Effect of vanadate on force and myosin light chain phosphorylation in skinned aortic smooth muscle.

The effects of vanadate were examined on Ca2+-activated force and phosphorylation of 20-kDa myosin light chain in membrane-permeabilized rabbit aortic smooth muscle strips. Addition of vanadate during maximum contraction reduced the force in a dose-dependent manner, and inhibited it almost completely at 1 mM. Two-dimensional polyacrylamide gel electrophoretic analyses revealed that vanadate also reduced the phosphorylation of 20- kDa myosin light chain in a dose-dependent manner from approximately 50% in the absence of vanadate to approximately 20% in the presence of 1 mM vanadate. The effects of 1 mM vanadate on purified myosin light chain kinase and phosphatase were then examined using purified myosin as substrate, and it was found that vanadate neither inhibited myosin light chain kinase nor activated myosin light chain phosphatase. These results indicate that the reduction in the 20-kDa myosin light chain phosphorylation level by vanadate may be effected through its inhibition of the force generation in skinned smooth muscle strip, as evidenced by the finding that vanadate eliminated the enhancement of myosin light chain kinase activity brought about by the interaction between purified myosin and actin.     

Structure and function of the two heads of the myosin molecule. I. Binding of adenosine diphosphate to myofibrils during the adenosinetriphosphatase reaction.

1. The myosin content of myofibrils was found to be 51% by SDS-gel electrophoresis. 2. The initial burst of Pi liberation of the ATPase [EC 3.6.1.3] of a solution of myofibrils in 1 M KCl was measured in 0.5 M KCl, and found to be 0.93 mole/mole of myosin. 3. The amount of ADP bound to myofibrils during the ATPase reaction and the ATPase activity were measured by coupling the myofibrillar ATPase reaction with sufficient amounts of pyruvate kinase [EC 2.7.1.40] and PEP to regenerate ATP. The maximum amount of ADP bound to myofibrils in 0.05M KCl and in the relaxed state was about 1.5 mole/mole of myosin. On the other hand, the ATPase activity exhibited substrate inhibition, and the amount of ATP required for a constant level of ATPase activity was smaller than that required for the maximum binding of ADP to myofibrils. 4. The maximum amount of ADP bound to myofibrils in 0.5 M KCl was about 1.9 mole/mole of myosin. When about one mole of ADP was found to 1 mole of myosin in myofibrils, the myofibrillar ATPase activity reached the saturated level, and with further increase in the concentration of ATP one more mole of ADP was found per mole of myosin.     

Nonuniform distribution of myosin light chains within the thick filaments of lobster slow muscle: Immunocytochemical study.

The in situ distribution of the alpha and beta myosin light chains was investigated at the subsarcomeric and subfilament levels in individual fibers of the superficial flexor muscle (SFM) of the lobster, Homarus americanus. Polyclonal antibodies were produced against the two classes of myosin light chains and used for subsequent immunolocalization on thin sections of sarcomeres and on isolated filaments from both the medial and lateral fiber bundles of the SFM. The beta myosin light chains were uniformly distributed within the crossbridge region of sarcomeres of both medial and lateral bundles. The alpha myosin light chains were uniformly distributed within the crossbridge region of sarcomeres from the medial bundle, but were nonuniformly distributed over the crossbridge region of lateral bundle sarcomeres. In the latter, the number of alpha myosin light chains was highest toward the center of the thick filaments, diminishing towards the ends. Similar distributions of alpha light chains were found in isolated myosin filaments. These data demonstrate that heterogeneity in protein composition extends to the level of the myosin filament and suggest that the myosin filament substructure in lobster may be different than that found in vertebrate skeletal muscle.     

Temperature dependence of speed of actin filaments propelled by slow and fast skeletal myosin isoforms.

It was shown that the temperature sensitivity of shortening velocity of skeletal muscles is higher at temperatures below physiological (10-25 degrees C) than at temperatures closer to physiological (25-35 degrees C) and is higher in slow than fast muscles. However, because intact muscles invariably express several myosin isoforms, they are not the ideal model to compare the temperature sensitivity of slow and fast myosin isoforms. Moreover, temperature sensitivity of intact muscles and single muscle fibers cannot be unequivocally attributed to a modulation of myosin function itself, as in such specimen myosin works in the structure of the sarcomere together with other myofibrillar proteins. We have used an in vitro motility assay approach in which the impact of temperature on velocity can be studied at a molecular level, as in such assays acto-myosin interaction occurs in the absence of sarcomere structure and of the other myofibrillar proteins. Moreover, the temperature modulation of velocity could be studied in pure myosin isoforms (rat type 1, 2A, and 2B and rabbit type 1 and 2X) that could be extracted from single fibers and in a wide range of temperatures (10-35 degrees C) because isolated myosin is stable up to physiological temperature. The data show that, at the molecular level, the temperature sensitivity is higher at lower (10-25 degrees C) than at higher (25-35 degrees C) temperatures, consistent with experiments on isolated muscles. However, slow myosin isoforms did not show a higher temperature sensitivity than fast isoforms, contrary to what was observed in intact slow and fast muscles.     

Myosin II phosphorylation and the dynamics of stress fibers in serum-deprived and stimulated fibroblasts.

The actin-based cytomatrix generates stress fibers containing a host of proteins including actin and myosin II and whose dynamics are easily observable in living cells. We developed a dual-radioisotope-based assay of myosin II phosphorylation and applied it to serum-deprived fibroblasts treated with agents that modified the dynamic distribution of stress fibers and/or altered the phosphorylation state of myosin II. Serum-stimulation induced an immediate and sustained increase in the level of myosin II heavy chain (MHC) and 20-kDa light chain (LC20) phosphorylation over the same time course that it caused stress fiber contraction. Cytochalasin D, shown to cause stress fiber fragmentation and contraction, had little effect on myosin II phosphorylation. Okadaic acid, a protein phosphatase inhibitor, induced a delayed but massive cell shortening preceded by a large increase in MHC and LC20 phosphorylation. Staurosporine, a kinase inhibitor known to effect dissolution but not contraction of stress fibers, immediately caused an increase in MHC and LC20 phosphorylation followed within minutes by the dephosphorylation of LC20 to a level below that of untreated cells. We therefore propose that the contractility of the actin-based cytomatrix is regulated by both modulating the activity of molecular motors such as myosin II and by altering the gel structure in such a manner as to either resist or yield to the tension applied by the motors.     

Motor function and regulation of myosin X.

Myosin X is a member of the diverse myosin superfamily that is ubiquitously expressed in various mammalian tissues. Although its association with actin in cells has been shown, little is known about its biochemical and mechanoenzymatic function at the molecular level. We expressed bovine myosin X containing the entire head, neck, and coiled-coil domain and purified bovine myosin X in Sf9 cells. The Mg(2+)-ATPase activity of myosin X was significantly activated by actin with low K(ATP). The actin-activated ATPase activity was reduced at Ca(2+) concentrations above pCa 5 in which 1 mol of calmodulin light chain dissociates from the heavy chain. Myosin X translocates F-actin filaments with the velocity of 0.3 microm/s with the direction toward the barbed end. The actin translocating activity was inhibited at concentrations of Ca(2+) at pCa 6 in which no calmodulin dissociation takes place, suggesting that the calmodulin dissociation is not required for the inhibition of the motility. Unlike class V myosin, which shows a high affinity for F-actin in the presence of ATP, the K(actin) of the myosin X ATPase was much higher than that of myosin V. Consistently nearly all actin dissociated from myosin X in the presence of ATP. ADP did not significantly inhibit the actin-activated ATPase activity of myosin X, suggesting that the ADP release step is not rate-limiting. These results suggest that myosin X is a nonprocessive motor. Consistently myosin X failed to support the actin translocation at low density in an in vitro motility assay where myosin V, a processive motor, supports the actin filament movement.     

Regulation of the actin-activated ATPase and in vitro motility activities of monomeric and filamentous Acanthamoeba myosin II.

The actin-activated Mg(2+)-ATPase activity of filamentous Acanthamoeba myosin II is inhibited by phosphorylation of 3 serine residues at the tip of the tail of each heavy chain. From previous studies, it had been concluded that the activity of each molecule in the filament was regulated by the global state of phosphorylation of the filament and was independent of its own phosphorylation state. The actin-activated Mg(2+)-ATPase activity of monomeric phosphorylated myosin II was not known because it polymerizes under the ionic conditions necessary for the expression of this activity. We have now found conditions to maintain myosin II monomeric and active during the enzyme assay. The actin-activated Mg(2+)-ATPase activities of monomeric dephosphorylated and phosphorylated myosin II were found to be the same as the activity of filamentous dephosphorylated myosin II. These results support the conclusion that phosphorylation regulates filamentous myosin II by affecting filament conformation. Consistent with their equivalent enzymatic activities, monomeric and filamentous dephosphorylated myosin II were equally active in an in vitro motility assay in which myosin adsorbed to a surface drives the movement of F-actin. In contrast to their very different enzymatic activities, however, filamentous and monomeric phosphorylated myosin II had similar activities in the in vitro motility assay; both were much less active than monomeric and filamentous dephosphorylated myosin II. One interpretation of these results is that the rate-limiting steps in the two assays are different and that, while the rate-limiting step for actin-activated Mg(2+)-ATPase activity is regulated only at the level of the filament, the rate-limiting step for motility can also be regulated at the level of the monomer.     

Rho-dependent agonist-induced spatio-temporal change in myosin phosphorylation in smooth muscle cells.

Agonist-induced translocation of RhoA and the spatio-temporal change in myosin regulatory light chain (MLC20) phosphorylation in smooth muscle was clarified at the single cell level. We expressed green fluorescent protein-tagged RhoA in the differentiated tracheal smooth muscle cells and visualized the translocation of RhoA in a living cell with three-dimensional digital imaging analysis. The stimulation of the cells by carbachol initiated the translocation of green fluorescent protein-tagged wild type RhoA to the plasma membrane within a minute. The change in MLC20 phosphorylation level after carbachol stimulation was monitored by using phospho-Ser-19-specific antibody recognizing the phosphorylated MLC20 in single cells. Cells expressing the dominant negative form (T19N) of RhoA significantly suppressed sustained MLC20 phosphorylation during the prolonged phase (>300 s), whereas the maximum phosphorylation level (reached at 10 s after stimulation) of these cells was not significantly different from the control cells. The kinetics of RhoA translocation was consistent with that of sustained myosin phosphorylation, suggesting the involvement of a RhoA pathway. Carbachol stimulation increased myosin phosphorylation within a minute both at the cortical and the central region. On the other hand, during prolonged phase, myosin phosphorylation was sustained at the cortical region of the cells but not at the central fibers. A myosin light chain kinase-specific inhibitor, ML-9, diminished myosin phosphorylation at the central region of the cells after the stimulation but not at the cortical area. On the other hand, Y-27632, a Rho kinase-specific inhibitor, diminished myosin phosphorylation at the cortical region but not the central region. The results clearly show that the myosin light chain kinase pathway and the Rho pathway distinctly change myosin phosphorylation in smooth muscle cells in both a temporal and spatial manner.     

Characterization of a muscle-associated antigen from Wuchereria bancrofti.

A recombinant clone, WbN1, isolated from a genomic expression library of Wuchereria bancrofti and showing restricted specificity at the DNA level (Southern and PCR analyses) for Wuchereria bancrofti and Brugia malayi has been previously described. Sequence analysis of WbN1 indicated that it had notable similarity to myosin. Further characterization using in situ hybridization has localized the mRNA in the muscle of the adult parasite and in the microfilariae. Rabbit polyclonal antiserum, raised against the recombinant WbN1 fused to the maltose-binding protein, recognized a 200-kDa polypeptide in immunoblots containing B. malayi antigen extracts. The same antibody also recognized myosin extracted from Brugia pahangi, Onchocerca volvulus, and Caenorhabditis elegans. Localization using the rabbit antiserum revealed the presence of the antigen in the adult muscle tissue and in the microfilariae; the same antibody inhibited the binding of a monoclonal antibody 28.2 (directed toward MHC B of C. elegans myosin) to the recombinant WbN1 antigen and also to purified C. elegans myosin. Based on homology data, structural location, competitive ELISA, and immunoblot we conclude that WbN1 is related to myosin or a similar myofibrillar protein.     

Role of actin in the myosin-linked Ca(2+)-regulation of ATP-dependent interaction between actin and myosin of a lower eukaryote, Physarum polycephalum.

Actin-activated ATPase activity of myosin from Physarum polycephalum decreases when it binds Ca2+ and increases when it loses Ca2+. This Ca-inhibition is observed with phosphorylated myosin [Kohama, K. (1990) Trend, Pharmacol. Sci. 11, 433-435]. The activity of dephosphorylated myosin remained at a low level both in the presence and absence of Ca2+, although Ca(2+)-binding ability was much the same as that of the phosphorylated myosin. The effect of phosphorylation has been studied at a conventional actin concentration, which is comparable with that of myosin by weight. When the concentration of actin was increased by 10 times, the dephosphorylated myosin became actin-activatable in the absence of Ca2+, and Ca-inhibition was recovered. As actin exists quite abundantly in non-muscle cells of Physarum, myosin phosphorylation plays virtually no role in regulating actin-myosin-ATP interaction in vivo. Physiologically the interaction may be regulated by Ca2+ by binding to and subsequent release from myosin. Latex beads coated by either phosphorylated or dephosphorylated myosin moved ATP-dependently on the actin cables of Characeae cells to the same extent in the absence of Ca2+, but the movement was abolished by increasing Ca2+. When the interaction was examined by monitoring the movement of actin filaments on myosin fixed on a coverslip, the movement and Ca-inhibition of the movement were detected with phosphorylated, not dephosphorylated, myosin [Okagaki, T., Higashi-Fujime, S., & Kohama, K. (1989) J. Biochem. 106, 955-957].(ABSTRACT TRUNCATED AT 250 WORDS)     

Myosin light chain phosphatase. Effect on the activation and relaxation of gizzard smooth muscle skinned fibers

Skinned cells of chicken gizzard were used to study the effect of a smooth muscle phosphatase (SMP-IV) on activation and relaxation of tension. SMP-IV has previously been shown to dephosphorylate light chains on myosin. When this phosphatase was added to submaximally Ca2+-activated skinned cells, tension increased while phosphorylation of myosin light chains decreased. In contrast, when the myosin phosphatase was added to cell bundles activated in the absence of Ca2+ by a Ca2+-insensitive myosin light chain kinase, tension and phosphorylation of the myosin light chains both decreased. These data suggest that Ca2+ inhibits the deactivation of tension even when myosin light chains are dephosphorylated to a low level. Furthermore, comparison of Ca2+-activated cells caused to relax in CTP, in the presence or absence of Ca2+, shows that cells in the presence of Ca2+ do not relax completely, whereas in the absence of Ca2+ cells completely relax. Solutions containing Ca2+ and CTP, however, are incapable of generating tension from the resting state. Endogenous myosin light chain kinase is not active in solutions containing CTP and dephosphorylation of myosin light chains occurs in CTP solutions both in the presence and absence of Ca2+. These data imply that Ca2+ inhibits relaxation even though myosin light chains are dephosphorylated. These data are consistent with a model wherein an obligatory Ca2+-activated myosin light chain phosphorylation is followed by a second Ca2+ activation process for further tension development or maintenance.     

Effects of ATP and Actin-Filament Binding on the Dynamics of the Myosin II S1 Domain

Actin and myosin interact with one another to perform a variety of cellular functions. Central to understanding the processive motion of myosin on actin is the characterization of the individual states along the mechanochemical cycle. We present an all-atom molecular dynamics simulation of the myosin II S1 domain in the rigor state interacting with an actin filament. We also study actin-free myosin in both rigor and post-rigor conformations. Using all-atom level and coarse-grained analysis methods, we investigate the effects of myosin binding on actin, and of actin binding on myosin. In particular, we determine the domains of actin and myosin that interact strongly with one another at the actomyosin interface using a highly coarse-grained level of resolution, and we identify a number of salt bridges and hydrogen bonds at the interface of myosin and actin. Applying coarse-grained analysis, we identify differences in myosin states dependent on actin-binding, or ATP binding. Our simulations also indicate that the actin propeller twist-angle and nucleotide cleft-angles are influenced by myosin at the actomyosin interface. The torsional rigidity of the myosin-bound filament is also calculated, and is found to be increased compared to previous simulations of the free filament.     

Degradation of rat cardiac myofibrils and myofibrillar proteins by a myosin-cleaving protease.

The degradation of rat cardiac myofibrils and their constituent proteins with a myosin-cleaving protease was studied. Electrophoretograms of the digestion products of myofibrils showed that myosin,M-protein, C-protein, and troponin were degraded, but actin and tropomyosin were not. Degradation of these constituents resulted in losses of the Mg2+-ATPase activity and its Ca2+-sensitivity of myofibrils. Incubation of myofibrils with the protease induced the release of alpha-actinin without degradation. Susceptibilities of myosin, actin, troponin, and alpha-actinin purified from rat and pig hearts to the protease were essentially identical to those of the assembled forms in myofibrils. Although the purified tropomyosin was readily degraded into five fragments with the protease, the tropomyosin assembled in myofibrils and actin-tropomyosin complex were insusceptible to the protease. Digestion of myosin in the filamentous state with the protease resulted in the disappearance of myosin heavy chain and light chain 2, producing two fragments having molecular weights of 130,000 and 94,000 which originated from the degradation of heavy chain. The Ca2+- and EDTA-ATPase activities of the degradation products remained unchanged during incubation for 22 h. The actin-activated ATPase activity of myosin was reduced by 30% during incubation for 6 h, and recovered to the original level on adding actin to give a ratio of actin to myosin of 2:1. The pH optima for degradation of myosin in the soluble and filamentous states were 8.5 and 7.0, respectively. The results indicate that cardiac myosin in the filamentous state was more readily degraded with the protease than the myosin in the soluble state.     

Effects of ATP and Actin-Filament Binding on the Dynamics of the Myosin II S1 Domain

Actin and myosin interact with one another to perform a variety of cellular functions. Central to understanding the processive motion of myosin on actin is the characterization of the individual states along the mechanochemical cycle. We present an all-atom molecular dynamics simulation of the myosin II S1 domain in the rigor state interacting with an actin filament. We also study actin-free myosin in both rigor and post-rigor conformations. Using all-atom level and coarse-grained analysis methods, we investigate the effects of myosin binding on actin, and of actin binding on myosin. In particular, we determine the domains of actin and myosin that interact strongly with one another at the actomyosin interface using a highly coarse-grained level of resolution, and we identify a number of salt bridges and hydrogen bonds at the interface of myosin and actin. Applying coarse-grained analysis, we identify differences in myosin states dependent on actin-binding, or ATP binding. Our simulations also indicate that the actin propeller twist-angle and nucleotide cleft-angles are influenced by myosin at the actomyosin interface. The torsional rigidity of the myosin-bound filament is also calculated, and is found to be increased compared to previous simulations of the free filament.     

Cooperativity of thiol-modified myosin filaments. ATPase and motility assays of myosin function.

The effects of chemical modifications of myosin's reactive cysteines on actomyosin adenosine triphosphatase (ATPase) activities and sliding velocities in the in vitro motility assays were examined in this work. The three types of modifications studied were 4-[N-[(iodoacetoxy)ethyl]-N-methylamino]-7-nitrobenz-2-oxa-1,3- diazole labeling of SH2 (based on Ajtai and Burghart. 1989. Biochemistry. 28:2204-2210.), phenylmaleimide labeling of SH1, and phenylmaleimide labeling of myosin in myofibrils under rigor conditions. Each type of modified myosin inhibited the sliding of actin in motility assays. The sliding velocities of actin over copolymers of modified and unmodified myosins in the motility assay were slowest with rigor-modified myosin and most rapid with SH2-labeled myosin. The actin-activated ATPase activities of similarly copolymerized myosins were lowest with SH2-labeled myosin and highest with rigor-modified myosin. The actin-activated ATPase activities of myosin subfragment-1 obtained from these modified myosins decreased in the same linear manner with the fraction of modified heads. These results are interpreted using a model in which the sliding of actin filaments over myosin filaments decreases the probability of myosin activation by actin. The sliding velocity of actin over monomeric rigor-modified myosin exceeded that over the filamentous form, which suggests for this myosin that filament structure is important for the inhibition of actin sliding in motility assays. The fact that all cysteine modifications examined inhibited the actomyosin ATPase activities and sliding velocities of actin over myosin poses questions concerning the information about the activated crossbridge obtained from probes attached to SH1 or SH2 on myosin.     

Susceptibility of Chara myosin to SH reagents

Cellular and intracellular motile events in plants are susceptible to SH reagents such as N-ethylmaleimide (NEM). It has long been believed that the target of the reagent is myosin. We compared the effect of NEM on the motile and ATPase activities of skeletal muscle myosin with that on plant myosin using characean algal myosin. It was found that the motile activity of myosin prepared from NEM-treated C. corallina decreased to a level accountable for the decrease in the velocity of cytoplasmic streaming but it was also found that Chara myosin was far less susceptible to NEM than skeletal muscle myosin.     

The effect of mechanical stretching of the myosin rod component (fragment LMMMM S-2) on the ATPase activity of myosin.

The binding of myosin to nylon fiber gives immobilized myosin with a considerable ATPase activity. Treatment of immobilized enzyme with papain results in the entire ATPase activity (known to be concentrated in myosin heads, (fragment HMM S-1)) being replaced from the fiber into the solution; this means that myosin is chemically bound to the fiber via its rod part (fragment LMM+HMM S-2). When nylon fiber is mechanically stretched, the ATPase activity of myosin attached to it sharply decreases; after relaxation of the fiber the enzymatic activity returns to the initial level. The detailed study of this phenomenon has shown that reversible inactivation of myosin upon fiber stretching is not the result of an altered microenvironment of the enzyme. The discovered regulatory effect is ascribed to deformation of myosin molecules induced by support stretching. Thus deformation of the myosin tail (not indispensable for ATPase since its cleaving-off does not alter the enzymatic activity) leads to decrease in the ATPase activity of the enzyme. The possible role of the above phenomenon in the mechanism of muscle contraction is discussed.     

Macrophage myosin. Regulation of actin-activated ATPase, activity by phosphorylation of the 20,000-dalton light chain.

Myosin was purified from rabbit alveolar macrophages in a form that could not be activated by actin. This myosin could be phosphorylated by an endogenous myosin light chain kinase, up to 2 mol of phosphate being incorporated/mol of myosin. The site phosphorylated was located on the 20,000-dalton myosin light chain. Phosphorylation of macrophage myosin was found to be necessary for actin activation of myosin ATPase activity. Moreover, the actin-activated ATPase activity was found to vary directly with the extent of myosin phosphorylation, maximal phosphorylation (2 mol of Pi/mol of myosin) resulting in an actin-activated MgATPase activity of approximately 200 nmol of Pi/mg of myosin/min at 37 degrees C. These results establish that phosphyoyration of the 20,000-dalton light chain of myosin is sufficient to regulate the actin-activated ATPase activity of macrophage myosin.     

Rabbit cardiac myosin. II. Proteolytic fragmentation with insolubilized papain.

The substructure of the cardiac myosin molecule was examined by the limited proteolytic digestion of the parent molecule with (dialdehyde starch)-methylenedianiline-mercuripapain, S-MDA-mercuripapain, at low temperatures and neutral pH, using moderate enzyme to myosin rations. Pertinent properties of the insoluble enzyme complex were also examined. Kinetic, ultracentrifugal, and chromatographic observations of the fragmentation process revealed that a single type of lytic reaction occurs during the early stages, predominately releasing heavy meromyosin subfragment 1 (HMM-S1) and myosin rods. With further time digestion, the rods are additionally cleaved yielding light meromyosin and HMM-S2, and HMM-S1 is found to be partially degraded. The major proteolytic subfragments were isolated, purified, and characterized with respect to their enzymatic, optical, amino acid, and physicochemical properties. Only HMM-S1 exhibited Ca-2+-activated ATPase activity, and at a level three- to fourfold higher than that of native myosin. Moreover, its hydrohynamic properties suggest that it is globular in structure. On the other hand, light meromyosin-A (LMM-A) (which consists mainly of rods), and HMM-S2 appear to be highly asymmetric, rigid, alpha-helical molecules devoid of the amino acid proline. Strong similarities were evident in all aspects upon comparison of these results with documented information concerning the skeletal system. On the basis of the physical and chemical properties of the proteolytic subfragments relative to that of native myosin, it was further concluded that the cardiac myosin molecule is a double-stranded, alpha-helical rod ending in tow subfragment 1 globules, of which only one may be enzymatically active at a time.     

The modulatory effect of MgATP on heterotrimeric smooth muscle myosin phosphatase activity.

Regulation of the enzymatic activity of heterotrimeric smooth muscle myosin phosphatase (SMMP) by MgATP was examined using phosphorylated myosin (P-myosin), heavy meromyosin (P-HMM), subfragment-1 (P-S1), and 20 kDa myosin light chain (P-MLC(20)) as substrates. The activity toward P-myosin and P-HMM was dose-dependently reduced by MgATP, whereas that toward P-S1 or P-MLC(20) was unchanged. The reduction was mainly due to a decrease in the affinity of SMMP for the substrate with the unchanged maximum activity. This regulation is entirely new in the respect that the responsible molecule is the substrate, not SMMP. Because P-myosin derived from myosin stored in 50% glycerol at -20 degrees C was insensitive to MgATP, the proper integrity of P-myosin is required. Coexisting myosin did not affect this regulation, but it inhibited the SMMP activity in the absence of MgATP. With P-myosin, the enzyme activity was biphasically steeply dependent on the ionic strength. This requires that determinations are conducted with a fixed ionic strength. The Q(10) value was about 2, which was quite similar to that for myosin light chain kinase. These results suggest that the rate of dephosphorylation of P-myosin is lowered at rest, but that it may reach a value comparable to the rate of phosphorylation of myosin in the sarcoplasm with the increased level of P-myosin during muscle activation. This regulation by MgATP may underlie the "latch mechanism" in some respects.