Amino acid sequence of the amino-terminal 24 kDa fragment of the heavy chain of chicken gizzard myosin

Chicken gizzard myosin was modified with N-iodoacetyl-N'-(5-sulfo-1-naphthyl)-ethylenediamine (IAEDANS) in the presence of ATP and in 0.15 M KCl, where the myosin assumed 10S conformation. From the tryptic digest of the modified myosin, a fluorescent fragment (24 kilodaltons) was isolated by gel filtration on a Sephadex G-100 column followed by chromatography on a CM 52 column. The amino acid sequence of the fragment was analyzed by conventional methods, and was: (S,Z)K-P-L-S-D-D-E-K-F-L-F-V-D-K-N-F-V-N-N-P-L-A-Q-A-D-W-S-A-K-K- L-V-W-V-P-S-E-K-H-G-F-E-A-A-S-I-K-E-E-K-G-D-E-V-T-V-E-L-Q-E-N-G-K-K- V-T-L-S-K-D-D-I-Q-K-M-N-P-P-K-F-S-K-V-E-D-M-A-E-L-T-C-L-N-E-A-S-V-L- H-N-L-R-E-R-Y-F-S-G-L-I-Y-T-Y-S-G-L-F-C-V-V-I-N-P-Y-K-Q-L-P-I-Y-S-E-K-I- I-D-M-Y-K-G-K-K-R-H-E-M-P-P-H-I-Y-A-I-A-D-T-A-Y-R-S-M-L-Q-D-R-E-D-Q- S-I-L-C-T-G-E-S-G-A-G-K-T-E-N-T-K-K-V-I-Q-Y-L-A-V-V-A-S-S-H-K-G-K. The amino-terminus was blocked, and the fragment was assigned as an amino-terminal part of the heavy chain of gizzard myosin. Position 127 was occupied by epsilon-N-trimethyllysine. Trp-130 of rabbit skeletal myosin heavy chain, which was reported to cross-link to an azide derivative of ATP by Okamoto and Yount (Proc. Natl. Acad. Sci. U.S. 82, 1575-1579 (1985], was replaced by glutamine in gizzard myosin. Cys-93 of the fragment is the amino acid residue whose reaction with IAEDANS alters the ATPase activity of gizzard myosin (Onishi, H. (1985) J. Biochem. 98, 81-86).     

Correlation between the papain digestibility and the conformation of 10s-myosin from chicken gizzard

In our previous reports, ATP was shown to induce a drastic change in the conformation of gizzard myosin molecules. For example, the sedimentation constant of unphosphorylated myosin (UM) increased from 6S to 10S although an ATP-induced change in the sedimentation constant did not occur with phosphorylated myosin (Suzuki et al. (1978) J. Biochem. 84, 1529). We now report the finding that the ATP-induced formation of 10S-myosin is associated with a drastic change in the papain digestibility of gizzard UM. With 10S-myosin, the cleavage by papain was strongly inhibited at two regions on heavy chains and at one region on light chains; that is, the junction between the 72K dalton and 22K dalton fragments (i.e., a cleavable site in myosin head), the one between the 22K dalton and 130K dalton fragments (i.e., a head-tail junction), and the one between the 3K dalton and 17K dalton fragments of 20K dalton light chains. An even more intimate correlation between the myosin conformation and the papain digestibility of myosin was demonstrated by using thiophosphorylated myosin (thioPM); the cleavages by papain at the 72K-22K dalton junction and the 22K-130K dalton junction were not inhibited when thioPM was digested.     

Reaction of thiol groups of gizzard myosin heavy chains with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole

Chicken gizzard myosin treated with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) resulted in a 65% inhibition of the K(+)-ATPase (myosin ATP phosphohydrolase (actin translocating), EC 3.6.1.32) activity and 3.5 mol of the reagent was bound per 4.7 x 10(5) g protein. The labeling was limited to the heavy chain region and none of the light chains were lost. MgATP had no effect on the inactivation or labeling pattern. Thiolysis of NBD-myosin with dithiothreitol restored the K(+)-ATPase activity and concurrently, 1 mol of the NBD group was removed from the heavy chain region. Cysteine residues were modified in NBD-myosin at sites other than the active site when the enzyme activity was inhibited. There was a difference in the extent of NBD-Cl modification of gizzard myosin at 0.6 M KCl (6 S elongated state) when compared to that at 0.15 M KCl (10 S folded state). This was also seen in the heavy meromyosin-like chymotryptic fragments and tryptic fragments of NBD-myosin. The reagent NBD-Cl can detect changes in the conformation of gizzard myosin by way of its reaction with thiol groups of the heavy chain region.     

Thiol reactivity as a sensor of rotation of the converter in myosin

Smooth muscle myosin has two reactive thiols located near the C-terminal region of its motor domain, the “converter”, which rotates by ∼70° upon the transition from the “nucleotide-free” state to the “pre-power stroke” state. The incorporation rates of a thiol reagent, 5-(((2-iodoacetyl)amino)ethyl)aminonaphthalene-1-sulfonic acid (IAEDANS), into these thiols were greatly altered by adding ATP or changing the myosin conformation. Comparisons of the myosin structures in the pre-power stroke state and the nucleotide-free state explained why the reactivity of both thiols is especially sensitive to a conformational change around the converter, and thus can be used as a sensor of the rotation of the converter. Modeling of the myosin structure in the pre-power stroke state, in which the most reactive thiol, “SH1”, was selectively modified with IAEDANS, revealed that this label becomes an obstacle when the converter completely rotates toward its position in the pre-power stroke state, thus resulting in incomplete rotation of the converter. Therefore, we suggest that the limitation of the converter rotation by modification causes the as-yet unexplained phenomena of SH1-modified myosin, including the inhibition of 10S myosin formation and the losses in phosphorylation-dependent regulation of the basic and actin-activated Mg-ATPase activities of myosin.     

Correlation of intrinsic fluorescence and conformation of smooth muscle myosin

The addition of ATP to turkey gizzard myosin causes an enhancement of the intrinsic tryptophan fluorescence. The level of fluorescence enhancement is determined by the myosin conformation. The transition of myosin from the folded (10 S) state to the extended (6 S) state is accompanied by a decrease in the fluorescence level. Phosphorylation-dephosphorylation of myosin does not directly influence fluorescence and will induce changes only if the myosin conformation is altered. Under the appropriate conditions, phosphorylation of myosin favors the transition of 10 S to 6 S. The phosphorylation dependence of the associated fluorescence decrease is not linear, and it is proposed that the phosphorylation of both light chains is required for the full transition. The tryptophan residues involved respond to the binding of ATP at the hydrolytic sites. Since the fluorescence properties of gizzard myosin are influenced by the myosin conformation, it is reasonable to assume that the active sites are also modified by the shape of the myosin molecule.     

SH1 (cysteine 717) of smooth muscle myosin: its role in motor function.

To determine if a thiol group called SH1 has an important role in myosin's motor function, we made a mutant heavy meromyosin (HMM) without the thiol group and analyzed its properties. In chicken gizzard myosin, SH1 is located on the cysteine residue at position 717. By using genetic engineering techniques, this cysteine was substituted with threonine in chicken gizzard HMM, and that mutant HMM and unmutated HMM were expressed in biochemical quantities using a baculovirus system. The basal EDTA-, Ca(2+)-, and Mg(2+)-ATPase activities of the mutant were similar to those of HMM whose SH1 was modified by N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (IAEDANS). However, while the chemically modified HMM lost the function of the light chain phosphorylation-dependent regulation of the actin-activated ATPase activity, the mutant HMM exhibited the normal light chain-regulated actin-activated ATPase activity. Using an in vitro motility assay system, we found that the IAEDANS-modified HMM was unable to propel actin filaments but that the mutant HMM was able to move actin filaments in a manner indistinguishable from filament sliding generated by unmutated HMM. These results indicate that SH1 itself is not essential for the motor function of myosin and suggest that various effects observed with HMM modified by thiol reagents such as IAEDANS are caused by the bulkiness of the attached probes, which interferes with the swinging motion generated during ATP hydrolysis.     

Proteolysis and actin-binding properties of 10S and 6S smooth muscle myosin: identification of a site protected from proteolysis in the 10S conformation and by the binding of actin

It was shown previously [Ikebe, M., & Hartshorne, D. J. (1985) Biochemistry 24, 2380-2387] that the conformation of gizzard myosin, either 10S or 6S, influences proteolysis of myosin at two regions designated sites A and B. The studies reported here are focused on site A, which is located approximately 68,000 daltons from the N-terminus of the myosin heavy chain. With papain, Staphylococcus aureus protease, and actinidin, it is shown that the formation of 10S myosin reduces proteolysis at site A. Binding of actin to 6S myosin also hinders cleavage at site A for each of these proteases. To investigate binding of actin to 6S and 10S myosins, adenosine 5'-(beta,gamma-imidotriphosphate) (AMPPNP) is used as a substitute for ATP. In the presence of AMPPNP, it is shown that the 6S to 10S transition occurs and that 10S myosin binds actin with lower affinity than 6S myosin. For 6S myosin at high salt (0.35 M KCl) the dissociation constant of actin from the actin-myosin-nucleotide complex (K3) is approximately the same for phosphorylated (1.9 mol of P/mol of myosin) and dephosphorylated myosin, i.e., 1.3-2.4 microM, respectively. At lower ionic strength (0.17 M KCl) K3 for dephosphorylated myosin (10S myosin) is 42 microM and K3 for phosphorylated myosin (6S myosin) is 0.3 microM. These data show that the conformation of myosin influences the actin-myosin interaction. The constant (K4) for the dissociation of nucleotide from the actin-myosin-nucleotide complex varies slightly (in the range of 0.2-1.3 mM), but there is no marked change as a result of either a conformational change or phosphorylation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Location of the sites of reaction of N-ethylmaleimide in papain and chymotryptic fragments of the gizzard myosin heavy chain

The thiol of the gizzard myosin heavy chain, which reacts most rapidly with N-ethylmaleimide (MalNEt), has been located in the subfragment 2 region of myosin rod by fragmentation of [14C]-MalNEt-labeled myosin with papain and chymotrypsin. MalNEt reacts more slowly with thiols present in the 70- and 25-kilodalton (kDa) papain fragments of subfragment 1. The reaction of MalNEt with thiols present in these regions is increased on addition of ATP by factors of 2 and 10, respectively, when myosin is modified in 0.45 M NaCl where it is present in the extended, 6S conformation. The rate of increase of Mg2+-activated adenosinetriphosphatase (ATPase) activity, which reflects the loss of ability of myosin to assume the folded, 10S conformation, and the rate of loss of K+-EDTA-activated activity produced by MalNEt are both accelerated 5- to 10-fold on addition of ATP. The rates at which ATPase activities change agree closely to the reaction rates of MalNEt with the 25-kDa region of subfragment 1; therefore, the changes in these activities can be attributed to modification of a thiol of the 25-kDa segment. An increase in actin-activated ATPase activity produced by reaction of myosin with MalNEt in 0.45 M NaCl is accelerated by ATP by a factor of at least 4. Reaction with [14C]MalNEt in the presence of MgATP and 0.2 M NaCl, where myosin is in the 10S form, inhibits the incorporation of radioactive MalNEt into the 25-kDa papain fragment of subfragment 1. It also prevents the increase in actin-activated ATPase activity and preserves the ability of myosin to assume the 10S form.     

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.     

Structure and function of chicken gizzard myosin.

In our previous study (Onishi, H., Susuki, H., Nakamura, k., and Watanabe, S. J. Biochem. 83, 835-847, 1978), we found it to be characteristic of chicken gizzard myosin that thick filaments of gizzard myosin are readily disassembled by a stoichiometric amount of ATP (3 mol of ATP per mol of myosin), and that the ATPase activity of gizzard myosin in the ATP-disassembled state is much lower than that of gizzard myosin disassembled by a high concentration of KCl. We now report the following findings: (1) Thick filaments of (unphosphorylated) gizzard myosin can be in a bipolar structure or in a non-polar structure, depending on the method of preparing the thick filaments. (2) Thick filaments of (unphosphorylated) gizzard myosin in either the bioplar or the non-polar structure are readily disassembled by ATP. (3) Addition of rabbit skeletal C-protein does not confer ATP resistance on thick filaments of (unphosphorylated) gizzard myosin. (4) Unphosphorylated) gizzard myosin in the ATP-disassembled state is in a dimeric form as determined by ultracentrifugation. Moreover, 0.2 M KCl-dissociated gizzard myosin in monomeric form is converted to a dimeric form by ATP. (5) The Mg-ATPase activity of (unphosphorylated) gizzard myosin is much lower in its dimeric form (less than one-tenth) than in its monomeric form. The activity depression observed around 0.15 M KCl is therefore due to the formation of myosin dimers. (6) Skeletal L-meromyosin can increase the very low activity of (unphosphorylated) gizzard myosin ATPase at low ionic strength (0.13 M KCl) by forming ATP-resistant hybrid filaments with (unphosphorylated) gizzard myosin, preventing the formation of myosin dimers. (7) Gizzard myosin in which one of the light-chain components is phosphorylated by myosin light-chain kinase can form thick filaments which are resistant to the disassembling action of ATP. (8) Even in the presence of ATP, thick filaments of phosphorylated gizzard myosin do not disassembled into myosin dimers. Accordingly, the ATPase activity of phosphorylated gizzard myosin does not show activity depression at low ionic strength.     

A quasi-elastic light scattering study of smooth muscle myosin in the presence of ATP.

We have investigated the hydrodynamic properties of turkey gizzard smooth muscle myosin in solution using quasi-elastic light scattering (QELS). The effects of ionic strength (0.05-0.5 M KCl) and light chain phosphorylation on the conformational transition of myosin were examined in the presence of ATP at 20 degrees C. Cumulant analysis and light scattering models were used to describe the myosin system in solution. A nonlinear least squares fitting procedure was used to determine the model that best fits the data. The conformational transition of the myosin monomer from a folded form to an extended form was clearly demonstrated in a salt concentration range of 0.15-0.3 M KCl. Light chain phosphorylation regulates the transition and promotes unfolding of the myosin. These results agree with the findings obtained using sedimentation velocity and electron microscopy (Onishi and Wakabayashi, 1982; Trybus et al., 1982; Trybus and Lowey, 1984). In addition, we present evidence for polymeric myosin coexisting with the two monomeric myosin species over a salt concentration range from 0.05 to 0.5 M KCl. The size of the polymeric myosin varied with salt concentration. This observation supports the hypothesis that, in solution, a dynamic equilibrium exists between the two conformations of myosin monomer and filaments.     

Modification of gizzard myosin and reconstituted actomyosin with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole at low and high ionic strength.

Treatment of reconstituted gizzard actomyosin at 0.15 M or 0.6 M KCl with the fluorescent adenine analog 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, NBD-Cl, resulted in a significant decrease in the labeling of the myosin from actomyosin compared to that of myosin alone. Actin protected partially the K(+)-ATPase activity of myosin from modified actomyosin. The reagent was able to detect changes in the conformation of myosin as the distribution of the label in the heavy and light chains of myosin and actin was different at 0.15 M and 0.6 M KCl. The 6S and 10S transition, unique to smooth muscle myosin, can be monitored with the aid of this reagent.     

Correlation of conformation and phosphorylation and dephosphorylation of smooth muscle myosin

The rate of phosphorylation and dephosphorylation of smooth muscle myosin by myosin light chain kinase and by two myosin light chain phosphatases (gizzard phosphatase IV and aorta phosphatase) are measured in various conditions; the relationship between the rate of phosphorylation and dephosphorylation of myosin and the myosin conformation is also studied. The rate of dephosphorylation of myosin was completely inhibited in the presence of 1 mM MgCl2 and ATP at low ionic strength where phosphorylated myosin forms a folded conformation. The inhibition was released when myosin formed either an extended monomer or filaments. The rate of phosphorylation of myosin was also affected by the conformation of myosin. The rate for a folded myosin was slower than those for an extended monomer and filamentous myosin. The phosphorylation and dephosphorylation of heavy meromyosin, subfragment-1, and the isolated 20,000-dalton light chain are not inhibited at low ionic strength, and the rate of phosphorylation and dephosphorylation was decreased with increasing ionic strength. KCl dependence of the rate of phosphorylation and dephosphorylation of myosin was normalized by using KCl dependence of subfragment-1, and it was found that the marked inhibition of the rate of phosphorylation and dephosphorylation of myosin is closely related to the change from an extended to a folded conformation of myosin.     

Phosphorylation of chicken gizzard myosin: Myosin filament hypothesis of calcium regulation

Myosin from chicken gizzard smooth muscle was found to be characteristically different from rabbit skeletal striated myosin: i) ATP induced a profound change in the conformation of gizzard myosin molecules. ii) ATP also induced disassembling of gizzard myosin filaments. iii) Enzymic phosphorylation of gizzard myosin light chains rendered both the myosin conformation and the myosin filaments resistant to the actions of ATP. iv) Very high concentrations of magnesium were required for formation of the ATP-resistant filaments as well as for superprecipitation (a model contraction) of actomyosin suspensions. v) ITP was a very poor substrate for MLCK, and was accordingly incapable of inducing “Ca-tension” in glycerinated fibers of gizzard muscle, but it did induce “Mg-tension.” Primarily from these findings, it was proposed that tje mechanism of gizzard muscle contraction involves ATP-induced changes in the morphology of myosin filaments which are reversibly altered by enzymic phosphorylation and dephosphorylation of myosin light chains in the presence of relatively high concentrations of magnesium.     

Amino acid sequence of the 203-residue fragment of the heavy chain of chicken gizzard myosin containing the SH1-type cysteine residue

A fluorescent fragment of Mr = 23,800 was obtained by the papain digestion of N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylene diamine (abbreviated as IAEDANS)-modified chicken gizzard myosin. The fragment was isolated by gel filtration on a Sephadex G-100 column in the presence of 5 M guanidine-HCl followed by anion exchange chromatography on a QAE Sephadex A-50 column. This fragment contained 203 amino acid residues which could be assigned as a COOH-terminal part of the S-1 heavy chain based on the homology with the known sequence of rabbit skeletal myosin fragment. The amino acid sequence was K-G-M-F-R-T-V- G-Q-L-Y-K-E-Q-L-T-K-L-M-T-T-L-R-N-T-N-P-N-F-V-R-C-I-I-P-N-H-E-K-R-A- G-K-L-D-A-H-L-V-L-E-Q-L-R-C-N-G-V-L-E-G-I-R-I-C-R-Q-G-F-P-N-R-I-V-F-Q- E-F-R-Q-R-Y-E-I-L-A-A-N-A-I-P-K-G-F-M-D-G-K-Q-A-C-I-L-M -I-K-A-L-E-L- D-P-N-L-Y-R-I-G-Q-S-K-I-F-F-R-T-G-V-L-A-H-L-E-E-E-R-D-L-K- I-T-D-V-I-I-A- F-Q-A-Q-C-R-G-Y-L-A-R-K-A-F-A-K-R-Q-Q-Q-L-T-A-M-K-V-I-Q-R-N-C-A -A-Y-L-K-L-R-N-W-Q-W-W-R-L-F-T-K-V-K-P-L-L-Q-V-T-R. The cysteine residue which was modified with IAEDANS was of the SH1 type (Cys-65). Pro-197 was suggested to be the NH2-terminal boundary of the alpha-helical coiled-coil rod sequence of gizzard myosin, based on the homology with the nematode sequence reported by MacLachlan and Karn (Proc. Natl. Acad. Sci. U.S. 80, 4253-4257 (1983)). Three different COOH-terminal peptides (Val-Lys-Pro-Leu-Leu-Gln-Val-Thr-Arg, Val-Lys-Pro-Leu-Leu-Gln, and Val-Lys-Pro-Leu-Leu) were isolated from the tryptic digest of this fragment.(ABSTRACT TRUNCATED AT 400 WORDS)     

Modification of thiols of gizzard myosin alters ATPase activity, stability of myosin filaments, and the 6-10 S conformational transition

The pattern of incorporation of [14C]N-ethylmaleimide (MalNEt) into gizzard myosin indicates the presence of two classes of thiols: rapidly and slowly modified. The first class contains two thiol residues, SH-A and SH-B, located in the myosin rod and the 17-kDa light chain, respectively, while the second contains at least two thiols located in the myosin heavy chain. Changes in ATPase activities upon modification occur rapidly or slowly, paralleling reaction of either the first or second class of thiols. Rapid changes include increases in the Ca2+- and Mg2+-activated activities of myosin alone, measured at ionic strengths below 0.3 M, and an increase and a decrease in the actin-activated activity of dephosphorylated and phosphorylated myosin, respectively. Modification of SH-A and SH-B with MalNEt is accompanied by stabilization of myosin filaments, seen as an increase in light-scattering intensity, and by destabilization of the folded, 10 S conformation of the myosin monomer. In the presence of 0.175 M NaCl and 1 mM MgATP, unmodified and MalNEt-modified myosin sediment in the ultracentrifuge as single components at 10.0 S and 6.0 S, respectively. The MalNEt-induced increase in the Ca2+- or Mg2+-activated ATPase activity, measured in the absence of actin, can be attributed either to stabilization of filaments or to destabilization of the 10 S conformation, depending on the ionic strength of the assay. Modification of the second class of thiols is accompanied by a decrease in K+-EDTA-activated activity and an increase in Ca2+-activated activity measured above 0.3 M NaCl, where myosin neither forms filaments nor assumes the 10 S conformation. These slow changes are characteristic of blocking the SH-1 thiols of skeletal-muscle myosin, but in gizzard myosin are attributable to modification of a less reactive thiol, SH-C.     

Conformational studies of myosin phosphorylated by protein kinase C

Smooth muscle myosin from chicken gizzard is phosphorylated by Ca2+-activated phospholipid-dependent protein kinase, protein kinase C, as well as by Ca2+/calmodulin-dependent kinase, myosin light chain kinase (Endo, T., Naka, M., and Hidaka, H. (1982) Biochem. Biophys. Res. Commun. 105, 942-948). We have now demonstrated the effect of phosphorylation by protein kinase C on the smooth muscle myosin molecule. In glycerol/urea polyacrylamide gel electrophoresis the 20,000-dalton light chain phosphorylated by protein kinase C co-migrated with that phosphorylated by myosin light chain kinase. Moreover, the light chain phosphorylated by both kinases migrated more rapidly than did the light chain phosphorylated by either myosin light chain kinase or protein kinase C alone. Myosin phosphorylated by protein kinase C formed a bent 10 S monomer while that phosphorylated by myosin light chain kinase was an unfolded and extended 6 S monomer in the presence of 0.2 M KCl. In addition, myosin phosphorylated by kinases had a sedimentation velocity of 7.3 S, thereby suggesting that the myosin was partially unfolded. The unfolded myosin was visualized electron microscopically. The fraction in the looped form was higher when for myosin phosphorylated by both kinases higher than for that phosphorylated by light chain kinase alone. Therefore, phosphorylation by protein kinase C does not lead to the change in myosin conformation seen with myosin light chain kinase.     

Phosphorylation of a second site for myosin light chain kinase on platelet myosin

The 20,000-dalton light chain of bovine platelet myosin is phosphorylated at two sites by myosin light chain kinase. The first and second phosphorylation sites are at a serine and a threonine residue, respectively. The location of the phosphorylation sites was determined by using limited proteolysis. The N-terminal sequence of the 17,000-dalton tryptic fragment of platelet myosin 20,000-dalton light chain was found to be identical with that of gizzard 20,000-dalton light chain from Ala-17 to Phe-33. On the basis of these results and the distribution of 32P among the proteolytic fragments, it was concluded that serine-19 and threonine-18 were the two phosphorylation sites. Phosphorylation at the threonine residue markedly increases the actin-activated ATPase activity of myosin. It was found that platelet myosin forms 10S and 6S conformations and its Mg2+-ATPase activity parallels the transition from the 6S to the 10S conformation. The conformational transition was influenced by phosphorylation at both sites, and the phosphorylation at the threonine residue further shifted the equilibrium toward the 6S conformation. The phosphorylation at the threonine residue also induced thick filament formation in the presence of ATP. These results suggest that the phosphorylation at the threonine residue as well as at the serine residue may play an important role in the contractility of nonmuscle cells.     

Actin-induced local conformational change in the myosin molecule. I. Effect of metal ions and nucleotides on the conformational change around a specific thiol group (S2) of heavy meromyosin.

As previously reported when a specific thiol group, S2, of myosin reacts with N-ethylmaleimide (NEM), its Ca2+-ATPase activity is decreased. Therefore, the reactivity of S2 can be estimated by measuring the decrement of the enzymatic activity. Using the change in the reactivity as a structural probe, we investigated whether F-actin affects the conformation around the region containing S2 under physiological conditions (at neutral pH and low ionic strength). 1. Experiments were carried out with heavy meromyosin (HMM), S1 of which had heen blocked with NEM, to observe the reactivity of S2 alone. In the experiments done in the presence of F-actin, the Ca2+-ATPase activity was measured using the heavy meromyosin fraction after actin had been removed by centrifugation and gel filtration. 2. ATP and other nucleotides activated the reactivity of S2 in the presence of Mg2+. On the other hand, F-actin markedly activated the reactivity of S2 which had been increased by ATP, but not by the other nucleotides. 3. The above cooperative action of F-actin with ATP was not observed in the presence of Ca2+ instead of Mg2+, or above 0.2 M KCl. These results suggest that the S2 region of the myosin molecule is a key region in the molecular interaction of the actin myosin-ATP system under physiological conditions.     

Effect of ionic strength on the conformation of myosin subfragment 1-nucleotide complexes.

The effect of ionic strength on the conformation and stability of S1 and S1-nucleotide-phosphate analog complexes in solution was studied. It was found that increasing concentration of KCl enhances the reactivity of Cys(707) (SH1 thiol) and Lys(84) (reactive lysyl residue) and the nucleotide-induced tryptophan fluorescence increment. In contrast, high KCl concentration lowers the structural differences between the intermediate states of ATP hydrolysis in the vicinity of Cys(707), Trp(510) and the active site, possibly by increasing the flexibility of the molecule. High concentrations of neutral salts inhibit both the formation and the dissociation of the M**.ADP.Pi analog S1.ADP.Vi complex. High ionic strength profoundly affects the structure of the stable S1.ADP.BeF(x) complex, by destabilizing the M*.ATP intermediate, which is the predominant form of the complex at low ionic strength, and shifting the equilibrium to favor the M**.ADP.Pi state. The M*.ATP intermediate is destabilized by perturbation of ionic interactions possibly by disruption of salt bridges. Two salt-bridge pairs, Glu(501)-Lys(505) in the Switch II helix and Glu(776)-Lys(84) connecting the catalytic domain to the lever arm, seem most appropriate to consider for participating in the ionic strength-induced transition of the open M*.ATP to the closed M**.ADP.Pi state of S1.