The rate constant for dissociation of ADP from actomyosin subfragment 1 (Si) has been measured in this laboratory and elsewhere for a variety of vertebrate muscle types. We have made the following observations: (i) In solution, the dissociation of ADP from actomyosin-Sl limits the rate of dissociation of actomyosin-Sl-ADP by ATP and, presumably, also limits the rate of crossbridge detachment in contracting muscle. (it) For muscle types in which the rate of ADP dissociation from actomyosin-Sl is slow enough to measure using stopped-flow methods, the rate constants are nearly the same as the theoretical value for the minimum allowable rate constant for dissociation of an attached crossbridge. Therefore, ADP dissociation is sufficiently slow to be the molecular step that limits the maximum shortening velocity of these muscles. (i) Variation with muscle type of the rate constant for ADP dissociation may be a general phylogenetic mechanism for regulating shortening velocity.that one or more of the rate constants of the ATP hydrolysis mechanism limits the muscle contraction rate. For a molecular step to limit the shortening velocity of muscle, it must have the following attributes:(i) The rate constant for such a step must be consistent with both the maximum working length of an individual crossbridge and the rate of axial displacement of the thick and thin filaments observed in contracting muscle. The minimum allowable rate constant, kmin, for the conversion of an attached crossbridge state in muscle (corresponding to the AM states in the top line of Eq. 1) to other attached or detached states (corresponding to the M states in the bottom line of Eq. 1) can be estimated from the maximum rate of contraction (the unloaded shortening velocity) and the distance over which a crossbridge can remain attached, using Eq. 2, Muscle contraction is thought to occur as the result of a cyclic association and dissociation of crossbridges formed between myosin molecules in the thick filaments and F-actin molecules in the thin filament, involving concomitant hydrolysis of ATP. This cycle can lead to relative sliding of the actin and myosin filaments and result in the production of work. A rationale for studying the kinetic mechanism of ATP hydrolysis by actomyosin-Sl in solution is that it may directly relate to the observed physiological properties of muscle. A condensed version of the kinetic mechanism, consistent with recent observations (1, 2), is shown in Eq. 1. The second-order reactions of nucleotide and phosphate binding have been shown to be two-step reactions (3-5), but they are condensed here to single steps:where M represents myosin subfragment-1 (Si) and AM represents actomyosin S1. Different types of vertebrate muscles have shortening velocities that vary by almost two orders of magnitude. Barany showed that the steady-state rate of hydrolysis of MgATP by actomyosin is correlated with muscle shortening velocity (6).However, it has not been demonstrated that the rate of ATP hydrolysis directly limits shortening ve...

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1. Hydrolysis of the myosins from smooth and from skeletal muscle by a rat trypsin-like serine proteinase and by bovine trypsin at pH 7 is compared. 2. Proteolysis of the heavy chains of both myosins by the rat enzyme proceeds at rates approx. 20 times faster than those obtained with bovine trypsin. Whereas cleavage of skeletal-muscle myosin heavy chain by both enzymes results in the generation of conventional products i.e. heavy meromyosin and light meromyosin, the heavy chain of smooth-muscle myosin is degraded into a fragment of mol. wt. 150000. This is dissimilar from heavy meromyosin and cannot be converted into heavy meromyosin. It is shown that proteolysis of the heavy chain takes place in the head region. 3. The 'regulatory' light chain (20kDa) of smooth-muscle myosin is degraded very rapidly by the rat proteinase. 4. The ability of smooth-muscle myosin to have its ATPase activity activated by actin in the presence of a crude tropomyosin fraction on introduction of Ca2+ is diminished progressively during exposure to the rat proteinase. The rate of loss of the Ca2+-activated actomyosin ATPase activity is very similar to the rate observed for proteolysis of the heavy chain and 3-4 times slower than the rate of removal of the so-called 'regulatory' light chain. 5. The significance of these findings in terms of the functional organization of the smooth muscle myosin molecule is discussed. 6. Since the degraded myosin obtained after exposure to very small amounts of the rat proteinase is no longer able to respond to Ca2+, i.e. the functional activity of the molecule has been removed, the implications of a similar type of proteolysis operating in vivo are considered for myofibrillar protein turnover in general, but particularly with regard to the initiation of myosin degradation, which is known to take place outside the lysosome (i.e. at neutral pH).

The effects of the Ca2+-activated cysteine proteinase, the rat trypsin-like serine proteinase and bovine trypsin on myofibrillar proteins from rabbit skeletal muscle are compared. 2. Myofibrils that had been treated at neutral pH with the Ca2+-dependent proteinase and with the rat enzyme were (a) analyzed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and (b) examined in the electron microscope. Treatment with each proteinase resulted in the loss of the Z-discs, but the rat enzyme caused much more extensive disruption of the ultrastructure and degraded more of the myofibrillar proteins. 3. Purified F-actin was almost totally resistant to the proteinases, whereas G-actin was degraded by the rat trypsin-like proteinase at a rate approx. 15 times faster than was obtained with bovine trypsin. 4. Similar results were obtained with alpha-actinin, whereas tropomyosin was degraded more readily by bovine trypsin than by the rat trypsin-like proteinase. 5. The implications of these findings for the non-lysosomal breakdown of myofibrillar proteins in vivo are considered.

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