Influence of Mg2+ and Ca2+ bound to 1,5-IAEDANS-labeled phosphorylated and dephosphorylated heavy meromyosin complexed with F-actin on polarized fluorescence of the fluorophore

Dephosphorylated and phosphorylated heavy meromyosin, fluorescently labeled with 1,5-IAEDANS attached at the SH1 group, was introduced into myosin-free ghost fibres and the polarized fluorescence of the bound label was measured. The results depended on whether the divalent cation binding sites on heavy meromyosin were saturated with Mg2+ or Ca2+. The calculated angles of absorption and emission dipoles and the amount of random fluorophores were significantly changed, indicating that the random mobility and orientation of the fluorophores of phosphorylated and dephosphorylated heavy meromyosin heads complexed with F-actin in the ghost fibre depend on saturation of heavy meromyosin with Ca2+ or Mg2+. The presence of bound Ca2+ has an opposite effect on the polarized fluorescence of phosphorylated and dephosphorylated 1,5-IAEDANS-heavy meromyosin.     

A spin-label study of phosphatidylcholine exchange protein. Regulation of the activity by phosphatidylserine and calcium ion.

The effects of the divalent cations Mg2+, Mn2+ and Ca2+ on the Brownian rotational motion of fluorescently labeled myosin, heavy meromyosin and myosin subfragment-1 were measured by the method of time-resolved fluorescence depolarization. When Mg2+ was added to solutions of myosin or heavy meromyosin and EDTA, their rotational mobility increased. Ca2+ had no effect. Mn2+ increased the mobility of heavy meromyosin but decreased that of myosin. None of these divalent cations effected the mobility of subfragment-1. The binding of heavy meromyosin to actin was affected very little by Mg2+ or EDTA over a wide range of conditions. Divalent cations appear to change the swivel about which the heads of myosin rotate, presumably by binding to light chain 2 (also called DTNB light chain). However, the heads are still able to bind actin in nearly the same way whether Mg2+ is present or not. The concentration of free Mg2+ for the mid-point of the change in heavy meromyosin mobility is in good agreement with that for EDTA activation of ATPase activity. This suggests that EDTA activation is due to removal of Mg2+ bound to myosin itself.     

Proteolysis of smooth muscle myosin by Staphylococcus aureus protease: preparation of heavy meromyosin and subfragment 1 with intact 20 000-dalton light chains

The proteolysis of gizzard myosin by Staphylococcus aureus protease produces both heavy meromyosin and subfragment 1 in which the 20 000-dalton light chains are intact, and conditions are suggested for the preparation of each. Cleavage of the myosin heavy chain to produce subfragment 1 is dependent on the myosin conformation. Proteolysis of myosin in the 10S conformation yields predominantly heavy meromyosin, and myosin in the 6S conformation yields mostly subfragment 1 and some heavy meromyosin. Two sites are influenced by myosin conformation, and these are located at approximately 68 000 and 94 000 daltons from the N-terminus of the myosin heavy chain. The latter site is thought to be located at the subfragment 1-subfragment 2 junction, and cleavage at this site results in the production of subfragment 1. The time courses of phosphorylation of both heavy meromyosin and subfragment 1 can be fit by a single exponential. The actin-activated Mg2+-ATPase activity of heavy meromyosin is markedly activated by phosphorylation of the 20 000-dalton light chains. From the actin dependence of Mg2+-ATPase activity the following values are obtained: for phosphorylated heavy meromyosin, Vmax approximately 5.6 s-1 and Ka (the apparent dissociation constant for actin) approximately 2 mg/mL; for dephosphorylated heavy meromyosin, Vmax approximately 0.2 s-1 and Ka approximately 7 mg/mL. The actin-activated ATPase activity of subfragment 1 is not influenced by phosphorylation, and Vmax and Ka for both the phosphorylated and dephosphorylated forms are 0.4 s-1 and 5 mg/mL, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calcium ions modulate regulation of smooth muscle contraction mediated by phosphorylation of myosin regulatory light chains

The effect of calcium ions on conformational changes of F-actin initiated by decoration of thin filaments with phosphorylated and dephosphorylated heavy meromyosin from smooth muscles was studied by fluorescence polarization spectroscopy. It is shown that heavy meromyosin with phosphorylated regulatory light chains (pHMM) promotes structural changes of F-actin which are typical for the "strong" binding of actin to the myosin heads. Heavy meromyosin with dephosphorylated regulatory light chains (dpHMM) causes conformational changes of F-actin which are typical for the "weak" binding of actin to the myosin heads. The presence of calcium enhances the pHMM effect and attenuates the dpHMM effect. We propose that a Ca2+-dependent mechanism exists in smooth muscles which modulates the regulation of actin--myosin interaction occurring via phosphorylation of myosin regulatory light chains.     

Heavy meromyosin binds actin with negative cooperativity.

The association of fluorescently labeled heavy meromyosin (HMM) and F-actin was measured by time-resolved fluorescence depolarization. The effects of varying the protein concentrations, temperature, KCl concentration, and pH were determined. Measurements of HMM mobility supported a model of no interaction between the two heads in the absence of actin. Measurements of actin binding, when compared with results for myosin subfragment I, indicated that the two heads of HMM do not bind independently in the rigor complex. This could result from actin-transmitted negative cooperativity or from steric inhibition due to the structure of HMM. For HMM and actin in 0.15 7 kcl at 25 degrees C: Ka = 3.9 X 10(7) M-1, deltaHco' = 36 +/- 2 J M-1, deltaSco' = 0.26 +/- 0.02 kJ M-1 K-1; the slope of ln Ka vs. [KCl]1/2 = -3.88 and the pH of maximum association was 6.9.     

The purification and characterization of a globular subfragment of Acanthamoeba myosin II that is fully active when cross-linked to F-actin

Acanthamoeba myosin II contains two heavy chains of Mr 185,000 and two pairs of light chains of Mr 17,500 and 17,000. We now report the purification of a globular proteolytic 103-kDa subfragment of myosin II which contained a 68-kDa NH2-terminal segment of the heavy chain and one pair of intact light chains. The myosin II head fragment expressed full Ca2+-ATPase activity but its actin-activated Mg2+-ATPase activity had a Vmax of only 0.07 s-1 compared to 1.9 s-1 (per head) for filaments of native unphosphorylated myosin II. The head fragment had a similar KATPase to that of filaments (5 versus 4 microM) and about 75% of the head fraction could bind to F-actin in the presence of ATP with a Kbinding of 5.6 microM. The Kbinding of the head fragment may be similar to that of individual heads in the native myosin II filaments although the experimentally determined apparent Kbinding for filaments is much lower, 0.3 microM. The head fragment was covalently cross-linked to F-actin in the absence of nucleotide using the zero length cross-linker 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide. The cross-linked actin-myosin head complex hydrolyzed MgATP at a rate equivalent to Vmax for the active dephosphorylated native myosin II. These data indicate that the isolated head fragment had intact catalytic and actin-binding domains but that it bound to F-actin in the presence of ATP in a relatively inactive conformation. When covalently cross-linked to F-actin the head fragment was apparently locked into a catalytically fully active conformation.     

Phosphorylation of light chains of myosin from rabbit skeletal muscles affects the type of conformation changes of F-actin induced by heavy meromyosin

The changes in F-actin conformation in myosin-free single ghost fiber induced by the binding of heavy meromyosin (HMM) with dephosphorylated or phosphorylated light chains-2 (LC2) have been studied by measuring intrinsic tryptophan polarized fluorescence of F-actin. It has been found that at low concentrations of Ca2+ (pCa greater than or equal to 8), the binding of HMM with dephosphorylated LC2 to F-actin in ghost fibres increases, whereas the binding of HMM with phosphorylated LC2 decreases the anisotropy of polarized tryptophan fluorescence. The effect is reversed at high concentrations of Ca2+ (pCa = 5). It has been assumed that this effect of myosin light chains phosphorylation may be due to its influence on the type of myosin head binding to F-actin.     

Rotational dynamics of spin-labeled F-actin in the sub-millisecond time range.

The rotational motions of F-actin filaments and myosin heads attached to them have been measured by saturation transfer electron paramagnetic resonance spectroscopy using spin-labels rigidly bound to actin, or to the myosin head region in intact myosin molecules, heavy meromyosin, and subfragment-1. The spin-label attached to F-actin undergoes rotational motion having an effective correlation time of the order of 10^?4 seconds. This cannot be interpreted as rotation of the entire F-actin filament or local rotation of the spin-label, but must represent an internal rotational mode of F-actin, possibly a bending or flexing motion, or a rotation of an actin monomer or a segment of it. The rate of this rotational motion is reduced approximately fourfold by myosin, HMM or S-1; HMM and S-1 are equally effective, on a molar basis, in slowing this rotation and both produce their maximal effect at a ratio of about one molecule of HMM or S-1 per ten actin monomers. With chymotryptic S-1, the effect is partially reversed at higher concentrations. With S-1 prepared with papain in the presence of Mg²⁺, the reversal is smaller, while with HMM or myosin there is no reversal at higher concentrations. Tropomyosin slightly decreases the actin rotational mobility, and the addition of HMM to the actin-tropomyosin complex produces a further slowing. The rotational correlation time for acto-HMM is the same whether the spin-label is on actin or HMM, indicating that the rotation of the head region of HMM when bound to F-actin is controlled by a mode of rotation within the F-actin filaments.     

Further studies on the interaction of actin with heavy meromyosin and subfragment 1 in the presence of ATP.

It has been postulated that, during the hydrolysis of ATP, both normal and SH1-blocked heavy meromyosin undergo a rate-limiting transition from a refractory state which cannot bind to actin to a nonrefractory state which can bind to actin. This model leads to several predictions which were studied in the present work. First, the fraction of heavy meromysin or subfragment 1 which remains unbound to actin when the ATPase equals Vmax should have the same properties as the original protein. In the present study it was determined that the unbound protein has normal ATPase activity which suggests that it is unbound to actin for a kinetic reason rather than because it is a permanently altered form of the myosin. Second, if the heavy meromyosin heads act independently half as much subfragment 1 as heavy meromyosin should bind to actin. Experiments in the ultracentrifuge demonstrate that about half as much subfragment 1 as heavy meromyosin sediments with the actin at Vmax. Third, the ATP turnover rate per actin monomer at infinite heavy meromyosin concentration should be much higher than the ATP turnover rate per heavy meromyosin head at infinite actin concentration. This was found to be the case for SH1-blocked heavy meromyosin since, even at very high concentrations of SH1-blocked heavy meromyosin, in the presence of a fixed actin concentration, the actin-activated ATPase rate remained proportional to the SH1-blocked heavy meromyosin concentration. All of these results tend to confirm the refractory state model for both SH1-blocked heavy meromyosin and unmodified heavy meromyosin and subfragment 1. However, the nature of the small amount of heavy meromyosin which does bind to actin in the presence of ATP at high actin concentration remains unclear.     

Effects of actin and calcium ion on chymotryptic digestion of skeletal myosin and their implications to the function of light chains

Experiments have been carried out to assess the involvement of the myosin light chains [obtained by treatment of myosin with 5,5'-dithiobis(2-nitrobenzoic acid) (Nbs2)] in the control of cross-bridge movement and actomyosin interactions. Chymotryptic digestions of myosin, actomyosin, and myofibrils do not detect any Ca2+-induced change in the subfragment 2 region of myosin. Actin, like Ca2+, protects the in situ Nbs2 light chains from proteolysis and causes a partial switch in the digestion product of myosin from subfragment 1 to heavy meromyosin. This effect is independent of the state of aggregation of myosin, and it persists in acto heavy meromyosin and in actinomyosin in 0.6 M NaCl. Digestions and sedimentation studies indicate that there is no direct acto light chain interaction. Proteolysis of myosin shows a gradual transition from production of heavy meromyosin to subfragment 1 with lowering of the salt level. In the presence of Ca2+ heavy meromyosin is generated both in digestions of polymeric and of monomeric myosin. These results are explained in terms of localized changes within the Nbs2 light chains and subfragment 1. Subunit interactions in the myosin head lead to a Ca2+-induced reduction in the affinity of heavy meromyosin for actin in the presence of MgATP. The resulting Ca2+ inhibition of the actin-activated ATPase of myosin can be detected at high salt concentrations(75 mM KCl).     

The effect of phosphorylation of myosin light chains on the structural state of tropomyosin in thin filaments, decorated with heavy meromyosin

The structural state of tropomyosin (TM) modified by 5-(iodoacetamidoethyl)-aminonaphthalene-1-sulfonate (1.5-IAEDANS) upon F-actin decoration with myosin subfragment 1 (S1) and heavy meromyosin (HMM) in glycerinated myosin- and troponin-free muscle fibers was studied. HMM preparations contained native phosphorylated myosin light chains, while S1 preparations did not. The changes in the polarized fluorescence of 1.5-IAEDANS-TM during the F-actin interaction with S1 were independent of light chains phosphorylation and Ca2+ concentration, but were dependent on these factors during the F-actin interaction with HMM. The binding of myosin heads to F-actin is supposed to initiate conformational changes in TM which are accompanied by changes in the flexibility and molecular arrangement of TM. In the presence of light chains, the structural changes in TM depend on light chains phosphorylation and Ca2+ concentration. The conformational changes in TM seem to be responsible for the mechanisms of coupling of the myosin and tropomyosin modulation system during the actin-myosin interaction in skeletal muscles.     

Difference between subfragment-1 and heavy meromyosin in their interaction with F-actin

To elucidate the difference between subfragment-1 and heavy meromyosin in their interaction with F-actin, we used limited tryptic digestion and cross-linking with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide. The binding of actin to subfragment-1 lowers the susceptibility of the 50K-20K junction of its heavy chain to tryptic digestion. At a molar ratio of one actin to one subfragment-1, all the sites were gradually cleaved by trypsin whereas the sites were completely protected in the presence of a 2-fold molar excess of actin over subfragment-1. In the case of heavy meromyosin, nearly half of the sites were protected completely by the presence of an equimolar amount of actin to its heads suggesting that the two heads of heavy meromyosin bound actin in a different manner. The rate of the cross-linking reaction between subfragment-1 heavy chain and actin with 1-ethyl-3-[3-(dimethylamino) propyl]carbodiimide also depended on the molar ratio of actin to subfragment-1. The rate was maximum at a molar ratio of about 5 actin to 1 subfragment-1. When heavy meromyosin was cross-linked to actin, the maximum rate was observed at a molar ratio of about 3 actin to 1 heavy meromyosin head, the level being about 60% that for subfragment-1 and actin. It was suggested that the presence of the subfragment-2 portion of heavy meromyosin caused these differences by restricting the motion of the two heads.     

Interaction of myosin subfragments with F-actin.

The effect of ionic strength, temperature, and divalent cations on the association of myosin with actin was determined in the ultracentrifuge using scanning absorption optics. The association constant (Ka) for the binding of heavy meromyosin (HmM) to F-actin was 1 X 10(7) M-1 at 20 degrees C, in 0.10 M KCl, 0.01 M imidazole (pH 7.0), 5 MM potassium phosphate, 1 mM MgCl2, and 0.3 mM ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid. Ka was the same for HMM prepared by trypsin or chymotrypsin. The affinity of subfragment 1 (S1) for actin under the same ionic conditions was 3 X 10(6) M-1. Varying the preparative procedure for S1 had little effect on Ka. The small difference in binding energy between HMM and S1 suggests that either only one head can bind strongly to actin at a time or that free energy is lost during the sterically unfavorable attachment of the two heads to actin.     

The covalent modification of myosin's proteolytic fragments by a purine disulfide analog of adenosine triphosphate. Reaction at a binding site other than the active site.

A purine disulfide analog of ATP, 6,6'-dithiobis(inosinyl imidodiphosphate), forms mixed disulfide bonds between the 6 thiol group on the purine ring and certain key cysteines on myosin, heavy meromyosin, and subfragment one. The EDTA ATPase activities of myosin and heavy meromyosin were completely inactivated when 4 mol of thiopurine nucleotide was bound. When similarly inactivated, subfragment one, depending on its method of preparation, incorporated either 1 or 2 mol of thiopurine nucleotide. Modification of a single cysteine on subfragment one resulted in an inhibition of both the Ca2+ and the EDTA ATPase activities, but the latter always to a greater extent. Modification of two cysteines per head of heavy meromyosin had the same effect suggesting that the active sites were not blocked by the thiopurine nucleotides. Direct evidence for this suggestion was provided by equilibrium dialysis experiments. Heavy meromyosin and subfragment one bound 1.9 and 0.8 mol of [8-3H]adenylyl imidodiphosphate per mol of enzyme, respectively, with an average dissociation constant of 5 X 10(-7) M. Heavy meromyosin with four thiopurine nucleotides bound or subfragment one with two thiopurine nucleotides bound retained 65-80% of these tight adenylyl imidodiphosphate binding sites confirming the above suggestion. Thus previous work assuming reaction of thiopurine nucleotide analogs at the active site of myosin must be reevaluated. Ultracentrifugation studies showed that heavy meromyosin which had incorporated four thiopurine nucleotides did not bind to F-actin while subfragment one with one thiopurine nucleotide bound interacted only very weakly with F-actin. Thus reaction of 6,6'-dithiobis(inosinyl imidodiphosphate) at nucleotide binding sites other than the active sites reduces the rate of ATP hydrolysis and inhibits actin binding. It is suggested that these second sites may function as regulatory sites on myosin.     

Activation of heavy meromyosin adenosine triphosphatase by various states of actin.

F-actin monomer (F-monomer) is formed upon the addition of neutral salt to G-actin. Since F-monomer has a digestibility similar to that of F-actin and much lower than that of G-actin, it has been proposed that F-monomer has a conformation different from that of G-actin and similar to the conformation of the subunits in F-actin. To examine whether F-monomer will enhance the magnesium-activated myosin adenosine triphosphatase (Mg2+-ATPase) as much as F-actin, the ability of partially polymerized actin populations at equilibrium to activate the Mg2+-ATPase of heavy meromyosin was investigated. Correlations were made between ATPase activities and the polymerization state of actin as determined by measurements of viscosity and digestibility. No significant activation of the heavy meromyosin ATPase was observed under conditions where G-actin or mixtures of G-actin and F-monomer were present. As polymer formation occurred at higher actin concentrations, or with increased KCl concentrations, substantial activation characteristic of F-actin was observed. The data suggest that F-monomer may undergo a further conformational change as it forms nuclei or joins onto polymers. Alternatively, the site of actin which activates the myosin ATPase may involve the crevice between two adjacent actin subunits.     

Polarization microfluorimetry study of interaction between myosin head and F-actin in muscle fibers.

Changes in conformation of F-actin induced by the binding of myosin molecule subfragment 1 were studied in myosin-free single ghost muscle fibers with the method of polarization microfluorimetry. The modification of the structure of subfragment 1 by proteolytic digestion with one or two cuts in subfragment 1 or degradation of 50 kDa domain did not influence the character of changes in the conformation of F-actin. The use of preparations of subfragment 1 devoid of the 20 kDa domain or both cross-linked SH1 and SH2-groups changed the character of conformational rearrangements in F-actin. The present data show that a site of interaction with actin in the 20 kDa domain plays a key role in inducing the changes in actin conformation corresponding to a "strong" form of the binding. It is supposed that transmission of changes in the conformation of the myosin head to F-actin might be important for muscle contraction.     

Effect of Ca2+ on the motility of myosin head in the F-actin-HMM complex

Using polarization microfluorimetry, the effect of Ca2+ on the mode of interaction between heavy meromyosin labeled with 1.5-IAEDANS and F-actin was studied. The results obtained suggest that both the orientation and motility of myosin heads in the F-actin-heavy meromyosin complex depend on Ca2+ concentration. The experimental results are suggestive of the existence of a myosin-coupled regulatory system in skeletal muscles.     

Cooperativity in F-actin filaments on binding of myosin subfragments, demonstrated by fluorescence of 1, N6- ethenoadenosine diphosphate.

The fluorescence lifetime of 1,N⁶-ethenoadenosine diphosphate (?-ADP) is 33 ns when bound to F-actin at 4 °C. When heavy meromyosin or myosin subfragment-1 binds to the F-actin filament, the lifetime of ?-ADP drops, reaching 29 ns when every actin monomer is bound to a myosin head. The change in lifetime is a consequence of cooperative conformational changes among the actin monomers. The results of these experiments support the contention that there are differences in the ways in which the two heads of the myosin molecule interact with the actin filament.     

Effect of F-actin upon the binding of ADP to myosin and its fragments.

The effect of F-actin upon the binding of ADP to rabbit skeletal muscle myosin, heavy meromyosin, and subfragment 1 was studied by equilibrium dialysis, ultracentrifuge transport, and light scattering techniques. Both myosin and H-meromyosin (HMM) bind a maximum of approximately 1.6 mol of ADP/mol of protein, while S-1 binds approximately 0.9 mol of ADP/mol of protein. The affinity for ADP of all three preparations was similar at a given ionic strength (approximately 10(6) M-1 at 0.05 M KCl) and decreased with increasing ionic strength. Under conditions similar to those used for the measurement of ADP binding, the binding sites of myosin, HMM, and subfragment 1 (S-1) are saturated with actin at molar ratios of 2, 2, and 1 mol of actin monomer/mol of protein, respectively, as determined by light scattering, ultracentrifuge transport, and in the case of myosin by ATPase measurements. F-actin was found to inhibit ADP binding, but even at an actin concentration at least twice that required for saturation of myosin, HMM, or S-1, significant ADP binding remained. This ADP binding was inhibited by 10(-4) M pyrophosphate. The observations are consistent with the formation of an actomyosin-ADP complex in which actin and ADP are bound to myosin at distinct but interacting sites.     

Interhead distances in myosin attached to F-actin estimated by fluorescence energy transfer spectroscopy.

Fluorescence resonance energy transfer (FRET) spectroscopy has been used to determine distances between probes attached to the most reactive sulfhydryl (SH1) group on individual myosin "heads." We measured intramolecular and intermolecular interhead distances as well as the distance between one head of heavy meromyosin (HMM) mixed with subfragment-1 (S1) heads attached to F-actin under rigor conditions. The SH1 cysteine was specifically labeled with either a donor (5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid) or an acceptor probe (5-iodoacetamidofluorescein). In free solution, the distance between these probes was too large to allow significant FRET, but in the rigor complex with F-actin, intermolecular interhead distances between S1 molecules, HMM molecules, or S1 and HMM were determined to be 6.0-6.3 nm. The radial coordinate of the labels relative to F-actin was 5.0-6.4 nm. However, the intramolecular interhead distance in HMMs in which the two heads were labeled with D and A probes was estimated to be larger. The binding affinity of the second head of HMM(D/A) to F-actin may be reduced because of heterogeneous modification of the SH1 groups, such that the probability of single-head binding is increased.