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.     

The widespread distribution of alpha-N-trimethylalanine as the N-terminal amino acid of light chains from vertebrate striated muscle myosins

Identical tripeptides of the sequence X-Pro-Lys, where X is an unknown blocking group, were isolated from trypsin digests of bovine cardiac alkali light chain and the LC2 light chain of rabbit fast muscle. Chemical, electrophoretic and 1H-NMR evidence characterized X as an unusual amino acid, alpha-N-trimethylalanine (Me3Ala), which was earlier reported as the N-terminal amino acid of the A1 alkali light chain of rabbit fast muscle [Henry et al. (1982) FEBS Lett. 144, 11-15]. The narrow line width and chemical shift position (delta = 3.23 ppm) of the--N+-(CH3) protons of Me3Ala made 1H-NMR spectroscopy a convenient method to search for this residue in other light chains. A survey of many different light chains showed that this signal was present in all vertebrate striated muscle light chains of the A1-type (LC1, 'essential' light chains) and LC2-type ('DTNB'-light chains, 'phosphorylatable' light chains) but was absent from all invertebrate muscle and vertebrate smooth muscle light chains tested. It was also absent from the vertebrate fast-muscle-specific A2-type (LC3) light chains. The spectral characteristics of these signals were consistent with their having arisen from the protons of an--N+-(CH3)3 grouping. Since no epsilon-trimethyllysine could be detected in acid hydrolysates of these proteins, it appears that Me3Ala is a general feature as the N-terminal amino acid in these light chains. 1H-NMR studies on bovine cardiac myosin subfragment 1 (S1) showed that the Me3Ala methyl proton signal was clearly visible and that the spectrum more closely resembled that of a rabbit S1 isoenzyme, S1(A1), than S1(A2), suggesting that the 40-residue N-terminal segment of the alkali light chain in cardiac S1 also possesses a high segmental mobility. Addition of actin caused the same gross changes to the cardiac S1 spectrum as noted earlier for rabbit S1(A1) [Prince et al. (1981) Eur. J. Biochem. 121, 213-219]. In particular, a marked reduction in the segmental mobility of the N-terminal region of the alkali light chain was noted, consistent with a direct interaction of this area with actin.     

Interaction of isoforms of subfragment-1 of myosin, containing fluorescently labelled alkaline light chains, with muscle fiber actin

Using polarization microfluorimetry, the interaction of myosin subfragment 1 (S1) isoforms containing alkali light chains A1 and A2 respectively (S1(A1) and S1(A2] with F-actin of single glycerinated rabbit skeletal muscle fibers was studied. The alkali light chains of S1 were substituted by reassociation for A1 or A2 chains modified by a fluorescent label (1.5-IAEDANS) at the single SH-group located in the C-terminus. It was found that in S1(A1) bound to muscle fiber F-actin the mobility of the fluorescent label is lower than in S1(A2). At the same time the S1(A1) and S1(A2) interaction with F-actin induces similar changes in polarized fluorescence of rhodamine linked to falloidine which, in turn, is specifically bound to F-actin. It is concluded that the both S1 isoforms bind to F-actin and produce similar effects on the conformational state of actin filaments in muscle fibers. Local differences between S1(A1) and S1(A2) seem to be due to the interaction of the N-terminus of A1 within S1(A1) with the C-terminal region of actin.     

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.     

Possible presence of the difference peptide in alkali light chain 1 of fish fast skeletal myosin

1. Presence of N-terminal peptide ("difference peptide") in alkali light chain 1 (A1) of fish fast skeletal myosin was examined by comparing two kinds of light chain-based myosin subfragment 1 (S1) isozymes from the yellowtail Seriola quinqueradiata. 2. On tryptic digestion, A1 was cleaved to a smaller fragment (mol. wt decrement by 2000) along with the cleavage of S1 heavy chain, while A2 was resistant to trypsin. Two-dimensional gel electrophoresis showed that A1 released a basic peptide by tryptic digestion. 3. Both S1 isozymes showed clear kinetic differences in actin-activated Mg-ATPase activity, suggesting a higher affinity of A1 for actin. Affinity of A2 for heavy chain was also estimated to be about 2-fold higher than that of A1, as judged by the model experiments in which rabbit S1 isozymes were hybridized with heterologous alkali light chains.     

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.     

Characterization of the ATPase active site in myosin subfragment-1 with the use of vanadate plus ADP as a reversible "affinity-labeling" reagent: evidence for heterogeneity in the active sites

Our previous work showed that the active site heterogeneity in heavy meromyosin (HMM) becomes evident when highly reactive SH-groups in HMM are modified by thimerosal (Kawamura, Higuchi, Emoto, & Tawada (1985) J. Biochem. 97, 1583-1593). The heterogeneity was revealed by "affinity-labeling" analysis with vanadate plus ADP, which was developed in the previous paper. To see whether this heterogeneity is due to the head-head interaction or two different alkali light chains present in HMM, we carried out similar studies with myosin subfragment-1 (S1) and one of the isozymes, S1(A1), which contains only the alkali light chain 1, and obtained essentially the same results as those previously obtained with HMM. The S1 results are easily explained by the same hypothesis previously used for explaining the HMM results: SH-modified S1 or S1(A1) contains two kinds of active site in a 1:1 ratio with almost the same ATPase activity: one hydrolyzes ATP by a mechanism giving a protein Trp fluorescence enhancement, whereas the other hydrolyzes ATP by another mechanism giving no fluorescence enhancement.     

Preparation of the alkali and P light chains of chicken gizzard myosin. Amino acid sequence of the alkali light chain

1. A simple method is described for the purification of the alkali and P light chains from chicken gizzard myosin. 2. The sequence of the alkali light chain has been unequivocally determined, except for the N-terminal dipeptide, by using the tryptic and CNBr peptides. 3. No evidence was obtained for any specific high-affinity Ca2+-binding sites on the alkali light chain. 4. Detailed evidence on which the sequence is based has been deposited as Supplementary Publication SUP 50120 (14 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7QB, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1983) 209, 5.     

Myosin subunit interactions. Localization of the alkali light chains

Myosin homodimers, molecules containing either the A1 or the A2 light chain, do not exchange their light chains under conditions approximating physiological temperature and ionic strength. Myosin heterodimers, molecules containing both A1 and A2 light chains, are therefore formed at the time of synthesis rather than by a labile subunit exchange. Antibodies specific for the amino-terminal region of the alkali light chains were used to localize these subunits in myosin by immunoelectron microscopy. The close proximity of the alkali light chain to the 5,5'-dithiobis-(2-nitrobenzoic acid) light chain in the "neck" region of the myosin head is consistent with the finding that the 5,5'-dithiobis-(2-nitrobenzoic acid) light chain influences subunit interactions between the alkali light chain and heavy chain in vertebrate skeletal muscle myosin.     

Separation of two HMM-S1 species from white muscle myosin

Heavy meromyosin subfragment 1 was resolved by chromatography on DEAE-cellulose into two fractions characterized by the nature of the alkali light chains present. It was shown that even in an HMM-S1 preparation with an extensive fragmentation of the heavy chain a polyacrylamide gel electrophoresis analysis differentiates alkali light chains among the light fragmentation components. A non-fragmented HMM-S1 was obtained from a papain digest of myofibrils and the chromatographic analysis supplied further evidence of the separation of the two species of HMM-S1 present in rabbit white muscle myosin.     

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).     

An efficient exchange method for alkali light chain in myosin subfragment

The alkali light chain, A2, in subfragment-1 (S-1) was exchanged with A1 added externally in NH4 + -NH3 buffer (pH 9.9). The exchange yield was higher than 80% using only 2-fold molar excess of A1 over S-1 containing A2. The ATPase activities of the exchanged S-1 (A1) were the same as those of untreated S-1 (A1). The method was also applicable to exchanging the alkali light chains in myosin.     

A comparison of the alkali light chain subunits of vertebrate skeletal muscle myosin in free and heavy chain associated states

The conformations of the alkali light-chain subunits A1 and A2 of vertebrate fast-twitch muscle myosin have been compared for these chains both in their free state and their heavy-chain-associated states by examining the fluorescence parameters of the extrinsic probe 2-(4′-maleimidylanilino)naphthalene-6-sulfonic acid attached covalently to the two light chains. The effect of temperature, salt concentration, and ligands such as Mg²⁺ ions, MgADP, and MgATP has also been investigated. In spite of the extensive sequence homology between the two light chains the data indicate that in their free states the fluorophore in the A2 chain resides in a considerably higher hydrophobic environment. It was also found that the presence of the bulky fluorophore on these light chains does not adversely affect their ability to hybridize with Subfragment 1 heavy chains to form ATPase active hybrids. This association to the heavy chains is accompanied by significant changes in the quantum yields of the 2-(4′-maleimidylanilino)naphthalene-6-sulfonic acid label indicating that conformational changes do occur during this transition. Mg²⁺ ions were found to cause either an enhancement or a decrease in fluorescence intensity depending on whether the alkali light chains were free or combined to the heavy chains, respectively. Fluorescence perturbation by nucleotide was only observed for the heavy-chain-associated state.     

An immunological approach to the role of the low molecular weight subunits in myosin. I. Physical--chemical and immunological characterization of the light chains.

The light chains of chicken breast muscle myosin (alkali 1 and 2, 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) 1.c.) have been isolated in pure form and characterized with respect to amino acid composition, uv and circular dichroism (CD) spectral properties, and molecular weight. Antibodies specific for each of the light chains have been used to demonstrate the similarity of alkali 1 and 2 (mol wt 21,000 and 16,000, respectively), and the distinctness of these from DTNB 1.c. (mol wt of 18,000). The DTNB 1.c. isolated by a variety of methods were all immunologically identical. Significant cross-reactivity was observed between corresponding rabbit and chicken light chains, confirming other indications of homology between these proteins in the two species. The immunological difference between alkali 1 and 2 was largely accounted for by an N-terminal peptide, rich in proline, alanine, and lysine, which is unique to alkali 1. The presence of antibodies to this peptide in anti-alkali 1 serum suggests an immunological approach to the question of how alkali 1.c. are distributed in myosin.     

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.     

Evidence that the N-terminal region of A1-light chain of myosin interacts directly with the C-terminal region of actin. A proton magnetic resonance study

Earlier 1H-NMR experiments on the myosin subfragment-1 (S1) light chain isoenzymes from rabbit fast muscle, containing either the A1 or the A2 alkali light chains [S1(A1) or S1(A2)], have shown that the 41-residue N-terminal extension of A1, rich in proline, alanine and lysine residues, is freely mobile in solution but that this mobility is constrained in the acto-S1(A1) complex [Prince et al. (1981) Eur. J. Biochem. 121, 213-219]. It is now established that this N-terminal region of the A1-light chain interacts directly with the C-terminal region of actin in the acto-S1(A1) complex. This was shown by covalently labelling the Cys-374 residue of actin with a spin-label and observing the enhanced relaxation this paramagnetic centre induced in the 1H-NMR spectrum of S1(A1). In particular, the signal arising from the -N+(CH3)3 protons of alpha-N-trimethylalanine (Me3Ala) were monitored as this residue is uniquely sited at the N-terminus of the A1 light chain [Henry et al. (1982) FEBS Lett. 144, 11-15]. Experiments using complexes of actin with either the N-terminal 37-residue peptide of A1, S1(A1) or heavy meromyosin indicate that the N-terminal region of A1 is binding in a similar manner to actin in each case, with the N-terminal Me3Ala residue within 1.5 nm of the spin label introduced to Cys-374 of actin. A similar strategy was adopted to show that the Me3Ala residue can also be found close (less than 1.5 nm) to the fast-reacting SH1 thiol group on the S1 heavy chain. These data, together with published work, have been used to suggest a possible organisation for the polypeptide chains in the myosin head.     

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.     

Mapping of fish myosin light chains by two-dimensional gel electrophoresis

1. Myosins were prepared from the ordinary muscle of 16 fish species as well as from rabbit fast muscle, and light chain subunits [alkali light chains A1, A2 and DTNB (5,5'-dithio-bis-2-nitrobenzoate) light chain] were separated on two-dimensional gel electrophoresis in combination with isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 2. A1 light chains showed mol. wts ranging from 21,000 to 22,900 and isoelectric points ranging from 4.51 to 4.62. DTNB light chains were spotted in a narrow area, with a mol. wt range of 16,800-17,600 and an isoelectric point range of 4.48-4.55. On the other hand, A2 light chains were most species-specific, with a mol. wt range of 14,000-19,500 and an isoelectric point range of 4.31-4.46. 3. It was suggested that the lower species-specificity in A1 as opposed to A2 is accounted for by the addition of an N-terminal peptide ("difference peptide") in the former. The properties and possible role of this peptide are discussed.     

Subunit interactions of skeletal muscle myosin and myosin subfragment 1. Evidence for heavy chain-alkali light chain association-dissociation equilibrium

Modification of the free alkali light chains of myosin by iodoacetylation results in a much lower extent of exchange into myosin subfragment 1 by the thermal hybridization procedure (Burke, M., and Sivaramakrishnan, M. (1981) Biochemistry 20, 5908-5913). As reported by others (Wagner, P. D., and Stone, D. B. (1983) J. Biol. Chem. 258, 8876-8882), free alkali light chains modified by iodoacetate at their single sulfhydryl residue exhibit minimal exchange into intact myosin. However, when unmodified alkali light chain is used to probe for exchange, close to the theoretical limit of exchange is observed for subfragment 1, and significant levels of exchange are found for myosin. It appears that modification of the free alkali light chain alters the structure of the protein, and this causes either a marked reduction in its affinity for the heavy chain or in its ability to enter the light chain binding site. This conclusion is supported by tryptic digestions done on the unmodified and modified free light chains where it is found that the latter is degraded at a much faster rate, indicating a more open structure for the modified protein. The observation that alkali light chain exchanges into myosin when unmodified alkali light chains are used indicates that the presence of the associated 5,5'-dithiobis-(2-nitrobenzoic acid) light chains does not preclude the reversible dissociation of this subunit from myosin under ionic and temperature conditions approaching the physiological state.     

[2-3H]ATP synthesis and 3H NMR spectroscopy of enzyme-nucleotide complexes: ADP and ADP.Vi bound to myosin subfragment 1

Summary The synthesis of [2-³H]ATP with specific activity high enough to use for ³H NMR spectroscopy at micromolar concentrations was accomplished by tritiodehalogenation of 2-Br-ATP. ATP with greater than 80% substitution at the 2-position and negligible tritium levels at other positions had a single ³H NMR peak at 8.20 ppm in 1D spectra obtained at 533 MHz. This result enables the application of tritium NMR spectroscopy to ATP utilizing enzymes.The proteolytic fragment of skeletal muscle myosin, called S1, consists of a heavy chain (95 kDa) and one alkali light chain (16 or 21 kDa) complex that retains myosin ATPase activity. In the presence of Mg²⁺, S1 converts [2-³H]ATP to [2-³H]ADP and the complex S1.Mg[2-³H]ADP has ADP bound in the active site. At 0°C, 1D ³H NMR spectra of S1.Mg[2-³H]ADP have two broadened peaks shifted 0.55 and 0.90 ppm upfield from the peak due to free [2-³H]ADP. Spectra with good signal-to-noise for 0.10 mM S1.Mg[2-³H]ADP were obtained in 180 min. The magnitude of the chemical shift caused by binding is consistent with the presence of an aromatic side chain being in the active site. Spectra were the same for S1 with either of the alkali light chains present, suggesting that the alkali light chains do not interact differently with the active site. The two broad peaks appear to be due to the two conformations of S1 that have been observed previously by other techniques. Raising the temperature to 20 °C causes small changes in the chemical shifts, narrows the peak widths from 150 to 80 Hz, and increases the relative area under the more upfield peak. Addition of orthovanadate (V_i) to produce S1.Mg[2-³H]ADP.V_i shifts both peaks slightly more upfield without chaning their widths or relative areas.