Interaction of isozymes of myosin subfragment 1 with actin: effect of ionic strength and nucleotide

Myosin subfragment 1 (S-1) can be fractionated into two isozymes, (A1)S-1 containing alkali light chain 1 and (A2)S-1 containing alkali light chain 2. The predominant difference in the behavior of the two isozymes of S-1 is that, at low ionic strength, the actin concentration required for half-maximal ATPase activity is considerably lower for (A1)S-1 than for (A2)S-1; that is, the apparent binding constant KATPase for (A1)S-1 is greater than KATPase for (A2)S-1 [Weeds, A.G., & Taylor, R.S. (1975) Nature (London) 257, 54-56]. This difference disappears at high ionic strength [Wagner, P. D., Slater, C. S., Pope, B., & Weeds, A.G. (1979) Eur. J. Biochem. 99, 385-394]. In the present study we investigated whether the difference in the KATPase values of (A1)S-1 and (A2)S-1 is due to a difference in the actual affinity of these S-1 isozymes for actin. Binding was measured in the presence of ATP and AMP-PNP and in the absence of nucleotide at varied ionic strengths. We found that at low ionic strength where KATPase is several times stronger for (A1)S-1 than for (A2)S-1, the binding of (A1)S-1 to actin is correspondingly stronger than that of (A2)S-1 irrespective of the nucleotide present. Furthermore, as the ionic strength is increased, just as the difference between the KATPase values for (A1)S-1 and (A2)S-1 disappears so too does the difference in the affinity of the two isozymes for actin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Involvement of C-terminal 14 residues of alkali light chain in binding to the heavy chain of myosin

The alkali light chain of rabbit skeletal muscle myosin, A1, was cyanylated with 2-nitro-5-thiocyanobenzoic acid, and the peptide bond at Cys 177 was subsequently cleaved in the presence of 0.05 M CaCl2. Two peptide fragments, from the N-terminal to the residue 176 (CF1) and from the residue 177 to the C-terminal (CF2), were obtained. The CD spectrum and the difference UV absorption spectrum induced by CaCl2 suggested that CF1 largely retained the higher order structure of A1. The CF1 fragment, however, could neither incorporate subfragment-1 (S-1) by an exchange reaction, nor bind with the renatured 20K fragment of S-1 heavy chain. On the other hand, the C-terminal fragment of 14 residues, CF2, could bind with the 20K fragment of S-1 heavy chain. These results indicate that the binding site of the alkali light chain for the heavy chain of myosin is located within the C-terminal 14 residues.     

Effect of calcium and F-actin on the rate of SH2 modification in skeletal myosin

The rate constant of modification of a specific thiol group, SH₂, with N-ethylmaleimide (NEM) has been used to estimate the conformational change in the local area containing SH₂ (SH₂ region) of skeletal myosin as a structural probe. The rate of Mg²⁺-ATP-induced SH₂ modification of subfragment-1 (S-l) isozymes was regulated by Ca²⁺ in the pCa range below 6.4 and was not regulated in the pCa range above 6.4. No substantial difference between S-1 containing alkali light chain, A1, (S-1(A1)) and S-1 containing alkali light chain, A2, (S-1(A2)) was observed in the Ca²⁺-dependent rate of SH₂ modification. Due to the presence of this Ca²⁺ regulation in myosin (absence in S-1 isozymes) in the pCa range above 6.4, absence of 5,5-dithiobis-(2-nitrobenzoic acid) (DTNB) light chain in S-1 isozymes, and high affinity of Ca²⁺ for DTNB light chain, this Ca²⁺ regulation in the pCa range above 6.4 is possibly related to the Ca²⁺ binding to DTNB light chain. F-Actin, which is entirely free from tropomyosin and troponin, enhanced the rate of Mg²⁺-ATP-induced SH₂ modification of S-1 isozymes equally and of myosin, and reduced the Ca²⁺ sensitivity with an increase in F-actin concentration.     

Binding of magnesium to myosin subfragment-1 ATPase

Tyr 180 of chicken breast muscle alkali light chain A1 was nitrated with tetranitromethane. The nitroA1 was incorporated into chicken breast muscle subfragment-1 (S-1) by exchange with the intrinsic alkali light chain. In the presence of adenylylimidodiphosphate (AMPPNP) or ADP, the S-1 containing nitroA1 showed a difference visible absorption spectrum by Mg2+ or Ca2+. The difference spectrum has a trough around 435 nm, indicating a blue shift of the absorption spectrum due to the nitrophenol chromophore of the modified A1. The plot of delta A at 435 nm versus concentration of free Mg2+ fitted a single binding curve, independent of the total concentration of AMPPNP. These results reveal that free Mg2+ binds to the active site of S-1 ATPase, but not as Mg-AMPPNP complex. The dissociation constants of magnesium from S-1 complex were different with the two nucleotides and were 1.25 X 10(-8) M and 1.24 X 10(-7) with AMPPNP and ADP, respectively. The difference spectrum was also obtained in the presence of ATP. The delta epsilon value after adding ATP changed with the ATPase reaction. The steady state rate of S-1 ATPase was measured at various concentrations of free Mg2+. The dissociation constant of magnesium from the steady state complex, EPADP(a), was estimated as 6 X 10(-8) M. These results suggest that the affinity of magnesium at the active site of ATPase changes with the intermediate states of ATPase reaction. The affinity of calcium was lower than that of magnesium.     

Location of the SH group of the alkali light chain on the myosin head as revealed by electron microscopy

The location of the single cysteinyl residue of the alkali light chain on the myosin head was determined by electron microscopy. The cysteinyl residue of isolated alkali light chain 2 was biotinylated and the light chain was exchanged with that of heavy meromyosin in 4.7 M-NH4Cl. Avidin was attached to the biotin in the heavy meromyosin and the complex was rotary shadowed and observed in the electron microscope. The distance from the head-rod junction to the centre of avidin was 8(+/- 3) nm (mean value +/- standard deviation: n = 105).     

Proximity of alkali light chains to 27K domain of the heavy chain in myosin subfragment 1

When myosin chymotryptic subfragment-1 was treated with dimethyl-suberimidate or dithiobis (succinimidylpropionate) under nearly physiological ionic conditions, the alkali light chains A1 and A2 were selectively and intramolecularly cross-linked to the 95K heavy chain. Experimental conditions were developed with both reagents for optimal production of A1 and A2-containing dimers. After conversion of reversibly cross-linked S-1 (A1+A2) into (27K-50K-20K)-S-1 derivative by restricted tryptic proteolysis, the light chains were found to be attached to the NH₂-terminal 27K segment of the heavy chain.     

Exchange of the fluorescence-labeled 20,000-dalton light chain of smooth muscle myosin.

The 20,000-dalton light chain of smooth muscle myosin was exchanged with exogenous light chain in a solution containing 0.5 M NaCl and 10 mM EDTA at 40 degrees C. The light chain was almost completely exchanged within 30 min under the above conditions. The exchange was markedly inhibited either below 37 degrees C or in the presence of Mg2+ concentrations higher than 10 microM. The 20,000-dalton light chain was selectively labeled of a single thiol (Cys-108) with 5-[[2-[(iodoacetyl)amino]ethyl]amino-naphthalene-1-sulfonic acid (1,5-IAEDANS). The labeled light chain was exchanged stoichiometrically into myosin and was used as a probe to investigate the conformation of smooth muscle myosin. The resulting myosin hybrids showed enzymatic properties virtually identical with those of the control, untreated myosin; i.e., actin-activated ATPase activity was dependent on the 20,000-dalton light-chain phosphorylation catalyzed by myosin light chain kinase, and the 10S-6S conformational transition of myosin correlating with the changes in ATPase was also affected either by the light-chain phosphorylation or by the change in the ionic strength. Steady-state fluorescence antisotropy measurements were performed by varying the temperature. The Perrin-Weber plots were constructed in order to obtain information about the average rotational mobility of the probe and to estimate the rotational correlation time for the AEDANS-myosin head. The fluorescence probe on the 20,000-dalton light chain was found to be quite immobile as indicated by its limiting anisotropy (A0 = 0.33).(ABSTRACT TRUNCATED AT 250 WORDS)     

Ca2+ bindings of pig cardiac myosin, subfragment-1, and g2 light chain.

Ca2+ binding to pig cardiac myosin, subfragment-1 (S-1), and g2 light chain were investigated by the equilibrium dialysis method. Two different S-1s, one of which can bind Ca2+ and another which cannot, were prepared. In order to calculate the free Ca2+ concentrations adequately, the amounts of Ca2+ included in various chemicals and proteins were measured by atomic absorption spectroscopy. Ca2+ contamination was greatest in KCl among the chemicals tested. In addition, the Ca2+ strongly bound to myosin and S-1 was released in the presence of Mg2+. When Mg2+ was not added, the Ca2+-binding constant of myosin was 4 x 10(5) M-1 and the maximum binding number was 1.8 mol per mol of myosin. Cooperativity between the 2 Ca2+ bindings could not be demonstrated. Mg2+ strongly inhibited the Ca2+ binding: at a free Ca2+ concentration of 1 x 10(-5) M, 1.3 mol Ca2+ was bound to myosin in the absence of Mg2+, but 0.6 and 0.2 mol were bound in the presence of 0.3 and 4.5 mM Mg2+, respectively. The Ca2+-binding constant of S-1, which contained a 15,000 dalton component, was 8.6 x 10(5) M-1, and the maximum binding number was 0.7 mol per mol of S-1. The 15,000 dalton component could be exchanged with extraneous g2. S-1 which lacked the 15,000 component could not bind Ca2+ at free Ca2+ concentrations less than 0.1 mM. The Ca2+ binding to free g2 light chain was about 100 times weaker than the binding to myosin, as indicated previously for skeletal myosin (Okamoto, Y. & Yagi, K. (1976) J. Biochem. 80, 111--120). The Ca2+-binding constant was obtained as 4.1 x 10(3) M-1 in the absence of added Mg2+. Phosphorylation of g2 light chain did not affect the Ca2+ binding to the free g2 light chain or to myosin. Ca2+ binding to cardiac native tropomyosin was also measured.     

Properties of the alkali light-chain-20-kilodalton fragment complex from skeletal myosin heads

We have developed a rapid and reproducible procedure widely applicable to the preparation of pure aqueous solutions of the complex between an alkali light chain and the COOH-terminal heavy-chain fragments of skeletal myosin chymotryptic subfragment 1 (S-1) split by various proteases. It was founded on the remarkable ethanol solubility of these complexes. A systematic study of the ethanol fractionation of the tryptic (27K-50K-20K)-S-1 (A2) showed the NH2-terminal 27K fragment to behave like a specific protein entity being quantitatively precipitated at a relatively low ethanol concentration. Only the 20K peptide-A2 complex remained in solution when the S-1 derivative was treated with exactly 4 volumes of ethanol in the presence of 6 M guanidinium chloride. At a lower ethanol concentration, a soluble mixture of 50K and 20K peptides together with the light chain was obtained. The isolated 20K fragment-A2 system containing a 1:1 molar ratio of each component was investigated by biochemical and 1H nuclear magnetic resonance (NMR) techniques to highlight its structure and the interaction of the 20K heavy-chain segment with F-actin and with the light chain. During the treatment of the complex with alpha-chymotrypsin, only the 20K peptide was fragmented in contrast to its stability within the whole S-1. The binding of F-actin to the complex led, however, to a strong inhibition of its chymotryptic degradation. 1-Ethyl-3-[3-(dimethylamino)propyl]carbodiimide cross-linking of F-actin to the complex produced covalent actin-20K peptide only, the amount of which was lower relative to that observed with the entire split S-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Alkali light chains are involved in stabilization of myosin head

1. Fast skeletal myosin subfragment 1 (S1) was separated into two isozymes, S1(A1) and S1(A2), based on the associated alkali light chain, and their thermostabilities were compared. 2. Inactivation rate constants of Ca2(+)-ATPase (at 30 and 35 degrees C) were higher and heat-induced turbidity increase at 340 nm (at 40 degrees C) was faster with S1(A1) than with S1(A2), indicating a higher stability of S1(A2). 3. When S1 isozymes were incubated in the presence of excess alkali light chain, turbidity increase was markedly reduced, depending on the amount of light chain added. 4. Results obtained strongly suggest that alkali light chains are involved in the maintenance of myosin head structure.     

A method to exchange alkali light chains on myosin subfragment 1

Exchange of bound alkali light chains on myosin by free alkali light chains is described. It was found that the yield of hybrid obtained was dependent on the incubation time in 4.7 M NH4Cl at pH 9.5. 60% recovery of S1(A1) from S1(A2) was obtained using only a 2-fold molar excess of A1 over S1(A2).     

Isolation and distribution of myosin isoenzymes in chicken pectoralis muscle

The preparation of rabbit antibodies uniquely specific for the alkali 1 (A1) and alkali 2 (A2) light chains of chicken pectoralis myosin has led to the direct isolation of two homodimeric species of myosin: A1-myosin and A2-myosin, molecules which contain the same light chain on each head. The existence of a heterodimeric species, containing both A1 and A2 light chains, was also inferred. The three types of alkali light chain isoenzymes occur in approximately equal amounts in adult chicken pectoralis muscle.The specificities of the antibodies were determined by modified Farr and solid phase radioimmunoassays, as well as by antibody-affinity chromatography. The determinants in myosin that are recognized by the purified antibodies appear to be confined to the N-terminal sequences of the alkali light chains. As a result of this narrow specificity, these immunological reagents can be used to characterize the distribution of A1 and A2 within the myosin molecule, and to localize the individual light chains within the muscle.By labeling the antibodies with a fluorescent marker we have shown that A1 and A2 are present within each myofibril, as well as within the same fiber (Lowey et al., 1979a). Moreover, by using goat anti-rabbit immunoglobulin to enhance the visualization of the primary antibodies against the light chains, we have demonstrated in the electron microscope that A1 and A2 co-exist along the length of each myofilament. This observation suggests that whatever functional differences may exist among the alkali light chain isoenzymes, they must operate within the constraints of a single filament.     

Formation and characterization of myosin hybrids containing essential light chains and heavy chains from different muscle myosins

Light chain exchange in 4.7 M NH4Cl was used to hybridize the essential light chain of cardiac myosin with the heavy chain of fast muscle myosin subfragment 1, S-1. The actin-activated ATPase properties of this hybrid were compared to those of the two fast S-1 isoenzymes, S-1(A1), fast muscle subfragment 1 which contains only the alkali-1 light chain, and S-1(A2), fast muscle myosin subfragment 1 which contains only the alkali-2 light chain. This hybrid S-1 behaved like S-1(A1)., At low ionic strength in the presence of actin, this hybrid had a maximal rate of ATP hydrolysis about the same as that of S-1(A1) and about one-half that of S-1(A2), while at higher ionic strengths the actin-activated ATPases of these three S-2 species were all similar. Light chain exchange in NH4Cl was also used to hybridize the essential light chains of fast muscle myosin with the heavy chains of cardiac myosin and to hybridize the essential light chains of cardiac myosin with the heavy chains of fast muscle myosin. In 60 and 100 mM KCl, the actin-activated ATPases of these two hybrid myosins were very different from those of the control myosins with the same essential light chains but were very similar to those of the control myosins with the same heavy chains, differing at most by one-third.     

Effect of myosin alkali light chains on myosin subfragment 1 interaction with actin in solution and in ghost muscle fiber

At low ionic strength (7-25 mM) Mg2(+)-ATPase of myosin subfragment 1 (S1) isoforms containing alkali light chain A1 [S1(A1)] is activated by actin 1.5-2.5 times as strongly as Mg2(+)-ATPase of S1 isoforms containing alkali light chain A2[S1(A2)]. Data from analytical ultracentrifugation suggest that at low ionic strength in the absence of ATP in solution S1(A1) displays a higher affinity for F-actin than S1(A2). Such a higher affinity of S1(A1) for F-actin was also demonstrated by experiments, in which the interaction of S1 isoforms fluorescently labeled by 1.5-IAEDANS with F-actin of ghost fibers (single glycerinated muscle fibers containing F-actin but devoid of myosin) was studied. Using polarization microfluorimetry, it was shown that the interaction of both S1 isoforms with ghost fiber F-actin induces similar changes in the parameters of polarized tryptophan fluorescence. At the same time the mobility of the fluorescent probe, 1.5-IAEDANS, specifically attached to the SH-group of Cys-374 in the C-terminal region of action is markedly decreased by S1(A1) and is only slightly affected by S1(A2). The data obtained suggest that S1(A1) and S1(A2) interact with the C-terminal region of the actin molecule in different ways, i.e. S1(A1) is attached more firmly than S1(A2). This may be due to the existence of contacts between the alkali light chain of A1 of S1(A1) and the C-terminal region of actin as well as to the absence of such contacts in the case of S1(A2).     

The interdomain motions in myosin subfragment 1.

The interdomain motions in myosin subfragment 1 (S1) were studied by steady-state and time-resolved fluorescence of tryptophan residues and N-(iodoacetyl)-N'-(5-sulfo-1-naphtyl)ethylenediamine (AEDANS) attached to Cys178 of alkali light chain 1 (A1) exchanged into S1. The efficiency of fluorescence resonance energy transfer (FRET) from tryptophan residues of motor domain to AEDANS at A1 decreased dramatically after addition of ATP to S1A1-AEDANS. The efficiency of FRET calculated from the crystal structure of chicken S1 corresponded to the experimental one measured in the presence of ATP. The results showed that AEDANS at Cys178 of A1 became more mobile and distant from the motor domain of S1 upon ATP binding. These findings led to the suggestion that a release of the products of ATP hydrolysis and power stroke might be associated with movement of light chain-binding domain towards the N-terminal domain of S1.     

Mapping myosin light chains by immunoelectron microscopy. Use of anti- fluorescyl antibodies as structural probes

The two classes of light chains in vertebrate fast muscle myosin have been selectively labeled with the thiol specific reagent 5- (iodoacetamido) fluorescein to determine their location in the myosin head. The alkali light chains (A1 and A2) were labeled at a single cysteine residue near the COOH terminus, whereas the regulatory light chain (LC2) was reacted at either cysteine 125 or 154. The two cysteines of LC2 appear to be near each other in the tertiary structure as evidenced by the ease of formation of an intramolecular disulfide bond. Besides having favorable spectral properties, fluorescein is a potent haptenic immunogen for raising high affinity antibodies. When anti-fluorescyl antibodies were added to the fluorescein-labeled light chains, the fluorescence was quenched by greater than 90%, thereby providing a simple method for determining an association constant. The interaction with antibody was the same for light chains exchanged into myosin as for free light chains. Complexes of antibody bound to light chain could be visualized in the electron microscope by rotary shadowing with platinum. By this approach we have shown that the COOH- terminal regions of the two classes of light chains are widely separated in myosin: the cysteine residues of LC2 lie close to the head/rod junction, whereas the single cysteine of A1 or A2 is located approximately 90 A distal to the junction. These sites correspond to the positions of the NH2 termini of the light chains mapped in earlier studies (Winkelmann, D. A., and S. Lowey. 1986. J. Mol. Biol. 188:595- 612; Tokunaga, M., M. Suzuki, K. Saeki, and T. Wakabayashi. 1987b. J. Mol. Biol. 194:245-255). We conclude that the two classes of light chains do not lie in a simple colinear arrangement, but instead have a more complex organization in distinct regions of the myosin head.     

The free heavy chain of vertebrate skeletal myosin subfragment 1 shows full enzymatic activity

Vertebrate skeletal fast-twitch muscle myosin subfragment 1 is comprised of a heavy polypeptide chain of 95,000 daltons and one alkali light chain of either 21,000 daltons (A1) or 16,500 daltons (A2). In the present study, the heavy chain of subfragment 1 has been separated from the alkali light chain under nondenaturing conditions resembling those in vivo. The heavy chain exhibits the same ATPase activity as myosin subfragment 1, indicating that the heavy chain alone contains the catalytic site for ATP hydrolysis and that the alkali light chains are nonessential for activity. The free heavy chain associates readily at 4 degrees C or 37 degrees C with free A1 or A2 to form the subfragment 1 isozymes SF1(A1) or SF1(A2) respectively. Actin activates the MgATPase activity of the heavy chain in the same manner as occurs with the native isozyme, indicating that the heavy chain possesses the actin binding domain.     

Immunochemical specificity of myosin light chains from mackerel ordinary and dark muscles

Five light chains were isolated from the ordinary and dark muscle myosins of mackerel Pneumatophorus japonicus japonicus, by a method consisting of DTNB and urea treatments, followed by DEAE-cellulose chromatography. Some physicochemical and immunochemical properties of the light chains thus obtained were analyzed. A1, A2, and DTNB light chains from ordinary muscle myosin resembled one another in ultraviolet absorption spectrum, as did D1 and D2 light chains from dark muscle myosin. However, the absorption spectra of the former three differed from those of the latter two. Amino acid compositions of A1 and A2 light chains resembled each other, except for a few amino acids such as lysine, proline, and alanine. Tryptophan was detected only in DTNB light chain. D1 and D2 light chains showed general similarity, except for a remarkably higher proline content in D1. Anti-A1 (or anti-A2) antiserum exhibited a cross-reaction against A2 (or A1) in both immunoelectrophoresis and ELISA, indicating an immunochemical similarity of these two alkali light chains. No precipitin line appeared when anti-A1 or anti-A2 antiserum was diffused against DTNB light chain in immunoelectrophoresis. In ELISA, however, each pair showed cross-reactivity values as high as 50-80%, values which were rather higher than those obtained with heterologous alkali light chains (10-40%). Anti-DTNB light chain antiserum reacted with either alkali light chain in both methods. Anti-D1 antiserum cross-reacted against D2, and anti-D2 antiserum did against D1. These myosin light chains exhibited a high immunochemical tissue-specificity.     

Distance measurement between the active site and cysteine-177 of the alkali one light chain of subfragment 1 from rabbit skeletal muscle

F?rster energy-transfer techniques have been applied to labeled myosin subfragment 1 from rabbit skeletal muscle to determine an intramolecular distance and whether this distance changes during magnesium-dependent ATPase activity. The alkali one light chain was labeled at Cys-177 with N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine (1,5-IAEDANS) and then exchanged into subfragment 1. High specificity of labeling was indicated by high-performance liquid chromatography analysis of a tryptic digest of the labeled light chain. 2'(3')-O-(2,4,6-Trinitrophenyl)adenosine 5'-diphosphate (TNP-ADP) was bound to the labeled protein at the ATPase active site. The efficiency of energy transfer between the probes was 0.09 when measured by both steady-state and time-resolved fluorescence. Anisotropy measurements of the bound AEDANS indicated considerable freedom of motion of the probe. The probable distance between the probes was 57 A. This distance was unchanged during triphosphatase activity. Two further sites of TNP-ADP interaction with subfragment 1 were found. The effect of these interactions on the energy-transfer measurements was reduced to a minimum by careful choice of reaction conditions.     

Hydrophobic interaction chromatography of myosin fragments: Potential use in purification

Myosin fragments were fractionated on columns of the hydrophobic gel phenyl-Sepharose CL-4B. In the presence of high NaCl concentrations the fragments bound tightly to the columns; they could be eluted by decreasing the ionic strength, by increasing the pH, or by applying various concentrations of ethylene glycol. In myosin subfragment-1 (S-1), the light chains underwent partial dissociation from the heavy chain and bound separately to the column matrix. The order of strength of binding of the various species to the column was heavy chain > A1 light chain > A2 light chain > native S-1 > denatured heavy chain or S-1. Thus the hydrophobic gel appears to be able to differentiate between enzymatically active and inactive S-1. Under appropriate elution conditions it was possible to obtain S-1 preparations depleted from nicked heavy chains and with specific ATPase activities 34–130% higher than those of untreated S-1. When S-1(A2) was fractionated on phenyl-Sepharose a fivefold enrichment of the heavy chain with respect to the light chains was obtained, while the ATPase activity was equal or larger than that of the original S-1, implying that the light chains are not essential for ATPase activity. Thus, it seems that chromatography of S-1 on phenyl-Sepharose is a potentially useful method for obtaining a purified myosin heavy-chain fragment with a high ATPase specific activity.