Correlation between multiple phosphorylation of gizzard myosin light chains and actin-activated myosin ATPase activity

With large amounts of gizzard Mr 135,000 calmodulin-binding protein (myosin light chain kinase), the phosphate incorporation into myosin light chains was determined to be 2 mol/mol of myosin light chain. The actin-activated ATPase activity was dramatically enhanced when myosin light chains were phosphorylated by more than 1 mol of phosphate incorporated/mol of myosin light chain.     

N-terminal region of gizzard myosin heavy chain is critical for the ATPase activity

Two different HMM species of gizzard myosin were prepared under conditions such that the phosphorylation of light chain was fully maintained. They were different in the N-terminal structure of the heavy chain but not in the light chain composition. A significant decrease in the Mg2+-ATPase activity was observed in one class of HMM which was proteolytically cleaved intramolecularly at site 1, 5 K daltons from the masked N terminus. Another class of HMM without the cleavage at site 1 showed ATPase activity similar to that of myosin. The decrease in ATPase activity was not caused by denaturation since similar amounts of initial burst of Pi liberation were observed with both HMMs and myosin. Kinetic and substructure analyses of HMM revealed that the activity change depended solely on the cleavage at site 1. The N-terminal region of gizzard myosin heavy chain may thus have an important role in maintaining the active site structure.     

Linear relationship between diphosphorylation of 20 kDa light chain of gizzard myosin and the actin-activated myosin ATPase activity

The addition of large amounts of myosin light chain kinase to the reconstituted gizzard actomyosin shows diphosphorylation of 20 kDa myosin light chain. Accompanying diphosphorylation, the actin-activated myosin ATPase activity was also enhanced. The extent of diphosphorylation and the myosin ATPase activity were clearly demonstrated to be in a linear relationship. From the time course experiment, the conversion of monophosphorylated light chain into one which was diphosphorylated seemed to be a sequential process. Moreover, analyzing phospho-amino acid by using a two-dimensional electrophoresis technique revealed that monophosphorylated light chain contained phosphoserine and diphosphorylated one contained phosphothreonine in addition to phosphoserine.     

Purealin, a novel stabilizer of smooth muscle myosin filaments that modulates ATPase activity of dephosphorylated myosin

Effects of purealin isolated from a sea sponge, Psammaplysilla purea, on the enzymatic and physiochemical properties of chicken gizzard myosin were studied. At 0.15 M KCl, 40 microM purealin increased the Ca2+- and Mg2+-ATPase activity of dephosphorylated gizzard myosin to 2.5- and 3-fold, respectively, but decreased the K+-EDTA-ATPase activity of the myosin to 0.25-fold. In contrast, purealin had little effect on the ATPase activities of phosphorylated gizzard myosin. The ATP-induced decrease in light scattering of dephosphorylated gizzard myosin at 0.15 M KCl was lessened by 40 microM purealin. Electron microscopic observations indicated that thick filaments of dephosphorylated myosin were disassembled immediately by addition of 1 mM ATP at 0.15 M KCl, although they were preserved by purealin for a long time even after addition of ATP. Upon ultracentrifugation, dephosphorylated myosin sedimented as two components, the 10 S species and myosin filaments, in the solution containing 0.18 M KCl and 1 mM Mg X ATP in the presence of 60 microM purealin. These results suggest that purealin modulates the ATPase activities of dephosphorylated gizzard myosin by enhancing the stability of myosin filaments against the disassembling action of ATP.     

A conformational transition in gizzard heavy meromyosin involving the head-tail junction, resulting in changes in sedimentation coefficient, ATPase activity, and orientation of heads

Gizzard heavy meromyosin (HMM) sediments in the ultracentrifuge as a single peak, whose sedimentation coefficient (S20,w) decreases from 9 to 7.5 S upon increasing the NaCl concentration from 0.02 to 0.3 M. This decrease is accompanied by a parallel increase in Mg2+-ATPase activity, suggesting that both changes have a common molecular basis. Phosphorylation decreases S20,w and increases ATPase activity, while ATP increases S20,w. Sedimentation equilibrium studies indicate that HMM undergoes no detectable aggregation at 0.02 or 0.4 M NaCl, remaining monomeric with a molecular weight of 3.4 X 10(5). In contrast, S20,w of subfragment 1 does not change with changes in ionic strength, and its ATPase activity does not decrease at low ionic strengths. Electron micrographs of samples of HMM prepared at low ionic strength show that up to half of the molecules are flexed, i.e. the heads are bent at the neck and project back toward the tail, while the remaining molecules have either one or both of the heads pointing away from the tail. In samples prepared at high ionic strength only about 10% of the molecules are flexed. There is a linear relationship between the fraction of flexed molecules and S20,w, with no significant bending or folding of the tail and no detectable change in the shape of the heads. This correlation suggests that the changes in ATPase activity and S20,w may be a result of the reorientation of the heads.     

Activation by actin of ATPase activity of chemically modified gizzard myosin without phosphorylation.

The ATPase activity of myosin from chicken gizzard measured in the presence of either Mg²⁺ or Ca²⁺ is increased in the absence of dithiothreitol or upon reaction with Cu²⁺, o-iodosobenzoate, or N-ethylmaleimide. Iodosobenzoate or Cu²⁺ produce no change in K⁺(EDTA)-ATPase while N-ethylmaleimide produces a decrease. These treatments also make the actin-activated ATPase insensitive to Ca²⁺ when assayed in the presence of tropomyosin and a partially purified myosin light chain kinase. Phosphorylation of N-ethylmaleimide modified myosin remains dependent on Ca²⁺ and therefore appears not to be required for activation by actin of the ATPase activity of modified myosin.     

Dinitrophenylated thiols in tryptic fragments of the heavy chain from chicken gizzard myosin

Trypsin digestion of phosphorylated and 3H-labeled dinitrophenylated chicken gizzard myosin released major fragments of Mr 29,000, 50,000 and 66,000 in a ratio of close to one to one. They contained 58% of the label bound to thiols of the heavy chains; 28% of the label was bound to the light chains. The heavy chain fragments of Mr 29,000 and Mr 66,000 were dinitrophenylated when the enzyme activity was inhibited. The 3H-labeled dinitrophenylated myosin alone followed a somewhat different pattern in that the label was bound to the light chains predominantly. Thiolysis of the phosphorylated and dinitrophenylated myosin with 2-mercaptoethanol restored the K+ -ATPase (ATP phosphohydrolase, EC 3.6.1.32) activity and the dinitrophenyl group was removed from the N-terminal fragment of Mr 29,000 of the heavy chain, predominantly. In contrast, restoration of the enzymic activity occurred in thiolyzed dinitrophenylated myosin alone when the label was removed from the light chains rather than the tryptic fragments of the heavy chain. Phosphorylation induced conformational changes in gizzard myosin that altered the reactivity of the thiols in fragments of the globular heavy chain region.     

Regulation of the actin-activated ATPase activity of Acanthamoeba myosin I by cross-linking actin filaments

The actin-activated Mg2+-ATPase activity of phosphorylated Acanthamoeba myosin I was previously shown to be cooperatively dependent on the myosin concentration (Albanesi, J. P., Fujisaki, H., and Korn, E. D. (1985) J. Biol. Chem. 260, 11174-11179). This observation was rationalized by assuming that myosin I contains a high-affinity and a low-affinity F-actin-binding site and that binding at the low-affinity site is responsible for the actin-activated ATPase activity. Therefore, enzymatic activity would correlate with the cross-linking of actin filaments by myosin I, and the cooperative increase in specific activity at high myosin:actin ratios would result from the fact that cross-linking by one myosin molecule would increase the effective F-actin concentration for neighboring myosin molecules. This model predicts that high specific activity should occur at myosin:actin ratios below that required for cooperative interactions if the actin filaments are cross-linked by catalytically inert cross-linking proteins. This prediction has been confirmed by cross-linking actin filaments with either of three gelation factors isolated from Acanthamoeba, one of which has not been previously described, or by enzymatically inactive unphosphorylated Acanthamoeba myosin I.     

The actomyosin ATPase of synthetic myosin minifilaments, filaments, and heavy meromyosin

The actin-activated ATPase activities of myosin minifilaments and heavy meromyosin are similar at high actin concentrations. Under low ionic strength conditions, the minifilaments in Tris citrate buffer yield the same maximal turnover rate (Vmax) and apparent dissociation constant of actin from myosin (Kapp) as heavy meromyosin in standard low salt conditions. The time course of actin-activated ATP hydrolysis of minifilaments is similar to that observed for standard myosin preparations. Depending on the exact protein composition of the assay mixture, either the ATPase activity declines continuously with time, or is accelerated at the onset of superprecipitation. In analogy with myosin filaments, the ATPase of minifilaments shows a biphasic dependence on actin concentration. Super-precipitation of minifilaments follows a well resolved clearing phase during which their structural integrity appears to be fully preserved. These results indicate that minifilaments or similar small assemblies of myosin can fulfill contractile functions.     

Cosolvent-induced aggregation inhibits myosin ATPase activity by stabilizing the predominant transition intermediate

High concentration of the cosolvent poly(ethylene glycol) (PEG) induces reversible aggregation of skeletal myosin subfragment 1 (S1) and inhibition of its Mg-ATPase activity [Highsmith et al. (1998) Biophys. J. 74, 1465-1472]. In the present work the effect of aggregation on the various steps of the ATPase cycle was studied. The isomerization and hydrolysis steps of the cycle were not affected by S1 aggregation since the formation of the "trapped" S1.MgADP.phosphate analogue complexes, which mimic the prehydrolysis M*ATP and posthydrolysis M**ADP.P(i) transition states, proceeded without any hindrance. Similar conclusions could be reached from the chemical modification of Lys-83 and Cys-707 in the presence of MgATP and MgATPgammaS, which indicated that the most populated intermediate of the cycle in solubilized and aggregated S1 is M**ADP.P(i). The dissociation of the trapped S1.MgADP.phosphate analogue complexes resembling the M**ADP.P(i) state was strongly inhibited by PEG-6000, showing that the transition from this intermediate is prevented by the aggregation. This step is presumably inhibited because the coupled swinging of the lever arm from the closed to the open position is constrained by the close packing of aggregated S1.     

Stability and photochemical properties of vanadate-trapped nucleotide complexes of gizzard myosin in the 6S and 10S conformations: identification of an active-site serine.

The properties of divalent metal.ADP.vanadate (V(i)) complexes of the 6S extended and 10S folded conformations of gizzard myosin before and after UV irradiation have been studied. The half-lives of both 6S and 10S myosin.MgADP.V(i) complexes in the dark at 0 degrees C are on the order of 2 weeks. Brief irradiation with UV light, however, photomodified the enzyme as suggested by changes in the NH(4+)-, K(+)-, and Ca(2+)-ATPase activities, and destabilized the complexes. The 6S complex, when irradiated, released ADP and V(i) rapidly (t1/2 less than or equal to 1 min) as has been observed in comparable experiments with skeletal myosin subfragment 1 (S1) [Grammer et al. (1988) Biochemistry 27, 8408-8415]. The irradiated 10S complex released approximately 20% of the ADP and V(i) rapidly (t1/2 less than or equal to 1 min), but the remainder stayed trapped, possibly as the vanadyl (VO2+).ADP complex, for much longer times (t1/2 approximately 8 h). The site of photomodification was sought by reducing both photomodified 6S and 10S myosin with NaB3H4. Amino acid composition analyses identified [3H]serine as the only labeled residue(s), suggesting that the hydroxymethyl group of serine had been oxidized to an aldehyde as shown previously for photomodified skeletal myosin S1 [Cremo et al. (1989) J. Biol. Chem. 264, 6608-6611]. The 29-kDa NH2-terminal tryptic peptide from the heavy chain was found to contain essentially all of the [3H]serine. Preparations of 6S and 10S [3H]myosin were digested exhaustively with trypsin. An identical [3H]peptide was purified from each preparation and its sequence determined to be Glu169-Asp-Gln-Ser-Ile-Leu-(Cys)-Thr-Gly-[3H]Ser-Gly-Ala-Gly-Ly s183.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

The relationship between ATPase activity, isometric force, and myosin light-chain phosphorylation and thiophosphorylation in skinned smooth muscle fiber bundles from chicken gizzard

Isometric force developed by skinned gizzard muscle fiber bundles and levels of phosphorylation and thiophosphorylation of the 20,000-dalton myosin light chain were determined. These data showed a highly non-linear relationship between isometric force and myosin light-chain phosphorylation. Maximum force was developed at approximately 0.2 mol of phosphate/mol of light chain as reported previously (Hoar, P. E., Kerrick, W. G. L., and Cassidy, P. S. (1979) Science 204, 503-506). In contrast, the relationship between isometric force and myosin light-chain thiophosphorylation was linear, with maximum force occurring at 1.0 mol of thiophosphate/mol of myosin light chain. These observations are consistent with the latch-bridge hypothesis for conditions of varying myosin light-chain phosphatase/myosin light-chain kinase activity ratios as discussed by Hai and Murphy [1988) Am. J. Physiol. 254, C99-C106). To further test the latch-bridge hypothesis, ATPase activity was also measured during isometric force development in these fiber bundles. The relationship between isometric force and ATPase activity was linear whether the myosin light chains were phosphorylated or thiophosphorylated. Thus the number of cycling myosin cross-bridges, as measured by ATPase activity, was directly proportional to the force the muscle developed, not to the level of myosin light-chain phosphorylation. This finding that high levels of tension generated at low levels of light-chain phosphorylation are associated with high levels of ATPase activity is inconsistent with the latch-bridge model (Hai and Murphy, 1988).     

Effects of light chain phosphorylation and skeletal myosin on the stability of non-muscle myosin filaments

The effect of light chain phosphorylation and the presence of skeletal muscle myosin on the stability of non-phosphorylated non-muscle myosin filaments was investigated. Purified skeletal, brush border and thymus myosins were assembled in vitro into hybrid filaments consisting of varying proportions of (1) non-muscle and skeletal myosins, or (2) phosphorylated and non-phosphorylated non-muscle myosins. The stability of these hetero- and homopolymers in the presence of MgATP was determined using sedimentation, gel electrophoresis and immunochemical techniques. In addition, the effect of a monoclonal antibody, binding to the tip of brush border myosin tail, on the assembly of the homo- and heteropolymers, was tested. Filamentous non-phosphorylated non-muscle myosin was disassembled by MgATP to the same extent whether in homo- or heteropolymers, indicating that skeletal myosin has no stabilising effect on the hybrid filaments. The presence of small amounts of phosphorylated non-muscle myosin was, however, found to prevent the complete disassembly by MgATP of non-phosphorylated non-muscle myosin filaments, indicating that light chain phosphorylation stabilizes co-operatively non-muscle myosin filaments. The monoclonal antibody prevented the assembly of brush border myosin into both homo- and heteropolymers, and its effect on the filaments was compared with that of MgATP.