Myosin mechanical experiments

Smooth muscle fibers generate as much isometric force per cross-sectional area as skeletal muscle fibers with only 20% as much myosin (Murphy et al., 1974). There have been various possible explanations for this, including different mechanical properties of the myosin itself. Experiments with the in vitro motility assay provide insight into this possibility. 

In this chapter we review the structure-function relationship of vertebrate smooth muscle myosin LCs. The LCs are the protein cofactors of the smooth muscle contractile machinery, and we will focus on the experiments that investigate the mechanisms of their functional role. We also describe basic methods for the study of the LCs, with special emphasis on the procedures needed for the characterization of the phospho-rylated LC20. 

Coupled to the folding of the myosin molecule is a change at the active site that results in "trapping" of nucleotide (Cross et al., 1986). The observed rate of phosphate release from single-turnover experiments, <0.0005 sec i, likely reflects product release from the small amount of extended myosin that is in equilibrium with the folded monomer (Cross et al., 1988). The structural basis for trapping is not known. It has been proposed that after MgATP is cleaved at the active site, phosphate leaves via a "backdoor" mechanism (Yount et al., 1995). Perhaps in the folded monomer the glycine-rich P-loop is stabilized in a conformation that prevents phosphate release. 

Possible caldesmon-induced movement of tropomyosin away from this site or competition with tropomyosin for the site may prevent potentiation from occurring. The work of Chacko and associates (Horiuchi and Chacko, 1989 Horiuchi et al., 1991), in fact, implies that caldesmon may modulate the magnitude of tropomyosin activation, and therefore may control actomyosin ATPase by inhibiting tropomyosin potentiation. This view would explain how a single caldesmon molecule could influence the reactivity of many actin molecules along thin filaments. Such a mechanism of modulation implies that caldesmon may fine-tune contractile activity but not act as an on-off switch per se. This view also fits with results of the elegant experiments of Fay and his collaborators (Itoh et al., 1989), who showed that myosin phosphoryla-... 

MLCK plays a central role in the initiation of smooth muscle contraction and many nonmuscle motile processes owing to its Ca2+/calmodulin-dependent phosphorylation of myosin RLC. Physiological experiments with rapid, synchronous activation of smooth muscle cells demonstrate that the significant 500-ms latency for RLC phosphorylation and force development is due to the time required for increases in cytosolic Ca + concentration and activation of MLCK. Once activated, MLCK rapidly (1 s i) phosphorylates RLC in a random mechanism leading to the rapid attachment of cross-bridges and subsequent force development. Although the general properties of this cellu-... 

Whenever possible the basic molecular events of physiological processes are studied in detail in solutions either in parallel with, or as a guide to, experiments on the organized system. For example, the study of the reactions of myosin and the associated proteins of the contractile cycle, by the methods used for the investigation of mechanisms of soluble enzymes (see section S.l), has helped in the planning of experiments and in the interpretation of the events observed in muscle fibres. The most useful methods for kinetic studies on physiological functions are those which can be applied to systems at different levels of organization. We shall return to them at the end of this chapter. 

From a practical point of view the major difference between the two approaches to enzyme kinetics, steady state and transient rate measurements, is in the concentrations of enzyme used. Steady state experiments are carried out with catalytic amounts of enzyme at concentrations negligible compared to those of the substrates or products. The rationale of transient kinetic experiments, discussed in section 5.1, will be seen to rest on the observation of complexes of enzymes with substrates and products. The importance of the direct observation and characterization of reaction intermediates for an understanding of mechanisms will be illustrated in that section. This requires enzyme concentrations sufficiently high for detection of intermediates by spectroscopic or other physical monitors. There are a number of interesting systems in vivo with enzyme and substrates at comparable concentrations and the potential kinetic consequences of such situations will be discussed in sections 5.2 and 5.3. Jencks (1989) comments in connection with a review of the transient kinetic behaviour and mechanism of one such system, the calcium pump of the sarcoplasmic reticulum, that steady state kinetics could make no contribution to an understanding of its ATPase linked reaction. The same can be said of the mechanism of myosin-ATPase, which has been elucidated in detail by transient kinetic studies (see section 5.1). 

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