Effects of phosphorylation, magnesium, and filament assembly on actin-activated ATPase of pig urinary bladder myosin

The relationship between the light-chain phosphorylation and the actin-activated ATPase activity of pig urinary bladder myosin was either linear or nonlinear depending on the free Mg2+ concentration. Varying the free [Mg2+] in the presence of 50 mM ionic strength (I) had a biphasic effect on the actin-activated ATPase. In 100 mM I, the activity increased on raising the free [Mg2+]. The activity of the phosphorylated myosin was 3-23-fold higher than that of the unphosphorylated myosin at all concentrations of free Mg2+, pH, and temperature used in this study. The increase in the turbidity and sedimentability of both phosphorylated and unphosphorylated myosins on raising the free [Mg2+] was associated with a rise in the actin-activated ATPase activity. However, myosin light-chain phosphorylation still had a remarkable effect on the actin activation. The myosin polymers formed under these conditions were sedimented by centrifugation. Experiments performed with myosin polymers formed in mixtures of unphosphorylated and phosphorylated myosins showed that the presence of phosphorylated myosin in these mixtures had a slight effect on the sedimentation of the unphosphorylated myosin but it had no effect on the actin-activated ATP hydrolysis. Electron microscopy showed that the unphosphorylated myosin formed unorganized aggregates while phosphorylated myosin molecules assembled into bipolar filaments with tapered ends. These data show that although the unphosphorylated and phosphorylated myosins have the same level of sedimentability and turbidity, the filament assembly present only with the phosphorylated myosin can be associated with the maximal actin activation of Mg-ATPase.     

Nonlinear dependence of actin-activated Mg2+-ATPase activity on the extent of phosphorylation of gizzard myosin and H-meromyosin

1. The actin-activated Mg2+-ATPase activity of gizzard HMM increased in proportion to the square of the extent of LC phosphorylation. This result indicates that the LCs of HMM are randomly phosphorylated, and the phosphorylation of both heads of HMM is required for the activation of HMM Mg2+-ATPase by F-actin. 2. In 75 mM KCl, the Mg2+-ATPase activity of gizzard myosin was activated by F-actin only slightly when a half of the total LC was phosphorylated. From 1 to 2 mol LC phosphorylation, the activity was enhanced by F-actin almost linearly. In 30 mM KCl, the activity of acto-gizzard myosin increased sigmoidally with increase in the extent of LC phosphorylation. On electron microscopy, side-by-side aggregates of myosin filaments were observed in 30 mM KCl, but not in 75 mM KCl. It was suggested that the activation of the Mg2+-ATPase activity of acto-gizzard myosin LC phosphorylation is modified by formation of myosin filaments and their aggregates. 3. The relationship between the actin-activated Mg2+-ATPase activity of HMM or myosin and the extent of LC phosphorylation was unaffected by tropomyosin.     

Phosphorylation of thymus myosin increases its apparent affinity for actin but not its maximum adenosinetriphosphatase rate

Vertebrate nonmuscle myosins contain two phosphorylatable light chains. The maximum rate, Vmax, of the actin-activated adenosinetriphosphatase (ATPase) of unphosphorylated calf thymus myosin was found to be about 100 nmol/(min X mg), the same as that of thymus myosin with two phosphorylated light chains. However, the Kapp (actin concentration required to achieve 1/2 Vmax) of the unphosphorylated myosin was 15-20-fold greater than that of the phosphorylated myosin. When actin complexed with either skeletal muscle tropomyosin or calf thymus tropomyosin was used, the values for Vmax were about the same as those obtained with F-actin. In the presence of skeletal muscle tropomyosin, the Kapp of the unphosphorylated myosin was only 2-3-fold greater than that of the phosphorylated myosin, and in the presence of thymus tropomyosin, there was about a 5-fold difference in their Kapp values. Thus, light chain phosphorylation regulates the actin-activated ATPase of thymus myosin not by increasing Vmax but rather by decreasing the Kapp of this myosin for actin. These rather small differences in Kapp suggest that other proteins may be involved in the regulation of the actin-activated ATPase of thymus myosin. Regulated actin (actin plus skeletal muscle troponin-tropomyosin) was used to examine possible effects of thin-filament regulatory proteins. In the presence of calcium, phosphorylation caused only a slight increase in Vmax and a 2-fold decrease in Kapp of the regulated actin-activated ATPase of thymus myosin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chicken gizzard heavy meromyosin that retains the two light-chain components, including a phosphorylatable one.

A method was developed to obtain a preparation of chicken gizzard heavy meromyosin (HMM) that retains the two light-chain components of parent myosin: the 20,000-dalton and 17,000-dalton light-chains. The HMM preparation was also shown to retain two characteristics of the ATPase activity of the parent myosin: the characteristic effect of phosphorylation of the 20,000-dalton light-chain component on the ATPase activity, and the characteristic dependence of the ATPase activity on the KCl concentration. 1. Two distinct stages were observed in the Mg-ATPase reaction catalyzed by gizzard HMM and rabbit skeletal actin in the presence of gizzard "native" tropomyosin (NTM) and Ca2+ ions: an early lag phase, in which the reaction rate gradually increased, and a subsequent steady state, in which the reaction proceeded at a high, constant rate. Urea-gel electrophoresis revealed that the 20,000-dalton light-chain component was gradually phosphorylated in the lag phase, and was fully phosphorylated in the steady state. It was also observed that addition of EGTA (to remove Ca2+ ions) at various times in the lag phase caused neither a further increase nor a decrease in the reaction rate, and that addition of EGTA in the steady state caused no change in the reaction rate. These observations imply that the ATPase activity increased as the amount of phosphorylated 20,000-dalton light-chain component increased, and also that Mg-ATPase of acto-phosphorylated HMM was no longer calcium-sensitive. 2. The Mg-ATPase activity of HMM in the presence of gizzard NTM and Ca2+ ions or EGTA was studied as a function of the concentration of rabbit skeletal actin. The maximal activity (Vmax) and the apparent affinity constant of acto-HMM (KA) were thus estimated from the double-reciprocal plot of Eisenberg-Moos: the Vmax and KA values for phosphorylated HMM (in the presence of Ca2+ ions) were 5 S(-1) and 5.5 mg/ml actin, respectively, and the Vmax value for unphosphorylated HMM (in the presence of EGTA) was 0.3 S(-1), assuming that the KA value with unphosphorylated HMM is equal to that with phosphorylated HMM.     

Phosphorylation of smooth muscle heavy meromyosin by calcium-activated, phospholipid-dependent protein kinase. The effect on actin-activated MgATPase activity

Smooth muscle heavy meromyosin (HMM) can serve as a substrate for the Ca2+-activated, phospholipid-dependent protein kinase (protein kinase C) as well as for the Ca2+/calmodulin-dependent kinase, myosin light chain kinase. When turkey gizzard HMM is incubated with protein kinase C, 1.7-2.2 mol of phosphate are incorporated per mol of HMM, all of it into the 20,000-Da light chain of HMM. Two-dimensional peptide mapping following tryptic hydrolysis revealed that protein kinase C phosphorylated a different site on the 20,000-Da HMM light chain than did myosin light chain kinase. Moreover, sequential phosphorylation of HMM by myosin light chain kinase and protein kinase C resulted in the incorporation of 4 mol of phosphate/mol of HMM, i.e. 2 mol of phosphate into each 20,000-Da light chain. When unphosphorylated HMM was phosphorylated by myosin light chain kinase, its actin-activated MgATPase activity increased from 4 nmol to 156 nmol of phosphate released/mg of HMM/min. Subsequent phosphorylation of this phosphorylated HMM by protein kinase C decreased the actin-activated MgATPase activity of HMM to 75 nmol of phosphate released/mg of HMM/min.     

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.     

Functional consequences of the proteolytic removal of regulatory serines from the nonhelical tailpiece of Acanthamoeba myosin II

The actin-activated Mg2(+)-ATPase activity of myosin II from Acanthamoeba castellanii is regulated by phosphorylation of 3 serines in its 29-residue, nonhelical, COOH-terminal tailpiece, i.e., serines-1489, -1494, and -1499 or, in reverse order, residues 11, 16, and 21 from the COOH terminus. To investigate the essential requirements for regulation, myosin II filaments in the presence of F-actin were digested by arginine-specific submaxillary gland protease. Two-dimensional peptide mapping of purified, cleaved myosin II showed that the two most terminal phosphorylation sites, serines-1494 and -1499, had been removed. Cleaved dephosphorylated myosin II retained full actin-activated Mg2(+)-ATPase activity (with no change in Vmax or Kapp) and the ability to form filaments similar to those of the native enzyme. However, higher Mg2+ concentrations were required for both filament formation and maximal ATPase activity. The one remaining regulatory serine in the cleaved myosin II was phosphorylatable by myosin II heavy-chain kinase, and phosphorylation inactivated the actin-activated Mg2(+)-ATPase activity, as in the case of the native myosin II. Also as in the case of the native myosin II, phosphorylated cleaved myosin II inhibited the actin-activated Mg2(+)-ATPase activity of dephosphorylated cleaved myosin II when the two were copolymerized. These results suggest that at least 18 of the 29 residues in the nonhelical tailpiece of the heavy chain are not required for either actin-activated Mg2(+)-ATPase activity or filament formation and that phosphorylation of Ser-1489 is sufficient to regulate the actin-activated Mg2(+)-ATPase activity of myosin II.     

Purification and characterization of a myosin heavy chain kinase from Dictyostelium discoideum

A Dictyostelium discoideum myosin heavy chain kinase has been purified 14,000-fold to near homogeneity. The enzyme has a Mr = 130,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and greater than 700,000 as determined by gel filtration on Bio-Gel A-1.5m. The enzyme has a specific activity of 1 mumol/min X mg when assayed at a Dictyostelium myosin concentration of 0.3 mg/ml. A maximum of 2 mol of phosphate/mol of myosin is incorporated by the kinase, and the phosphorylated amino acid is threonine. Phosphate is incorporated only into the myosin heavy chains, not into the light chains. The actin-activated Mg2+-ATPase of Dictyostelium myosin is inhibited 70-80% following maximal phosphorylation with the kinase. The myosin heavy chain kinase requires 1-2 mM Mg2+ for activity and is most active at pH 7.0-7.5. The activity of the enzyme is not significantly altered by the presence of Ca2+, Ca2+ and calmodulin, EGTA, cAMP, or cGMP. When incubated with Mg2+ and ATP, phosphate is incorporated into the myosin heavy chain kinase, perhaps by autophosphorylation.     

Inhibition of conformational change in smooth muscle myosin by a monoclonal antibody against the 17-kDa light chain

Monoclonal antibodies against gizzard smooth muscle myosin were generated and characterized. One of these antibodies, designated MM-2, recognized the 17-kDa light chain and modulated the ATPase activities and hydrodynamic properties of smooth muscle myosin. Rotary shadowing electron microscopy showed that MM-2 binds 51 (+/- 25) A from the head-rod junction. The depression of Ca2+- and Mg2+-ATPase activities of myosin and Ca2+-ATPase activity of heavy meromyosin at low KCl concentration were abolished by MM-2. Viscosity measurement indicated that MM-2 inhibits the transition of 6 S myosin to 10 S myosin. While the rate of the production of subfragment-1 by papain proteolysis of 6 S myosin was inhibited by MM-2, the rate of proteolysis of the heavy chain of 10 S myosin was enhanced by MM-2 and reached the same rate as that of 6 S myosin plus MM-2. These results suggest that MM-2 inhibits the formation of 10 S myosin by binding to the 17-kDa light chain which is localized at the head-neck region of the myosin molecule. MM-2 increased the Vmax of actin-activated Mg2+-ATPase activities of both dephosphorylated myosin and dephosphorylated heavy meromyosin about 10- and 20-fold, respectively. MM-2 also activated the actin-activated Mg2+-ATPase activity of phosphorylated myosin at a low MgCl2 concentration and thus abolished the Mg2+-dependence of acto phosphorylated myosin ATPase activity. These results suggest that MM-2 inhibits the formation of 10 S myosin, and this results in the activation of actin-activated Mg2+-ATPase activity even in the absence of phosphorylation.     

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.     

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.     

Cooperative dependence of the actin-activated Mg2+-ATPase activity of Acanthamoeba myosin II on the extent of filament phosphorylation

The actin-activated Mg2+-ATPase of myosin II from Acanthamoeba castellanii is regulated by phosphorylation of 3 serine residues at the tip of the tail of each of its two heavy chains; only dephosphorylated myosin II is active, whereas the phosphorylated and dephosphorylated forms have identical Ca2+-ATPase activities and Mg2+-ATPase activities in the absence of F-actin. We have now chemically modified phosphorylated and dephosphorylated myosin II with N-ethylmaleimide (NEM). The modification occurred principally at a single site within the NH2-terminal 73,000 Da of the globular head of the heavy chain. NEM-myosin II bound to F-actin and formed filaments normally, but the Ca2+- and Mg2+-ATPase activities of phosphorylated and dephosphorylated myosin II and the actin-activated Mg2+-ATPase activity of NEM-dephosphorylated myosin II were inhibited. Only filamentous myosin II has actin-activated Mg2+-ATPase activity. Native phosphorylated myosin II acquired actin-activated Mg2+-ATPase activity when it was co-polymerized with NEM-inactivated dephosphorylated myosin II, and the increase in its activity was cooperatively dependent on the fraction of NEM-dephosphorylated myosin II in the filaments. From this result, we conclude that the specific activity of each molecule within a filament is independent of its own state of phosphorylation, but is highly cooperatively dependent upon the state of phosphorylation of the filament as a whole. This enables the actin-activated Mg2+-ATPase activity of myosin II filaments to respond rapidly and extensively to small changes in the level of their phosphorylation.     

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.     

Increase in the actin-activated Mg2+-ATPase activity of smooth muscle myosin by increasing myosin concentration

The actin-activated Mg2+-ATPase activity of smooth muscle myosin was measured in 85 mM KCl, 6 mM MgCl2 in the absence of tropomyosin. The activity was dependent on myosin concentration. Vmax increased as myosin concentration was increased, while the Ka (the apparent dissociation constant for actin) remained the same. The extent of filament formation was also correlated with myosin concentration and most of the myosin monomers existed in 10S conformation. These results suggest that myosin concentration influences the actin-activated Mg2+-ATPase activity by changing the 10S-6S-filaments equilibrium.     

The phosphorylated L2 light chain of skeletal myosin is a modifier of the actomyosin ATPase

Incubation of rabbit skeletal myosin with an extract of light chain kinase plus ATP phosphorylated the L2 light chain and modified the steady state kinetics of the actomyosin ATPase. With regulated actin, the ATPase activity of phosphorylated myosin (P-myosin) was 35 to 181% greater than that of unphosphorylated myosin when assayed with 0.05 to 5 micro M Ca2+. Phosphorylation had no effect on the Ca2+ concentration required for half-maximal activity, but it did increase the ATPase activity at low Ca2+. With pure actin, the percentage of increase in the actomyosin ATPase activity correlated with the percentage of phosphorylation of myosin. Steady state kinetic analyses of the actomyosin system indicated that 50 to 82% phosphorylation of myosin decreased significantly the Kapp of actin for myosin with no significant effect on the Vmax. Phosphorylaton of heavy meromyosin similarly modified the steady state kinetics of the acto-heavy meromyosin system. Both the K+/EDTA- and Mg-ATPase activities of P-myosin and phosphorylated heavy meromyosin were within normal limits indicating that phosphorylaiion had not altered significantly the hydrolytic site. Phosphatase treatment of P-myosin decreased both the level of phosphorylation of L2 and the actomyosin ATPase activity to control levels for unphosphorylated myosin. It is concluded levels for unphosphorylated myosin. It is concluded from these results that the ability of P-myosin to modify the steady state kinetics of the actomyosin ATPase was: 1) specific for phosphorylation; 2) independent of the thin filament regulatory proteins.     

The effect of cleavage at site 1 of gizzard HMM in the interaction with skeletal muscle actin

Two different classes of gizzard heavy meromyosins (HMMs) were prepared from phosphorylated myosin by chymotryptic digestion in the presence and absence of ATP and were compared with respect to their actin-activated Mg2+-ATPase reactions. One class of HMM, named HMM(+), had a cleavage at site 1 in the N terminal portion of the heavy chain and the other class of HMM, named HMM(-), had no cleavage at this site. Maximum turnover rate (Vmax) of the skeletal acto-gizzard HMM Mg2+-ATPase reaction was obviously different between HMM(+) and HMM(-). The Vmax value of HMM(+) was 2.5-fold larger than that of HMM(-). On the other hand, the apparent association constants (Ka) of skeletal muscle actin for both HMMs which were deduced from double reciprocal plots (v-1 versus [actin]-1) seemed to be identical. The difference in Vmax value was attributed to the cleavage at site 1 since a following chymotryptic cleavage of HMM(-) at site 1 caused a 2.5-fold increase in the Vmax value. That site 1 in the N terminal portion of the gizzard myosin heavy chain was the key locus for the actin-myosin interaction was shown in addition to our previous finding of the effects of cleavage at site 1 on the ATPase activity and nucleotide binding ability of gizzard HMM (Okamoto, Y. & Sekine, T. (1981) J. Biochem. 90, 833-843; 843-949).     

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.     

Effects of phosphorylation of light chain residues threonine 18 and serine 19 on the properties and conformation of smooth muscle myosin

Smooth muscle myosin can be phosphorylated by myosin light chain kinase at the serine 19 and threonine 18 residues of the two 20,000-dalton light chains (Ikebe, M., Hartshorne, D. J., and Elizinga, M. (1986) J. Biol. Chem. 261, 36-39). These studies with myosin and heavy meromyosin (HMM) compare the effects induced by phosphorylation of serine 19 (M2P and HMM2P) and serine 19 plus threonine 18 (M4P and HMM4P). Formation of M4P altered the KCl dependence of viscosity and Mg2+-ATPase and higher values were maintained at lower ionic strengths, compared to M2P or dephosphorylated myosin (Mo). This is consistent with the stabilization of the 6 S conformation. The tendency for aggregation, as judged by light scattering, followed the sequence M4P greater than M2P greater than Mo. Filaments formed with M4P were more resistant to dissociation by ATP compared to filaments of M2P. Phosphorylation of HMM2P doubled Vmax of actin-activated ATPase with little effect on the apparent affinity for actin. The Mg2+-ATPase of HMM4P exhibited a higher activity at low ionic strength compared to HMM2P and HMMo. Hydrodynamic differences were detected at low ionic strength in the presence of ATP by sedimentation velocity measurements with HMM4P, HMM2P, and HMMo. Proteolysis by papain indicated an increased susceptibility of the head-neck junction of HMM4P compared to HMM2P. These data suggest that the phosphorylation of threonine 18 in addition to serine 19 change the conformation of myosin and HMM and this is associated with altered biological properties.     

Identification of three phosphorylation sites on each heavy chain of Acanthamoeba myosin II

It has been previously demonstrated that the actin-activated Mg2+-ATPase activity of Acanthamoeba myosin II is inhibited by phosphorylation of its two heavy chains (Collins, J. H., and Korn, E. D. (1980) J. Biol. Chem. 255, 8011-8014). In this paper, it is shown that a partially purified kinase preparation from Acanthamoeba catalyzes the incorporation of 3 mol of phosphate into each mole of myosin II heavy chain. Tryptic digestion of the 32P-myosin, followed by two-dimensional peptide mapping, indicates that two of the three sites phosphorylated by the kinase in vitro correspond to the two major phosphorylation sites on the myosin heavy chain in vivo. Phosphorylation of myosin II in vitro by the kinase fraction completely inhibits the actin-activated Mg2+-ATPase activity of myosin II. Myosin II can be isolated in a highly phosphorylated, enzymatically inactive form, then dephosphorylated to an active form, and finally rephosphorylated to an inactive form. The Acanthamoeba kinase fraction catalyzes the phosphorylation of all three sites on the heavy chain of myosin II at virtually the same rate. From a comparison of the decrease in actin-activated Mg2+-ATPase activity with the amount of phosphate incorporated into myosin II, and from the results obtained previously by dephosphorylating myosin II (Collins, J. H., and Korn, E. D., (1980) J. Biol. Chem. 255, 8011-8014), it can be inferred that two of the sites phosphorylated in vitro act in a synergistic manner to inhibit the actin-activated myosin II Mg2+-ATPase.     

Selective removal of the carboxyl-terminal tail end of the Dictyostelium myosin II heavy chain by chymotrypsin

Dictyostelium myosin II is a conventional myosin consisting of two heavy chains of 243,000 Da and two pairs of light chains of 16,000 and 18,000 Da. In this paper, we show that the heavy chain of myosin II can be rapidly and selectively cleaved by chymotrypsin to yield two fragments with molecular weights of 195,000 and 38,000 Da as estimated from sodium dodecyl sulfate-polyacrylamide gels. Chymotryptic cleavage at this site occurs most readily in the absence of salt and is greatly inhibited as the salt concentration is increased from 0 to 60 mM. Amino acid sequence analysis of the small fragment demonstrates that its amino terminus corresponds to lysine 1826 of the myosin II heavy chain. If the fragment extends to the carboxyl terminus of the myosin II heavy chain, it would have a molecular weight of 33,700. Upon digestion of myosin II which has been phosphorylated with a high molecular weight Dictyostelium myosin heavy chain kinase (C?té, G.P., and Bukiejko, U. (1987) J. Biol. Chem. 262, 1065-1072), all of the phosphate is recovered on the 33,700-Da tail-end fragment. Chymotrypsin-cleaved myosin II is shown to be capable of forming filaments at salt concentrations between 20 and 100 mM as judged by its ability to be sedimented by centrifugation. Only the large fragment of myosin II is found in the pellet; the 33,700-dalton fragment remains soluble. Chymotrypsin-cleaved myosin II can bind to actin and displays a high Ca2+-activated ATPase activity but has very low actin-activated ATPase activity.