Proteolytic fragmentation of brain myosin and localisation of the heavy-chain phosphorylation site

The heavy chains and the 19-kDa and 20-kDa light chains of bovine brain myosin can by phosphorylated. To localise the site of heavy-chain phosphorylation, the myosin was initially subjected to digestion with chymotrypsin and papain under a variety of conditions and the fragments thus produced were identified. Irrespective of the ionic strength, i.e. whether the myosin was monomeric or filamentous, chymotryptic digestion produced two major fragments of 68 kDa and 140 kDa; the 140-kDa fragment was further digested by papain to yield a 120-kDa and a 23-kDa fragment. These fragments were characterised by (a) a gel overlay technique using 125I-labelled light chains, which showed that the 140-kDa and 23-kDa polypeptides contain the light-chain-binding sites; (b) using myosin photoaffinity labelled at the active site with [3H]UTP, which showed that the 68-kDa fragment contained the catalytic site, and (c) electron microscopy, using rotary shadowing and negative-staining techniques, which demonstrated that after chymotryptic digestion the myosin head remains attached to the tail whereas on papain digestion isolated heads and tails were observed. Thus the 120-kDa polypeptide derived from the 140-kDa fragment is the tail of the myosin, and the 68-kDa fragment containing the catalytic site and the 23-kDa fragment, with the light-chain-binding sites, form the head (S1) portion of the myosin. When [32P]-phosphorylated brain myosin was digested with chymotrypsin and papain it was shown that the heavy-chain phosphorylation site is located in a 5-kDa peptide at the C-terminal end of the heavy chain, i.e. the end of the myosin tail. Using hydrodynamic and electron microscopic techniques, no significant effect of either light-chain or heavy-chain phosphorylation on the stability of brain myosin filaments was observed, even in the presence of MgATP. Brain myosin filaments appear to be more stable than those of other non-muscle myosins. Light-chain phosphorylation did, however, have an effect on the conformation of brain myosin, for example in the presence of MgATP non-phosphorylated myosin molecules were induced to fold into a very compact folded state.     

Metabolism of benzo[a]pyrene. Effect of substrate concentration and 3-methylcholanthrene pretreatment on hepatic metabolism by microsomes from rats and mice.

Digestion of insoluble myosin with soluble papain produces heavy meromyosin subfragment 1 (HMM-S-1) having ATPase activity and the ability to combine with actin. These fragments of myosin do not undergo appreciable changes in ATPase activity, chromatographic behavior, or actin combining ability during digestion up to 2 h but, as shown by sodium dodecyl sulfate gel electrophoresis, several splits occur in both the heavy and light polypeptide chains. The largest fragment of heavy chain present in fast, slow, cardiac and embryonic HMM-S-1 has a mass of 89,000 daltons. This fragment undergoes further degradation resulting in fragments having masses of the order of 70,000, 50,000, and 27,000 daltons. The latter fragment and other material resulting from the proteolysis of myosin appear as bands in that region of the gels where the light chains are found in electrophoretograms of the parent myosin. The precise size of the fragments and the rates of their formation depend on the type of myosin; slow and cardiac HMM-S-1 and their fragments show greater stability. Embryonic myosin has properties intermediate between those of fast skeletal and cardiac myosin. Experiments involving the combination of HMM-S-1 with actin and experiments with glutaraldehyde cross linking and chromatography on Sephadex G-200 indicate that the fragments separated by sodium dodecyl sulfate gel electrophoresis are held together by noncovalent forces in HMM-S-1.     

Isolation and characterization of the N-terminal 23-kilodalton fragment of myosin subfragment 1

The 23-kDa N-terminal tryptic fragment was isolated from the heavy chain of rabbit skeletal myosin subfragment 1 (S-1). The heavy-chain fragments were dissociated by guanidine hydrochloride following limited trypsinolysis, and the 23-kDa fragment was isolated by gel filtration and ion-exchange chromatography. Finally, the fragment was renatured by removing the denaturants. The CD spectrum of the renatured fragment shows the presence of ordered structure. The tryptophan fluorescence emission spectrum of the fragment is considerably shifted to the red upon adding guanidine hydrochloride which indicates that the tryptophans are located in relatively hydrophobic environments. The two 23-kDa tryptophans, unlike the rest of the S-1 tryptophans, are fully accessible to acrylamide as indicated by fluorescence quenching. The isolated 23-kDa fragment cosediments with F-actin in the ultracentrifuge and significantly increases the light scattering of actin in solution which indicates actin binding. The binding is rather tight (Kd = 0.1 microM) and ionic strength dependent (decreasing with increasing ionic strength). ATP, pyrophosphate, and ADP dissociate the 23-kDa-actin complex with decreasing effectiveness. The isolated 23-kDa fragment does not have ATPase activity; however, it inhibits the actin-activated ATPase activity of S-1 by competing presumably with S-1 for binding sites on actin. F-Actin binds to the 23-kDa fragment immobilized on the nitrocellulose membrane. The fragment was further cleaved, and one of the resulting peptides, containing the 130-204 stretch of residues, was found to bind actin on the nitrocellulose membrane, indicating that this region of the 23-kDa fragment participates in forming an actin binding site.     

Light chain dependent effects of actin binding on the S-1/S-2 swivel in myosin

The S-1/S-2 swivel in myosin provides a flexible link between the head and tail portions of the molecule. We have investigated the properties of the swivel by employing limited proteolysis methods. Our results indicate that the binding of actin to heavy meromyosin inhibits both the chymotryptic and papain cleavage of the S-1/S-2 swivel, and that this effect is dependent on the presence of intact LC-2 light chains. Actin did not slow digestions carried out using heavy meromyosin previously treated with proteases to nick the LC-2 chains to 17,000 or 14,000 Mr fragments. Although the integrity of the LC-2 light chain appears to be required to transmit the effects of actin binding from the myosin head to the S-1/S-2 swivel, the binding of Ca2+ to the 17,000 Mr LC-2 fragment can still affect the chemical reactivity of SH1 thiol groups. Both chymotryptic and papain digestions of heavy meromyosin containing intact or fragmented LC-2 light chain show substantial temperature sensitivity between 5 degrees C and 35 degrees C. Calculated apparent activation energies for this process indicate that the S-1/S-2 swivel in myosin can undergo temperature-dependent structural changes independently of the state of the LC-2 light chain. Thus, both actin binding and temperature variations can induce structural transitions in the S-1/S-2 swivel.     

Localization of a light-chain binding site on smooth muscle myosin revealed by light-chain overlay of sodium dodecyl sulfate-polyacrylamide electrophoretic gels

The interactions of smooth muscle myosin and its light chains have been examined by incubating sodium dodecyl sulfate-polyacrylamide gels of myosin with radioactively labeled regulatory or essential light chains. The technique involves sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fixation with methanol and acetic acid followed by an extensive series of washes. The gel is incubated overnight with labeled light chains in the presence of bovine serum albumin and then washed extensively to remove unbound protein. Following staining and destaining, the gel is autoradiographed to reveal which protein bands have bound light chain. The myosin heavy chain was able to rebind labeled regulatory or essential light chains despite the harsh procedure described above. By fragmenting the myosin heavy chain proteolytically, we were able to determine the binding site for both types of light chains to be within the 26,000-Da COOH-terminal segment of smooth muscle subfragment 1 (S-1) or the 20,000-Da COOH-terminal segment of skeletal muscle S-1. The extent of binding was 0.1-0.4 mol of light chain/mol of S-1 heavy chain. No binding was observed to portions of the myosin molecule which do not contain this segment such as myosin rod, light meromyosin, S-2, or the NH2-terminal 75,000-Da segment of S-1.     

Amino acid sequence of rabbit skeletal muscle myosin. 50-kDa fragment of the heavy chain

The amino acid sequence of the 50-kDa fragment that is released by limited tryptic digestion of the head portion of rabbit skeletal muscle myosin was determined by analysis and alignment of sets of peptides generated by digestion of the fragment at arginine or methionine residues. This fragment contains residues 205-636 of the myosin heavy chain; among the residues of particular interest in this fragment are N epsilon-trimethyllysine, one of four methyl-amino acids in myosin, and Ser-324, which is photoaffinity labeled by an ATP analogue (Mahmood, R., Elzinga, M., and Yount, R. G. (1989) Biochemistry 28, 3989-3995). Combination of this sequence with those of the 23- and 20-kDa fragments yields an 809-residue sequence that constitutes most of the heavy chain of chymotryptic S-1 of this myosin.     

Probing myosin head structure with monoclonal antibodies

Monoclonal antibodies that react with defined regions of the heavy and light chains of chicken skeletal muscle myosin have been used to provide a correlation between the primary and the tertiary structures of the head. Electron microscopy of rotary shadowed antibody-myosin complexes shows that the sites for three epitopes in the 25,000 Mr tryptic fragment (25k) of subfragment-1, including one within 4000 Mr of the amino terminus of the myosin heavy chain, are clustered 145(+/- 20) A from the head-rod junction. An epitope in the 50,000 Mr fragment maps even further out on the head. These antibodies bind to the head in several orientations, suggesting that each of the heads can rotate can rotate 180 degrees about the head-rod junction. The epitopes are accessible on subfragment-1 bound to actin when they were probed with Fab fragments; therefore, none of these heavy chain sites is is on the contact surface between the head and actin. Two of the anti-25k antibodies affect the K+-EDTA-and Ca2+-ATPase activities of myosin in a manner that mimics the effect on activity of the modification of the reactive thiol, SH-1. These two antibodies also inhibit the actin-activated ATPase non-competitively with respect to actin. None of the other eight antibodies tested had any marked effect on activity. A monoclonal antibody that reacts with an epitope in the amino-terminal third of myosin light chain 2 maps close to the head-rod junction. A polyclonal antibody specific for the amino terminus of light chain 3 binds further up in the "neck region" of the head, indicating that these portions of the two classes of light chains are located at different sites.     

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.     

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.     

Characterization of caldesmon binding to myosin

Caldesmon inhibits the binding of skeletal muscle subfragment-1 (S-1).ATP to actin but enhances the binding of smooth muscle heavy meromyosin (HMM).ATP to actin. This effect results from the direct binding of caldesmon to myosin in the order of affinity: smooth muscle HMM greater than skeletal muscle HMM greater than smooth muscle S-1 greater than skeletal muscle S-1 (Hemric, M. E., and Chalovich, J. M. (1988) J. Biol. Chem. 263, 1878-1885). We now show that the difference between skeletal muscle HMM and S-1 is due to the presence of the S-2 region in HMM and is unrelated to light chain composition or to two-headed versus single-headed binding. Differences between the binding of smooth and skeletal muscle myosin subfragments to actin do not result from the lack of light chain 2 in skeletal muscle S-1. In the presence of ATP, caldesmon binds to smooth muscle myosin filaments with a stoichiometry of 1:1 (K = 1 x 10(6) M-1). Similar results were obtained for the binding of caldesmon to smooth muscle rod as well as the binding of the purified myosin-binding fragment of caldesmon to smooth muscle myosin. The binding of caldesmon to intact myosin is ATP sensitive. The interaction of caldesmon with myosin is apparently specific and sensitive to the structure of both proteins.     

Thermal denaturation of the alkali light chain-20 kDa fragment complex obtained from myosin subfragment 1.

The thermal denaturation of the myosin subfragment 1 (S1) from rabbit skeletal muscle and of its derivatives obtained by tryptic digestion has been studied by means of differential scanning calorimetry. Two distinct thermal transitions were revealed in the isolated complex of the C-terminal 20 kDa fragment of the S1 heavy chain with the alkali light chain. These transitions were identified by means of a thermal gel analysis method. It has been shown that the thermal denaturation of the 20 kDa fragment of the S1 heavy chain correlates with the melting of the most thermostable domain in the S1 molecule. It is concluded that this domain is located in the C-terminal 20 kDa segment of the S1 heavy chain.     

Comparative structure of the protease-sensitive regions of the subfragment-1 heavy chain from smooth and skeletal myosins

The heavy chain fragments generated by restricted proteolysis of the smooth chicken gizzard myosin subfragment-1 (S-1) with trypsin, Staphylococcus aureus V8 protease, and chymotrypsin were isolated and submitted to partial amino acid sequencing. The comparison between the smooth and striated muscle myosin sequences permitted the unambiguous structural characterization of the two protease-vulnerable segments joining the three putative domain-like regions of the smooth head heavy chain. The smooth carboxyl-terminal connector is a serine-rich region located around positions 632-640 of the rabbit skeletal sequence and would represent the "A" site that is conformationally sensitive to the myosin 10 S-6 transition and to its interaction with actin (Ikebe, M., and Hartshorne, D. J. (1986) Biochemistry 25, 6177-6185). A third site which undergoes a nucleotide-dependent chymotryptic cleavage which inactivates the Mg2+-ATPase (Okamoto, Y., and Sekine, T. (1981) J. Biochem. (Tokyo) 90, 833-842, 843-849) was identified at Trp-31/Ser-32. It is vicinal to Lys-34 that is monomethylated in the skeletal heavy chain but not at all in the smooth sequence. However, the two trimethyl lysine residues present in the skeletal sequence are conserved in the same regions of the smooth S-1 and may play a general functional role in myosin. The smooth central 50-kDa segment could be selectively destroyed by a mild tryptic digestion in the absence of any unfolding agent, with a concomitant inhibition of the ATPase activities. This feature is in line with the proposed domain structure of the S-1 heavy chain and also suggests a relationship between the specific biochemical properties of the smooth S-1 and the particular conformation of its 50-kDa region.     

Preparation of subfragment-1 from abalone smooth muscle myosin

Myosin subfragment-1 (S1), which has one heavy chain (HC) (93 kDa) and two light chains (LC1 and LC2), was prepared by papain digestion of myosin from abalone-smooth muscle in the presence of Ca2+. The Ca-sensitivity of abalone S1 itself was not lost completely (about 30%). The tryptic digestion of S1 showed that in the presence of EDTA, S1 HC was split into 68, 55, and 23 kDa fragments, as in the presence of Ca2+, but 23 kDa was further degraded into 19 kDa. In contrast to the result in the presence of Ca2+, LCs disappeared in the early stage of reaction and Ca-ATPase activity decreased rapidly to about 70% of that of intact S1. This rapid decrease of Ca-ATPase activity seemed to be accompanied with the digestion of LCs. Therefore, LCs contribute to the protection of 23 kDa fragment from further digestion, to the maintenance of Ca-ATPase activity by stabilizing the structure of S1 to some extent in the presence of Ca2+. Since F-actin suppressed the cleavage of S1 HC to 68 and 23 kDa during tryptic digestion, it might be that 23 and 68 kDa corresponded to 20 kDa (C-terminal fragment) and to 50 + 25 kDa (N-terminal fragment) of skeletal myosin S1, respectively.     

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.     

Dinitrophenylated thiols in tryptic fragments of the heavy chain from chicken gizzard myosin

Trypsin digestion of phosphorylated and 3H-labeled dinitrophenylated chicken gizzard myosin released major fragments of Mr 29,000, 50,000 and 66,000 in a ratio of close to one to one. They contained 58% of the label bound to thiols of the heavy chains; 28% of the label was bound to the light chains. The heavy chain fragments of Mr 29,000 and Mr 66,000 were dinitrophenylated when the enzyme activity was inhibited. The 3H-labeled dinitrophenylated myosin alone followed a somewhat different pattern in that the label was bound to the light chains predominantly. Thiolysis of the phosphorylated and dinitrophenylated myosin with 2-mercaptoethanol restored the K+ -ATPase (ATP phosphohydrolase, EC 3.6.1.32) activity and the dinitrophenyl group was removed from the N-terminal fragment of Mr 29,000 of the heavy chain, predominantly. In contrast, restoration of the enzymic activity occurred in thiolyzed dinitrophenylated myosin alone when the label was removed from the light chains rather than the tryptic fragments of the heavy chain. Phosphorylation induced conformational changes in gizzard myosin that altered the reactivity of the thiols in fragments of the globular heavy chain region.     

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 nucleotide on interaction of the 567-578 segment of myosin heavy chain with actin

To probe the effect of nucleotide on the formation of ionic contacts between actin and the 567-578 residue loop of the heavy chain of rabbit skeletal muscle myosin subfragment 1 (S1), the complexes between F-actin and proteolytic derivatives of S1 were submitted to chemical cross-linking with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. We have shown that in the absence of nucleotide both 45 kDa and 5 kDa tryptic derivatives of the central 50 kDa heavy chain fragment of S1 can be cross-linked to actin, whereas in the presence of MgADP.AlF4, only the 5 kDa fragment is involved in cross-linking reaction. By the identification of the N-terminal sequence of the 5-kDa fragment, we have found that trypsin splits the 50 kDa heavy chain fragment between Lys-572 and Gly-573, the residues located within the 567-578 loop. Using S1 preparations cleaved with elastase, we could show that the residue of 567-578 loop that can be cross-linked to actin in the presence of MgADP.AlF4 is Lys-574. The observed nucleotide-dependent changes of the actin-subfragment 1 interface indicate that the 567-578 residue loop of skeletal muscle myosin participates in the communication between the nucleotide and actin binding sites.     

Regulatory light chain influences alterations of myosin head induced by actin.

The effect of magnesium-for-calcium exchange and phosphorylation of regulatory light chain (LC2) on structural organization of rabbit skeletal myosin head was studied by limited tryptic digestion. In the presence of actin, exchange of magnesium bound to LC2 by calcium in dephosphorylated myosin accelerates the digestion of myosin and heavy meromyosin heavy chain and increases the accumulation of a 50 kDa fragment. This effect is significantly diminished in the case of phosphorylated myosin. Thus, both phosphorylation and cation exchange influences the effect of actin binding on the structural organization of myosin head.     

Monoclonal antibodies distinguish between the head portions of two myosin isozymes from chicken breast muscle

Monoclonal antibodies against chicken breast myosin and its subfragment-1(S-1) were produced. One antibody, 2G41, reacted with S-1 containing a light chain 3 (LC3), but not with another S-1 containing a light chain 1 (LC1) or a mixture of the light chains. A structural difference can be assumed to exist between the head portions of the two myosin isozymes. Antigenicity of S-1 toward 2G41 could not be detected after tryptic digestion into three fragments of 50K, 27K, and 20K daltons. Another monoclonal antibody, M68, was obtained from mice immunized with myosin. M68 preferably recognized the heavy chain from S-1 containing LC3 rather than that from that containing LC1 or S-1. M68 reacted with the 27K fragment among the three.     

Regulatory and essential light-chain-binding sites in myosin heavy chain subfragment-1 mapped by site-directed mutagenesis

Site-directed mutagenesis of the cloned subfragment-1 (S-1) region of the unc-54 gene, encoding the myosin heavy chain B (MHC B) from Caenorhabditis elegans, has been used to locate binding sites for the regulatory and essential light chains. MHC B S-1 synthesized in Escherichia coli co-migrated with rabbit skeletal muscle myosin S-1 (Mr 90,000), was recognized by anti-nematode myosin antiserum on immunoblots, and specifically bound to 125I-labelled regulatory and essential light chains in a gel overlay assay. Deletion of 102 residues from the C terminus (mutant 655) reduced regulatory and essential light-chain binding to about 30% and 20% of wild-type levels, respectively. Similar reductions in relative binding of the two light chains were seen with mutant 534, in which 38 residues were deleted from the C terminus. Potential binding sites within 75 residues of the C terminus of S-1 were mapped by construction of five other mutant S-1 clones (398, 399, 400, 409 and 411) containing internal deletions of ten to 12 amino acid residues. These showed up to 30% reductions in their ability to bind essential light chains, but did not differ significantly from wild-type in their ability to bind regulatory light chains. Another mutant, 415, containing a deletion of a conserved acidic hexapeptide, E-D-I-R-D-E, showed enhancement of binding of regulatory and essential light chains to 150% and 165% of wild-type levels. Hence, the major binding sites for both light chains are within 38 amino acid residues of the C terminus.