Influence of trace amount of calponin on smooth muscle myosin in different states

The smooth muscle contraction and relaxation areprimarily regulated by the reversible Ca2 -calmodulin(CaM) dependent phosphorylation of myosin light chaincatalyzed by myosin light chain kinase (MLCK) [1–5].However, the detailed aspects of the regulation …     

Calcium regulation of porcine aortic myosin

Calcium regulation of actin-activated porcine aortic myosin MgATPase was studied. The MgATPase of the purified actomyosin was stimulated about 10-fold by 0.1 mM Ca2+. The 20,000 molecular weight light chain subunit (LC20) of myosin was phosphorylated by an endogenous kinase that required Ca2+. Half-maximal activation of both kinase and ATPase occurred at about 0.9 microM Ca2+. Phosphorylated and unphosphorylated myosins, free of actin, kinase, and phosphatase, were purified by gel filtration. The MgATPase of phosphorylated myosin was activated by rabbit skeletal muscle actin; unphosphorylated myosin was actin activated to a much lesser extent. Actin activation was maximal in the presence of Ca2+. Regulation of the aortic myosin MgATPase seems to involve both direct interaction of calcium with phosphorylated myosin and calcium activation of the myosin kinase. The MgATPase of trypsin-treated actomyosin did not require Ca2+ for full activity. The trypsin-treated actomyosin was devoid of LC20. When purified unphosphorylated aortic myosin was treated with trypsin, the LC20, was cleaved and the MgATPase, which was not appreciably actin activated before exposure to protease, was increased and was activated by skeletal muscle actin. After incubation of this light chain-depleted myosin with light chain from rabbit skeletal muscle myosin, the actin activation but not the increased activity, was abolished. Unphosphorylated LC20 seems to inhibit actin activation in this smooth muscle.     

Regulation of the actin-activated ATPase of aorta smooth muscle myosin

Phosphorylation of the 20,000-Da light chains, LC20, of vertebrate smooth muscle myosins is thought to be the primary mechanism for regulating the actin-activated ATPase activities of these myosins and consequently smooth muscle contraction. While actin stimulates the MgATPase activities of phosphorylated smooth muscle myosins, it is generally believed that the MgATPase activities of the unphosphorylated myosins are not stimulated by actin. However, under conditions where both unphosphorylated (5% phosphorylated LC20) and phosphorylated calf aorta myosins are mostly filamentous, the maximum rate, Vmax, of the actin-activated ATPase of the unphosphorylated myosin is one-half that of the phosphorylated myosin. While LC20 phosphorylation causes only a modest increase in Vmax, in the presence of tropomyosin, this phosphorylation does cause up to a 10-fold decrease in Kapp, the actin concentration required to achieve 1/2 Vmax. In the presence of low concentrations of tropomyosin/actin, a linear relationship is obtained between the fraction of LC20 phosphorylated and stimulation of the actin-activated ATPase. The relatively high actin-activated ATPase activity of unphosphorylated aorta myosin suggests that other proteins may be involved in the regulation of smooth muscle contraction. In contrast to the results presented here for aorta myosin, it has been reported that actin does not activate the MgATPase activity of unphosphorylated gizzard myosin and that the actin-activated ATPase of gizzard myosin increases more slowly than LC20 phosphorylation.     

Mechanism of regulation of cardiac actin-myosin subfragment 1 by troponin-tropomyosin

The effect of Ca2+ on the interaction of bovine cardiac myosin subfragment 1 (S-1) with actin regulated by cardiac troponin-tropomyosin was evaluated. The ratios of actin to troponin and to tropomyosin were adjusted to optimize the Ca2+-dependent regulation of the steady-state actin-activated magnesium adenosinetriphosphatase (MgATPase) rate of myosin S-1. At 25 degrees C, pH 6.9, 16 mM ionic strength, the extrapolated values for maximal adenosine 5'-triphosphate (ATP) turnover rate at saturating actin, Vmax, were 6.5 s-1 in the presence of Ca2+ and 0.24 s-1 in the absence of Ca2+. In contrast to this 27-fold regulation of ATP hydrolysis, there was negligible Ca2+-dependent regulation of cardiac myosin S-1 binding to actin. In the presence of ATP, the dissociation constant of regulated actin and cardiac myosin S-1 was 32 microM in the presence of Ca2+ and 40 microM in the presence of [ethylenebis(oxyethylenenitrilo)]tetraacetic acid. These dissociation constants are indistinguishable from the concentrations of actin needed to reach half-saturation of the myosin S-1 MgATPase rates, 37 microM actin in the presence of Ca2+ and 53 microM in its absence. Although there may be Ca2+-dependent regulation of cross-bridge binding in the intact heart, the present biochemical studies suggest that cardiac regulation critically involves other parts of the cross-bridge cycle, evidenced here by almost complete Ca2+-mediated control of the myosin S-1 MgATPase rate even when the myosin S-1 is actin-bound.     

Actin-activation of unphosphorylated gizzard myosin

The effect of light chain phosphorylation on the actin-activated ATPase activity and filament stability of gizzard smooth muscle myosin was examined under a variety of conditions. When unphosphorylated and phosphorylated gizzard myosins were monomeric, their MgATPase activities were not activated or only very slightly activated by actin, and when they were filamentous, their MgATPase activities could be stimulated by actin. At pH 7.0, the unphosphorylated myosin in the presence of ATP required 2-3 times as much Mg2+ for filament formation as did the phosphorylated myosin. The amount of stimulation of the unphosphorylated myosin filaments depended upon pH, temperature, and the presence of tropomyosin. At pH 7.0 and 37 degrees C and at pH 6.8 and 25 degrees C, the MgATPase activity of filamentous, unphosphorylated, gizzard myosin was stimulated 10-fold by actin complexed with gizzard tropomyosin. These tropomyosin-actin-activated ATPase activities were 40% of those of the phosphorylated myosin. Under other conditions, pH 7.5 and 37 degrees C and pH 7.0 and 25 degrees C, even though the unphosphorylated myosin was mostly filamentous, its MgATPase activity was stimulated only 4-fold by tropomyosin-actin. Thus, both unphosphorylated and phosphorylated gizzard myosin filaments appear to be active, but the cycling rate of the unphosphorylated myosin is less than that of the phosphorylated myosin. Active unphosphorylated myosin may help explain the ability of smooth muscles to maintain tension in the absence of myosin light chain phosphorylation.     

Filament assembly and regulation of the actin-activated ATPase activity of thymus myosin

The effects of light chain phosphorylation on the actin-activated ATPase activity and filament assembly of calf thymus cytoplasmic myosin were examined under a variety of conditions. When unphosphorylated and phosphorylated thymus myosins were monomeric, their MgATPase activities were not activated or only very slightly activated by actin, but when they were filamentous, their MgATPase activities were stimulated by actin. The phosphorylated myosin remained filamentous at lower Mg2+ concentrations and higher KC1 concentrations than did the unphosphorylated myosin, and the myosin concentration required for filament assembly was lower for phosphorylated myosin than for unphosphorylated myosin. By varying the myosin concentration, it was possible to have under the same assay conditions mostly monomeric myosin or mostly filamentous myosin; under these conditions, the actin-activated ATPase activities of the filamentous myosins were much greater than those of the monomeric myosins. The addition of phosphorylated myosin to unphosphorylated myosin promoted the assembly of unphosphorylated myosin into filaments. These results suggest that phosphorylation may regulate the actomyosin-based motile activities in vertebrate nonmuscle cells by regulating myosin filament assembly.     

ATPase activities and actin-binding properties of subfragments of Acanthamoeba myosin IA

Previous studies had led to the conclusion that the globular, single-headed myosins IA and IB from Acanthamoeba castellanii contain two actin-binding sites: one associated with the catalytic site and whose binding to F-actin activates the Mg2+-ATPase activity and a second site whose binding results in the cross-linking of actin filaments and makes the actin-activated ATPase activity positively cooperative with respect to myosin I concentration. We have now prepared a 100,000-Da NH2-terminal peptide and a 30,000-Da COOH-terminal peptide by alpha-chymotryptic digestion of the myosin IA heavy chain. The intact 17,000-Da light chain remained associated with the 100,000-Da fragment, which also contained the serine residue that must be phosphorylated for expression of actin-activated ATPase activity by native myosin IA. The 30,000-Da peptide, which contained 34% glycine and 21% proline, bound to F-actin with a KD less than 0.5 microM in the presence or absence of ATP but had no ATPase activity. The 100,000-Da peptide bound to F-actin with KD = 0.4-0.8 microM in the presence of 2 mM MgATP and KD less than 0.01 microM in the absence of MgATP. In contrast to native myosin IA, neither peptide cross-linked actin filaments. The phosphorylated 100,000-Da peptide had actin-activated ATPase activity with the same Vmax as that of native phosphorylated myosin IA but this activity displayed simple, noncooperative hyperbolic dependence on the actin concentration in contrast to the complex cooperative kinetics observed with native myosin IA. These results provide direct experimental evidence for the presence of two actin-binding sites on myosin IA, as was suggested by enzyme kinetic and filament cross-linking data, and also for the previously proposed mechanism by which monomeric myosins I could support contractile activities.     

Smooth muscle calponin. Inhibition of actomyosin MgATPase and regulation by phosphorylation

Calponin isolated from chicken gizzard smooth muscle inhibits the actin-activated MgATPase activity of smooth muscle myosin in a reconstituted system composed of contractile and regulatory proteins. ATPase inhibition is not due to inhibition of myosin phosphorylation since, at calponin concentrations sufficient to cause maximal ATPase inhibition, myosin phosphorylation was unaffected. Furthermore, calponin inhibited the actin-activated MgATPase of fully phosphorylated or thiophosphorylated myosin. Although calponin is a Ca2(+)-binding protein, inhibition did not require Ca2+. Furthermore, although calponin also binds to tropomyosin, ATPase inhibition was not dependent on the presence of tropomyosin. Calponin was phosphorylated in vitro by protein kinase C and Ca2+/calmodulin-dependent protein kinase II, but not by cAMP- or cGMP-dependent protein kinases, or myosin light chain kinase. Phosphorylation of calponin by either kinase resulted in loss of its ability to inhibit the actomyosin ATPase. The phosphorylated protein retained calmodulin and tropomyosin binding capabilities, but actin binding was greatly reduced. The calponin-actin interaction, therefore, appears to be responsible for inhibition of the actomyosin ATPase. These observations suggest that calponin may be involved in regulating actin-myosin interaction and, therefore, the contractile state of smooth muscle. Calponin function in turn is regulated by Ca2(+)-dependent phosphorylation.     

Effects of phosphorylated and unphosphorylated C-protein on cardiac actomyosin ATPase

C-protein, a component of the thick filaments of striated muscles, is reversibly phosphorylated and dephosphorylated in heart. It has been hypothesized that C-protein may be involved in regulating contraction, because the extent of C-protein phosphorylation correlates with the rate of cardiac relaxation. To test this hypothesis, the effects of phosphorylated and unphosphorylated C-protein on the actin-activated ATPase activity of myosin filaments prepared from DEAE-Sephadex-purified myosin were examined. Unphosphorylated C-protein (0.1 microM to 1.5 microM) stimulated actin-activated myosin ATPase activity in a dose-dependent manner. With a myosin: C-protein molar ratio of approximately 1, actin-activated myosin ATPase activity was elevated up to 3.2 times that of the control. Phosphorylated C-protein (2.5 mol PO4/mol C-protein) stimulated the activity somewhat less (2.5 times that of control). The stimulation of ATPase activity by C-protein was due to an increase in the Vmax value (from 0.25/second to 0.62/second) and a decrease in the Km value (from 11.9 microM to 6.7 microM). The addition of C-protein to actomyosin solutions produced an increase in the light-scattering of the actomyosin solution and a distinct precipitation of the actomyosin with time. Phosphorylated C-protein had a smaller effect on light-scattering than dephosphorylated C-protein. C-protein had a negligible effect on Ca-ATPase, EDTA-K-ATPase, or Mg-ATPase activities in the absence of actin. C-protein had only small effects on the actin-activated ATPase of heavy meromyosin. These results suggest that C-protein stimulates actin-activated myosin ATPase activity by enhancing the formation of stable aggregates between actin and myosin filaments.     

The effects of caldesmon on the ATPase activities of rabbit skeletal-muscle myosin.

We studied the effects of caldesmon, a major actin- and calmodulin-binding protein found in a variety of muscle and non-muscle tissues, on the various ATPase activities of skeletal-muscle myosin. Caldesmon inhibited the actin-activated myosin Mg2+-ATPase, and this inhibition was enhanced by tropomyosin. In the presence of the troponin complex and tropomyosin, caldesmon inhibited the Ca2+-dependent actomyosin Mg2+-ATPase; this inhibition could be partly overcome by Ca2+/calmodulin. Caldesmon, phosphorylated to the extent of approximately 4 mol of Pi/mol of caldesmon, inhibited the actin-activated myosin Mg2+-ATPase to the same extent as did non-phosphorylated caldesmon. Both inhibitions could be overcome by Ca2+/calmodulin. Caldesmon also inhibited the Mg2+-ATPase activity of skeletal-muscle myosin in the absence of actin; this inhibition also could be overcome by Ca2+/calmodulin. Caldesmon inhibited the Ca2+-ATPase activity of skeletal-muscle myosin in the presence or absence of actin, at both low (0.1 M-KCl) and high (0.3 M-KCl) ionic strength. Finally, caldesmon inhibited the skeletal-muscle myosin K+/EDTA-ATPase at 0.1 M-KCl, but not at 0.3 M-KCl. Addition of actin resulted in no inhibition of this ATPase by caldesmon at either 0.1 M- or 0.3 M-KCl. These observations suggest that caldesmon may function in the regulation of actin-myosin interactions in striated muscle and thereby modulate the contractile state of the muscle. The demonstration that caldesmon inhibits a variety of myosin ATPase activities in the absence of actin indicates a direct effect of caldesmon on myosin. The inhibition of the actin-activated Mg2+-ATPase activity of myosin (the physiological activity) may not be due therefore simply to the binding of caldesmon to the actin filament causing blockage of myosin-cross-bridge-actin interaction.     

Locking regulatory myosin in the off-state with trifluoperazine

Scallop striated adductor muscle myosin is a regulatory myosin, its activity being controlled directly through calcium binding. Here, we show that millimolar concentrations of trifluoperazine were effective at removal of all regulatory light chains from scallop myosin or myofibrils. More important, 200 microM trifluoperazine, a concentration 10-fold less than that required for light-chain removal, resulted in the reversible elimination of actin-activated and intrinsic ATPase activities. Unlike desensitization induced by metal ion chelation, which leads to an elevation of activity in the absence of calcium concurrent with regulatory light-chain removal, trifluoperazine caused a decline in actin-activated MgATPase activity both in the presence and absence of calcium. Procedures were equally effective with respect to scallop myosin, myofibrils, subfragment-1, or desensitized myofibrils. Increased alpha-helicity could be induced in the isolated essential light chain through addition of 100-200 microM trifluoperazine. We propose that micromolar concentrations of trifluoperazine disrupt regulation by binding to a single high-affinity site located in the C-terminal domain of the essential light chain, which locks scallop myosin in a conformation resembling the off-state. At millimolar trifluoperazine concentrations, additional binding sites on both light chains would be filled, leading to regulatory light-chain displacement.     

The binding of smooth muscle heavy meromyosin to actin in the presence of ATP. Effect of phosphorylation

Phosphorylation of the 20,000-dalton light chains of smooth muscle heavy meromyosin (HMM) from turkey gizzards results in a large increase in the actin-activated MgATPase activity over that observed with unphosphorylated HMM. In an attempt to define which step in the kinetic cycle is affected by phosphorylation, we have measured the binding of both unphosphorylated and phosphorylated HMM to actin in the presence of ATP using sedimentation. There was only a 4-fold difference in the actin binding constants of unphosphorylated HMM (5.35 x 10(3) M-1) and fully phosphorylated HMM (2.35 x 10(4) M-1). In contrast, the maximum rate of the actin-activated MgATPase activity (Vmax) of phosphorylated HMM was 25 times greater than that for unphosphorylated HMM. These data rule out a mechanism whereby the unphosphorylated light chain of myosin regulates actin-myosin interaction by directly or indirectly blocking the binding of HMM to actin. This implies that some step in the kinetic cycle other than the binding of HMM to actin must be regulated. We have also measured the rate constant for ATP hydrolysis (the initial phosphate burst) under the same conditions and found that this step was very fast compared to the steady state ATPase rate and was unaffected by phosphorylation. This suggests that the step which is regulated by phosphorylation is either phosphate release or a step preceding phosphate release but following ATP hydrolysis.     

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.     

Reversible phosphorylation of smooth muscle myosin, heavy meromyosin, and platelet myosin

Smooth muscle myosin was purified from turkey gizzards with the 20,000-dalton light chains in the unphosphorylated state. The actin-activated MgATPase activity was 4 nmol/min/mg at 25 degrees C. When the myosin was phosphorylated to 2 mol of Pi/mol of myosin using purified myosin light chain kinase, calmodulin, and ATP, the actin-activated MgATPase activity rose to 51 nmol/min/mg. Complete dephosphorylation of the same myosin by a purified phosphatase lowered the activity to 5 nmol/min/mg, and complete rephosphorylation of the myosin following inhibition of the phosphatase raised it again to 46 nmol/min/mg. Human platelet myosin could be substituted for turkey gizzard myosin, with similar results. A chymotryptic fragment of smooth muscle myosin which retains the phosphorylated site on the 20,000-dalton light chain of myosin was prepared. Using the same scheme for reversible phosphorylation, this smooth muscle heavy meromyosin was found to show the same positive correlation between phosphorylation of the myosin light chain and the actin-activated MgATPase activity. The results with smooth muscle heavy meromyosin show that the effect of phosphorylation on the actin-activated MgATPase activity can be separated from the effects of phosphorylation on myosin filament assembly.     

Effect of ADP and ionic strength on the kinetic and motile properties of recombinant mouse myosin V

Mouse myosin V is a two-headed unconventional myosin with an extended neck that binds six calmodulins. Double-headed (heavy meromyosin-like) and single-headed (subfragment 1-like) fragments of mouse myosin V were expressed in Sf9 cells, and intact myosin V was purified from mouse brain. The actin-activated MgATPase of the tissue-purified myosin V, and its expressed fragments had a high V(max) and a low K(ATPase). Calcium regulated the MgATPase of intact myosin V but not of the fragments. Both the MgATPase activity and the in vitro motility were remarkably insensitive to ionic strength. Myosin V and its fragments translocated actin at very low myosin surface densities. ADP markedly inhibited the actin-activated MgATPase activity and the in vitro motility. ADP dissociated from myosin V subfragment 1 at a rate of about 11.5 s(-1) under conditions where the V(max) was 3.3 s(-1), indicating that, although not totally rate-limiting, ADP dissociation was close to the rate-limiting step. The high affinity for actin and the slow rate of ADP release helps the myosin head to remain attached to actin for a large fraction of each ATPase cycle and allows actin filaments to be moved by only a few myosin V molecules in vitro.     

Effect of multiple phosphorylations of smooth muscle and cytoplasmic myosins on movement in an in vitro motility assay

The Nitella-based in vitro motility assay developed by Sheetz and Spudich (Sheetz, M.P., and Spudich, J. A. (1983) Nature 303, 31-35) is a quantitative assay for measuring the velocity of myosin-coated beads over an organized substratum of actin. We have used this assay to analyze the effect of phosphorylation of various sites on the 20,000-Da light chain of smooth muscle and cytoplasmic myosins. Phosphorylation by myosin light chain kinase at serine 19 on the 20,000-Da light chain subunit of smooth muscle myosin from turkey gizzard, bovine trachea and aorta, and of cytoplasmic myosin from human platelets was required for bead movement. The individual phosphorylated myosin-coated beads moved at characteristic rates under the same conditions (turkey gizzard myosin, 0.2 micron/s; aorta or trachea myosin, 0.12 micron/s; and platelet myosin, 0.04 micron/s; in contrast, rabbit skeletal muscle myosin, 2 micron/s). Myosin light chain kinase can also phosphorylate threonine 18 in addition to serine 19, and this phosphorylation resulted in an increase in the actin-activated MgATPase activity (Ikebe, M., and Hartshorne, D.J. (1985) J. Biol. Chem. 260, 10027-10031). Phosphorylation at this site had no effect on the velocity of smooth muscle myosin-coated beads. Protein kinase C (Ca2+/phospholipid-dependent enzyme) can also phosphorylate two to three sites on the 20,000-Da light chain, and this phosphorylation alone did not result in the movement of myosin-coated beads. When myosin that had been previously phosphorylated by myosin light chain kinase at serine 19 was also phosphorylated by protein kinase C, myosin-coated beads moved at the same velocity as beads coated with myosin phosphorylated by myosin light chain kinase alone. Tropomyosin binding to actin also had an activating effect on the actin-activated MgATPase activity through an effect on the Vmax and also resulted in an increase in the velocity of myosin-coated beads.     

Phosphorylation-dependent regulation of Limulus myosin

Myosin from Limulus, the horseshoe crab, is shown to be regulated by a calcium-calmodulin-dependent phosphorylation of its regulatory light chains. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of a Limulus myosin preparation reveals three light chain bands. Two of these light chains have been termed regulatory light chains based on their ability to bind to light chain-denuded scallop myofibrils (Sellers, J. R., Chantler, P. D., and Szent-Gy?rgyi, A. G. (1980) J. Mol. Biol. 144, 223-245). Ths other light chain does not bind to these myofibrils and is thus termed the essential light chain. Both Limulus regulatory light chains can be phosphorylated with a highly purified turkey gizzard myosin light chain kinase or with a partially purified myosin light chain kinase which can be isolated from Limulus muscle by affinity chromatography on a calmodulin-Sepharose column. Phosphorylation with both of these enzymes requires calcium and calmodulin. Limulus myosin is isolated in an unphosphorylated form. The MgATPase of this unphosphorylated myosin is only slightly activated by rabbit skeletal muscle actin plus tropomyosin. The calcium-dependent phosphorylation of the myosin results in an increase in the actin-activated MgATPase rate. Once phosphorylated, the actin-activated MgATPase rate is only slightly modified by calcium. This suggests that calcium operates mainly at the level of the myosin kinase-calmodulin system.     

Two new modes of smooth muscle myosin regulation by the interaction between the two regulatory light chains, and by the S2 domain

Previous studies indicated that single-headed smooth muscle myosin and S1 (a single head fragment) are not regulated through phosphorylation of the regulatory light chain (RLC). To investigate the importance of the double-headedness of myosin and of the S2 region for the phosphorylation-dependent regulation, we made three types of recombinant mutant smooth muscle HMMs with one intact head and an N-terminally truncated head. The truncated head of Delta MD lacked the motor domain, that of Delta(MD+ELC) lacked the motor and essential light chain binding domains, and single-headed HMM had one intact head alone. The basal ATPase activities of the three mutants decreased as the KCl concentration became less than 0.1 M. Such a decrease was not observed for S1, which had no S2 region, suggesting that S2 is necessary for this myosin behavior. This activity decrease also disappeared when RLCs of Delta MD and Delta(MD+ELC), but that of single-headed HMM, were phosphorylated. When their RLCs were unphosphorylated, the three mutants exhibited similar actin-activated ATPase levels. However, when they were phosphorylated, the actin-activated ATPase activities of Delta MD and Delta(MD+ELC) increased to the S1 level, while that of single-headed HMM remained unchanged. Even in the phosphorylated state, the actin-activated ATPase activities of the three mutants and S1 were much lower than that of wild-type HMM. We propose that S2 has an inhibitory function that is canceled by an interaction between two phosphorylated RLCs. We also propose that a cooperative interaction between two motor domains is required for a higher level of actin activation.     

A conserved negatively charged amino acid modulates function in human nonmuscle myosin IIA

A myosin surface loop (amino acids 391-404) is postulated to be an important actin binding site. In human beta-cardiac myosin, mutation of arginine-403 to a glutamine or a tryptophan causes hypertrophic cardiomyopathy. There is a phosphorylatable serine or threonine residue present on this loop in some lower eukaryotic myosin class I and myosin class VI molecules. Phosphorylation of the myosin I molecules at this site regulates their enzymatic activity. In almost all other myosins, the homologous residue is either a glutamine or an aspartate, suggesting that a negative charge at this location is important for activity. To study the function of this loop, we have used site-directed mutagenesis and baculovirus expression of a heavy meromyosin- (HMM-) like fragment of human nonmuscle myosin IIA. An R393Q mutation (equivalent to the R403Q mutation in human beta-cardiac muscle myosin) has essentially no effect on the actin-activated MgATPase or in vitro motility of the expressed HMM-like fragment. Three mutations, D399K, D399A, and a deletion mutation that removes residues 393-402, all decrease both the V(max) of the actin-activated MgATPase by 8-10-fold and the rate of in vitro motility by a factor of 2-3. The K(ATPase) of the actin-activated MgATPase activity and the affinity constant for binding of HMM to actin in the presence of ADP are affected by less than a factor of 2. These data support an important role for the negative charge at this location but show that it is not critical to enzymatic activity.     

The inhibitory Ca2+-regulation of the actin-activated Mg-ATPase activity of myosin from Physarum polycephalum plasmodia

Myosin was rapidly prepared from the slime mould, Physarum polycephalum to a high level of homogeneity (greater than 95%), in a high yield (about 10 mg/100 g tissue) and in a phosphorylated state (about 5 mol phosphate/mol of 500,000 Mr myosin). Actin activated the Mg-ATPase activity of this myosin in the absence of Ca2+ about 30-fold, and this actin-activated ATPase activity was reduced to about 20% of the original activity when Ca2+ concentration was increased to 50 microM, i.e., the actin-myosin-ATP interactions show Ca-inhibition. The Ca2+ concentration giving half-maximum inhibition was 1-3 microM. The Ca-inhibition was clearly observed at physiological concentrations of Mg2+ but was obscured at both lower and higher concentrations of Mg2+. The Ca-inhibitory effect on ATP hydrolysis by actomyosin reconstituted from skeletal actin and Physarum myosin was quick and reversible. Ca-binding measurement showed that myosin bound Ca2+ with half-maximal binding at 2 microM Ca2+ and maximum binding of 2 mol per mol myosin, indicating that Ca2+ may inhibit the ATPase activity by binding to myosin. The involvement of this myosin-linked regulatory system in the Ca2+ -control of cytoplasmic streaming is discussed.