I-protein forms cage-like aggregates of myosin in vitro

I-protein was mixed with myosin before or after myosin filaments were reconstituted. In both cases, I-protein seemed to accelerate the myosin assembly. The binding of I-protein to myosin filaments was tested by sedimentation experiments and SDS-polyacrylamide gel electrophoresis. In a low ionic strength solution at pH 6.5, the binding ratio of I-protein to myosin was 1:40 by molar ratio when the I-protein molecules highly specifically bound to myosin filaments. I-protein could maximally bind to myosin filaments at the molar ratio of 1:2.7. In this case, excess I-protein molecules remained in the supernatant after sedimentation, although the unbound I-protein could still bind to myosin filaments. Electron microscopic observations revealed that I-protein bundled myosin filaments in the low ionic strength solution (pH 6.5). Cage-like structures which were very similar to the Mg-paracrystals of non-muscle myosins were formed at pH 7.2. In gel filtration, the apparent molecular mass of I-protein was 100 kDa, while it was 50 kDa in SDS gel electrophoresis. Therefore, I-protein is regarded to be a homodimer of a 50 kDa subunit and can divalently bind to myosin molecules.     

Ca2+-sensitivity of actomyosin ATPase purified from Physarum polycephalum.

A contractile protein closely resembling natural actomyosin (myosin B) of rabbit skeletal muscle was extracted from plasmodia of the slime mold, Physarum polycephalum, by protecting the SH-groups with beta-mercaptoethanol or dithiothreitol. Superprecipitation of the protein induced by Mg2+-ATP at low ionic strength was observed only in the presence of very low concentrations of free Ca2+ ions, and the Mg2+-ATPase [EC 3.6.1.3] reaction was activated 2- to 6-fold by 1 muM of free Ca2+ ions. Crude myosin and actin fractions were separated by centrifuging plasmodium myosin B in the presence of Mg2+-PPi at high ionic strength. The crude myosin showed both EDTA- and Ca2+-activated ATPase activities. The Mg2+-ATPase activity of crude myosin from plasmodia was markedly activated by the addition of pure F-actin from rabbit skeletal muscle. Addition of the F-action-regulatory protein complex prepared from rabbit skeletal muscle as well as the actin fraction of plasmodium caused the same degree of activation as the addition of pure F-actin only in the presence of very low concentrations of Ca2+ ion     

Ca2+ regulation not associated with phosphorylation of myosin light chain in aortic intima smooth muscle. II. Effects of regulatory proteins on the actin-myosin interaction

Contractile and regulatory proteins were prepared from bovine aortic intima, and actin from bovine stomach smooth and rabbit skeletal muscles. In the desensitized and reconstituted actomyosin system, the superprecipitation activity was measured by the turbidity method. Superprecipitation of each system was not exhibited even in the presence of Ca ions, but was observable only in the presence of tropomyosin and Ca ions, while 20,000-dalton light chain of myosin remained dephosphorylated during the reaction. Addition of tropomyosin to the reconstituted acto-myosin digest system (trypsin-digested myosin was devoid of 20,000-dalton light chain) also restored the Ca2+-sensitivity. These results indicate that the phosphorylation of myosin light chain is not a crucial step in the contraction of aortic intima smooth muscle. For full activation of the actin-myosin-ATP interaction, additional factors other than the myosin light chain kinase are required, although some contribution of the kinase to the full activation cannot be ruled out.     

Phosphorylation and dephosphorylation of a light chain of the chicken gizzard myosin molecule.

Chicken gizzard myosin was incubated with ATP and/or "native" tropomyosin (NTM) of gizzard muscle in the presence or absence of calcium ions. One of the two light chains of the myosin molecule was phosphorylated in the presence of Ca, but not in its absence. The phosphorylated gizzard myosin was dephosphorylated by a crude preparation of myosin light-chain phosphatase obtained from gizzard muscle. In a superprecipitation test in the presence of EGTA, actomyosin reconstituted from dephosphorylated gizzard myosin did not superprecipitate, whereas actomyosin reconstituted from phosphorylated gizzard myosin showed superprecipitation activity which was inhibited by skeletal NTM and reactivated by Ca.     

Immobilization of actin and myosin.

Using glutaric dialdehyde, the muscle proteins myosin, actin, actomyosin and heavy meromyosin subfragment-1 (S-1) have been immobilized on capron fibers. The ATPase activity of myosin and its capability to interact with actin have been preserved whereas the ATPase activity of its subfragment decreased significnatly. Immobilization on capron fibers changes the pH dependence of the ATPase activity of myosin and of S-1 shifting the maximum towards the acid zone (pH 5.5) and increases the thermal stability of the enzyme. Calcium ions produce a stimulatory effect on ATPase; Mg2+ions yield no effect on myosin and S-1 but enhance the activity in the case of immobilized actomyosin though to a lesser degree than the ions of Ca2+. Immobilized actin retains its ability to form actomyosin complex.     

The kinetics of bivalent metal ion dissociation from myosin subfragments.

Bivalent metal ions have multiple roles in subunit association and ATPase regulation in scallop adductor-muscle myosin. To help elucidate these functions, the rates of Ca2+ and Mg2+ dissociation from the non-specific high-affinity sites on the regulatory light chains were measured and compared with those of rabbit skeletal-muscle myosin subfragments. Ca2+ dissociation had a rate constant of about 0.7 s-1 in both species, as measured by the time course of the pH change on EDTA addition. Mg2+ dissociation had a rate constant of 0.05 s-1, as monitored by its displacement with the paramagnetic Mn2+ ion. It is concluded that the exchange between Ca2+ and Mg2+ at the non-specific site, on excitation of both skeletal and adductor muscles, is too slow to contribute to the activation itself. The release of bivalent metal ions from the non-specific site is, however, the first step in release of the scallop regulatory light chain (Bennett & Bagshaw (1986) Biochem. J. 233, 179-186). In scallop myosin additional specific sites are present, which can bind Ca2+ rapidly, to effect activation of the ATPase. In the course of this work, Ca2+ dissociation from EGTA was studied as a model system. This gave rates of 1 s-1 and 0.3 s-1 at pH 7.0 and pH 8.0 respectively.     

Myosin-linked calcium regulation in squid mantle muscle. Light-chain components of squid myosin.

As reported by Kendrick-Jones et al. (1976), myosin from squid mantle muscle contains two types of light-chain components, different in size but similar in net charge. We were able to separate the two types of light chains by a five-step procedure, yielding LC-1 (17,000 daltons) and LC-2 (15,000 daltons). It was found that squid mantle LC-1 and LC-2 function exactly like SH-light chains and EDTA-light chains of scallop adductor myosin, respectively. In functional tests, we used "desensitized" myosin of scallop adductor muscle, simply because "EDTA washing" removed neither LC-1 nor LC-2 from squid mantle myosin. The removal and recombination of light chains were examined by gel electrophoresis, and Ca or Sr sensitivity was determined by measuring the Mg-ATPase activity of skeletal acto-scallop or squid myosin. It was found that EDTA washing readily released the EDTA-light chains of scallop myosin completely, and that the EDTA-washed scallop myosin was capable of regaining its full content of EDTA-LC as well as its full sensitivity to calcium. We also found that as regards combining with, and conferring calcium sensitivity on the EDTA-washed myosin of scallop adductor, squid mantle LC-2 could effectively replace scallop adductor EDTA-LC. In addition, calcium or strontium ions were found to induce changes in the UV absorption spectrum of scallop adductor EDTA-LC, although the apparent binding constants estimated from the difference spectrum were too low to account for the Ca or Sr sensitivity of scallop actomyosin-ATPase. The divalent cations also induced changes in the UV absorption spectrum of squid LC-2, and the apparent binding constants estimated from the difference spectrum were sufficiently high (1.5 X 10(5) M-1 for Ca binding, and 1.6 X 10(3) M-1 for Sr binding) to account for the Ca and Sr sensitivities of squid mantle myosin B-ATPase. The findings with scallop adductor myosin are in conflict with those reported by Kendrick-Jones et al., and must be accounted for in formulating the molecular mechanism of myosin-linked calcium regulation in molluscan muscles.     

Reversible changes in the ATPase activity and in the regulatory light chain content upon heat (30 degrees C)-treatment of "Akazara" striated adductor myosin

Myosin from striated adductor muscle of "Akazara" scallop was incubated at 30 degrees C for 5 min in a medium containing 2 mM MgCl2 and various concentrations of Ca2+ ions. It was observed that the 30 degrees C-treatment resulted in a decrease in the Ca2+-sensitivity of myosin-ATPase as well as in the release of the regulatory light chain (EDTA-LC) of myosin. The 30 degrees C-treated myosin was then subjected to a cooling treatment, being kept for 18 h at 0 degrees C. It was found that EDTA-LC recombined with myosin and that Ca2+-sensitivity of myosin-ATPase was restored. It was also found that Ca2+ alone was about 70 times more effective than Mg2+ alone in preventing the heat-induced release of EDTA-LC from occurring and also in recombination of EDTA-LC with the heat-treated myosin.     

Relationship between cytoplasmic free calcium and myosin light chain phosphorylation in intact platelets.

Human platelets were prepared and loaded with the fluorescent Ca2+ indicator quin2. The relation between cytoplasmic free calcium concentration, [Ca2+]i, and the extent of the phosphorylation of myosin light chains of Mr 20 000 could then be examined. When the calcium ionophore ionomycin is used to stimulate platelets, little phosphorylation is seen until [Ca2+]i exceeds 400 nM; half-maximal response occurs at 600 nM with a full response at about 1 microM-[Ca2+]i. Under optimal conditions, physiological stimuli such as platelet-activating factor and thrombin can increase [Ca2+]i to sufficiently high levels [Rink, Smith & Tsien (1982) FEBS Lett. 148, 21-26; Hallam, Sanchez & Rink (1984) Biochem. J. 218, 819-827] that Ca2+ ions could be the trigger for the myosin phosphorylation evoked by these agonists. However, in this paper we show that, in the absence of external calcium, platelet-activating factor and thrombin can stimulate myosin phosphorylation while [Ca2+]i remains at levels which are well below those needed when the calcium ionophore is the stimulus. This observation suggests that myosin light chain phosphorylation may be controlled by an additional pathway.     

Calcium sensitivity of contractile proteins from chicken gizzard muscle.

Actin, myosin, and "native" tropomyosin (NTM) were separately isolated from chicken gizzard muscle and rabbit skeletal muscle. With various combinations of the isolated contractile proteins, Mg-ATPase activity and superprecipitation activity were measured. It was thus found that gizzard myosin and gizzard NTM behaved differently from skeletal myosin and skeletal NTM, whereas gizzard actin functioned in the same wasy as skeletal actin. It was also found that gizzard myosin preparations were often Ca-sensitive, that is, that the two activities of gizzard myosin plus actin without NTM were activated by low concentrations of Ca2+. The Mg-ATPase activity of a Ca-insensitive preparation of gizzard myosin was not activated by actin even in the presence of Ca2+. When Ca-sensitive gizzard myosin was incubated with ATP (and Mg2+) in the presence of Ca2+, a light-chain component of gizzard myosin was phosphorylated. The light-chain phosphorylation also occurred when Ca-insensitive myosin was incubated with gizzard NTM and ATP (plus Mg2+) in the presence of Ca2+. In either case, the light-chain phosphorylation required Ca2+. Phosphorylated gizzard myosin in combination with actin was able to exhibit superprecipitation, and Mg-ATPase of the phosphorylated gizzard myosin was activated by actin; the actin activation and superprecipitation were found to occur even in the absence of Ca2+ and NTM or tropomyosin. The phosphorylated light-chain component was found to be dephosphorylated by a partially purified preparation of gizzard myosin light-chain phosphatase. Gizzard myosin thus dephosphorylated behaved exactly like untreated Ca-insensitive gizzard myosin; in combination with actin, it did not superprecipitate either in the presence of Ca2+ or in its absence, but did superprecipitated in the presence of NTM and Ca2+. Ca-activated hydrolysis of ATP catalyzed by gizzard myosin B proceeded at a reduced rate after removal of Ca2+ (by adding EGTA), whereas that catalyzed by a combination of actin, gizzard myosin, and gizzard NTM proceeded at the same rate even after removal of Ca2+. However, addition of a partially purified preparation of gizzard myosin light-chain phosphatase was found to make the recombined system behave like myosin B. Based on these findings, it appears that myosin light-chain kinase and myosin light-chain phosphatase can function as regulatory proteins for contraction and relaxation, respectively, of gizzard muscle.     

Myosin and actin from ascidian smooth muscle and their interaction

Myosin and actin were purified from ascidian smooth muscle. Ascidian myosin contained two classes of light chains and the pH dependence of Ca2+-activated ATPase and the KCl dependence of actin-activated ATPase of ascidian myosin differed from those of vertebrate skeletal myosin. Troponin-tropomyosin complex from ascidian increased the ATPase activity of ascidian reconstituted actomyosin in a Ca2+-dependent manner. Ascidian myosin provided the reconstituted actomyosin with the responsiveness to calcium ions. Two actin isoforms were present in ascidian, which were distinguished by isoelectric points.     

Phosphorylation in skeletal myosin light chain modulates the actin--myosin interaction in the presence of regulatory proteins in vitro

The effect of phosphorylation in skeletal myosin light chain (LC2) on the actomyosin and acto-heavymeromyosin (HMM) ATPase activities was investigated in the presence or absence of regulatory proteins (tropomyosin-troponin complex). Phosphorylation in LC2 did not modulate the actin-myosin and actin-HMM interactions over a wide range of KCl concentrations from 30 to 150 mM without regulatory proteins. In the presence of regulatory proteins, phosphorylation in myosin LC2 enhanced the ATPase activity of actomyosin with calcium ions, but the removal of calcium ions made little difference in the ATPase activity between phosphorylated and dephosphorylated myosins. Ca2+-sensitivity of the regulated actomyosin was slightly changed by phosphorylation in myosin LC2. However, both the ATPase activity and Ca2+-sensitivity of the regulated acto-HMM were unaffected by phosphorylation in HMM LC2.     

Dinitrophenylation of rabbit skeletal actomyosin.

Dinitrophenylated reconstituted or natural actomyosin effected changes in the Ca2+ sensitivity which were dependent upon the ionic strength of the reaction medium. Dinitrophenylation of reconstituted actomyosin in 0.6 M KCl led to the incorporation of 2-6 mol of the reagent per 5-10(5) g of protein and it possessed considerable Ca2+ sensitivity. Dinitrophenylated natural actomyosin under the same conditions lost most of its Ca2+ sensitivity when 1.3-5.4 mol of the dinitrophenyl group were bound. The myosin from these modified actomyosins did not lose Ca2+ sensitivity and the myosin was labeled only with 0.4-1.7 mol of the dinitrophenyl group. Dinitrophenylation of both kinds of actomyosin in 0.06 M KCl abolished the Ca2+ sensitivity; the myosin from the modified actomyosins also lost Ca2+ sensitivity. Myosin alone was more susceptible to a loss of Ca2+ sensitivity than myosin in actomyosin. Actin protected the ability of myosin to sense Ca2+ regulated actin in modified actomyosin at 0.6 M KCl but not at 0.06 M KCl. Actomyosin dinitrophenylated in the presence of ATP lost Ca2+ sensitivity. However, the myosin from this actomyosin possessed Ca2+ sensitivity. Thiolysis of the dinitrophenylated actomyosin by 2-mercaptoethanol at low ionic strength did not restore the Ca2+ sensitivity of this actomyosin or its myosin although there was a significant loss of the dinitrophenyl group.     

Octopus calmodulin. The trimethyllysyl residue is not required for myosin light chain kinase activation

Octopus calmodulin was purified to homogeneity and shown to contain 0.1 residue each of epsilon-N-monomethyl-lysine, epsilon-N-dimethyllysine, and epsilon-N-trimethyllysine/mol. With the exception of this partial methylation and of a single tyrosyl residue, it shared all the characteristic properties of mammalian calmodulin in terms of molecular weight, amino acid composition, electrophoretic behavior in the presence or absence of Ca2+ ions, and activation of calcium/calmodulin-dependent myosin light chain kinase. In fact, Octopus calmodulin proved to be slightly more effective than ram testis calmodulin in activating both skeletal and smooth muscle myosin light chain kinases in the presence of Ca2+. This provides conclusive evidence that (a) stoichiometric trimethylation of lysine 115 is not required for enzyme activation, and (b) the inability of troponin C to activate myosin light chain kinase (Walsh, M. P., Vallet, B., Cavadore, J. C., and Demaille, J. G.     

Intermolecular tuning of calmodulin by target peptides and proteins: differential effects on Ca2+ binding and implications for kinase activation.

Ca(2+)-activated calmodulin (CaM) regulates many target enzymes by docking to an amphiphilic target helix of variable sequence. This study compares the equilibrium Ca2+ binding and Ca2+ dissociation kinetics of CaM complexed to target peptides derived from five different CaM-regulated proteins: phosphorylase kinase. CaM-dependent protein kinase II, skeletal and smooth myosin light chain kinases, and the plasma membrane Ca(2+)-ATPase. The results reveal that different target peptides can tune the Ca2+ binding affinities and kinetics of the two CaM domains over a wide range of Ca2+ concentrations and time scales. The five peptides increase the Ca2+ affinity of the N-terminal regulatory domain from 14- to 350-fold and slow its Ca2+ dissociation kinetics from 60- to 140-fold. Smaller effects are observed for the C-terminal domain, where peptides increase the apparent Ca2+ affinity 8- to 100-fold and slow dissociation kinetics 13- to 132-fold. In full-length skeletal myosin light chain kinase the inter-molecular tuning provided by the isolated target peptide is further modulated by other tuning interactions, resulting in a CaM-protein complex that has a 10-fold lower Ca2+ affinity than the analogous CaM-peptide complex. Unlike the CaM-peptide complexes, Ca2+ dissociation from the protein complex follows monoexponential kinetics in which all four Ca2+ ions dissociate at a rate comparable to the slow rate observed in the peptide complex. The two Ca2+ ions bound to the CaM N-terminal domain are substantially occluded in the CaM-protein complex. Overall, the results indicate that the cellular activation of myosin light chain kinase is likely to be triggered by the binding of free Ca2(2+)-CaM or Ca4(2+)-CaM after a Ca2+ signal has begun and that inactivation of the complex is initiated by a single rate-limiting event, which is proposed to be either the direct dissociation of Ca2+ ions from the bound C-terminal domain or the dissociation of Ca2+ loaded C-terminal domain from skMLCK. The observed target-induced variations in Ca2+ affinities and dissociation rates could serve to tune CaM activation and inactivation for different cellular pathways, and also must counterbalance the variable energetic costs of driving the activating conformational change in different target enzymes.     

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

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

The sulfhydryl groups involved in the active site of myosin B adenosinetriphosphatase. II. Effect of modification of the Sa thiol group on superprecipitation and clearing.

The effect of Sa modification with NEM, which activates Mg2+-ATPase through an enhancement of the association of actin and myosin, was investigated on the superprecipitation, clearing and Mg2+-ITPase of myosin B with reference to the effect of S1-blocking. 1. Superprecipitation induced by ATP was markedly enhanced by Sa-blocking even at high concentrations of Mg2+ and substrate; this may be due to an increase in the affinity of myosin and actin on blocking Sa. 2. Nevertheless, neither ITP-induced superprecipitation nor Mg2+-ITPase was affected by Sa modification. 3. Blocking of S1 brought about the inhibition of ATP- and ITP-induced superprecipitation and Mg2+-ITPase activity, suggesting that S1-blocking decreases the affinity of myosin and actin. 4. Sa-blocked myosin B showed greater resistance to clearing by ATP, especially in the presence of Ca2+ ions, whereas in the clearing response of actomyosin gel to PPi no difference between Sa-blocked and unmodified myosins B was observed. On the other hand, the clearing response of myosin B became more sensitive to both ATP and PPi on blocking S1. Based on the above results and preliminary data suggesting that Sa is located in LMM, the interaction of myosin filaments and actin filaments under physiological conditions is discussed.     

多头绒泡菌肌球蛋白的纯化及性质的研究

从多头绒泡菌中纯化了肌球蛋白,并对其亚基组成及ＡＴＰ酶性质进行了研究。该肌球蛋白是由一种重链（２２５ｋＤ）和两种轻链（２０ｋＤ,１７．５ｋＤ）组成的大分子,其亚基之比为ＨＣ：ＬＣ１：ＬＣ２＝２：４：２。兔肌Ｆ－肌动蛋白能较大激活粘菌肌球蛋白ＡＴＰ酶活性,Ｃａ~（２＋）离子也能提高其活性,Ｍｇ~（２＋）离子无明显影响。钒酸盐,碘乙酸,对氯汞苯甲酸对其ＡＴＰ酶活性有显著抑制作用。     

Lysyl residues of cardiac myosin accessible to labeling with a fluorescent reagent, N-methyl-2-anilino-6-naphthalenesulfonyl chloride

Two mol of N-methyl-2-anilino-6-naphthalenesulfonyl (Mns) groups was preferentially incorporated into pig cardiac myosin in the absence of divalent metal ions, 1 mol rapidly and 1 mol slowly. In the presence of divalent metal ions. 1 mol was rapidly incorporated but subsequent incorporation was strongly suppressed. No substantial effect of incorporation of Mns groups in the presence or absence of divalent metal ions on the Ca2+- and K+-ATPase activities of myosin was found. However, the fluorescence spectra due to attached Mns groups were different in the two cases. Extensive pronase digestion of labeled myosin indicated that the Mns groups were attached predominantly to lysyl residues, regardless of the labeling conditions. Peptide mapping of the labeled myosin digested with subtilisin, pepsin or trypsin uniformly showed the selective incorporation of an Mns group into essentially one species of peptide. However, the peptide labeled in the absence of divalent metal ions was clearly different from that labeled in their presence. The present results confirm that pig cardiac myosin heavy chains contain two distinct lysyl residues, which are both accessible to labeling with Mns groups only when divalent metal ions are absent. The results also suggest that conformational changes occur around these residues when divalent metal ions are added.     

Ca2+ dependence of the ATPase activity of phosphorylated smooth muscle myosin: effects of tropomyosin and actin

Calcium ions produce a 3-4-fold stimulation of the actin-activated ATPase activities of phosphorylated myosin from bovine pulmonary artery or chicken gizzard at 37 degrees C and at physiological ionic strengths, 0.12-0.16 M. Actins from either chicken gizzard or rabbit skeletal muscle stimulate the activity of phosphorylated myosin in a Ca2+-dependent manner, indicating that the Ca2+ sensitivity involves myosin or a protein associated with it. Partial loss of Ca2+ sensitivity upon treatment of phosphorylated gizzard myosin with low concentrations of chymotrypsin and the lack of any change on similar treatment of actin supports the above conclusion. Although both actins enhance ATPase activity, activation by gizzard actin exhibits Ca2+ dependence at higher temperatures or lower ionic strengths than does activation by skeletal muscle actin. The Ca2+ dependence of the activity of phosphorylated heavy meromyosin is about half that of myosin and is affected differently by temperature, ionic strength and Mg2+, being independent of temperature and optimal at lower concentrations of NaCl. Raising the concentration of Mg2+ above 2-3 mM inhibits the activity of heavy meromyosin but stimulates that of myosin, indicating that Mg2+ and Ca2+ activate myosin at different binding sites.