoplasmic reticulum, calcium binds with a protein called calsequestrin, where it remains until the muscle is again stimulated by a neural impulse leading to calcium release into the cytosol and the cycle repeats. 

Figure 9.22 The relationship between ATP utilisation by myosin ATPase and ATP generation by Krebs cycle and electron transfer. This relationship between the two major energy systems in muscle is critical. The rate of the cycle and electron transfer is controlled, in part, by ATP utilisation by muscle contraction (see below). This is equivalent to a market economy so that the law of supply and demand applies. The greater the demand and hence the use of ATP, the greater is the rate of generation.

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