F-actin-binding synthetic heptapeptide having the amino acid sequence around the SH1 cysteinyl residue of myosin

The heptapeptide Ile-Arg-Ile-Cys-Arg-Lys-Gly-ethyl ester, having the amino acid sequence around the SH1 of myosin heavy chain, was coprecipitated with F-actin after ultracentrifugation. The heptapeptide inhibited the formation of acto-S-1 rigor complex by competing with S-1 for actin. Assuming a simple competitive inhibition, the dissociation constant of acto-heptapeptide complex was evaluated as 0.23 mM. An N-terminal tripeptide derivative from the heptapeptide Ile-Arg-Ile-methyl ester also formed a complex with F-actin with a dissociation constant of 1.1 mM. However, the other piece, Cys-Arg-Lys-Gly-ethyl ester, and a tetrapeptide, Val-Leu-Glu-Gly-ethyl ester, having the sequence adjacent to the N-terminal of the heptapeptide, scarcely bound with F-actin. These results suggest that part of the actin-binding site of myosin heavy chain around SH1 (Katoh, T., Katoh, H., and Morita, F. (1985) J. Biol. Chem. 260, 6723-6727) has the sequence of Ile-Arg-Ile and it is located adjacent to SH1 on its N-terminal side.     

Actin-binding peptide obtained by the cyanogen bromide cleavage of the 20-kDa fragment of myosin subfragment-1

The 20-kDa fragment of myosin subfragment-1 heavy chain was cleaved with cyanogen bromide. Gel electrophoresis of the fragmented peptides indicated the presence of 20-, 18-, 16-, 14-, 12-, and 10-kDa peptides in addition to two peptides smaller than 10 kDa. The renaturation procedure of Muhlrad and Morales (Muhlrad, A., and Morales, M. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 1003-1007) was applied to the mixture of these peptides. The peptides larger than 10 kDa, which contain both the reactive SH1 and SH2 groups, were precipitated with F-actin by ultracentrifugation. The 10-kDa peptide was purified and was identified as p10 of Elzinga and Collins (Elzinga, M., and Collins, J. H. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 4281-4284). The renaturation procedure was applied to the purified 10-kDa peptide. The 10-kDa peptide was also precipitated with F-actin by ultracentrifugation. Affinity of the 10-kDa peptide for F-actin was determined with an increase of turbidity, and the apparent dissociation constant was 0.94 microM. Results are consistent with our proposition that a binding site for F-actin exists around the SH1 and SH2 groups of subfragment-1 (Katoh, T., Imae, S., and Morita, F. (1984) J. Biochem. 95, 447-454; Katoh, T., and Morita, F. (1984) J. Biochem. 96, 1223-1230).     

Localization of the actin-binding sites of Acanthamoeba myosin IB and effect of limited proteolysis on its actin-activated Mg2+-ATPase activity

Acanthamoeba myosin IB contains a 125-kDa heavy chain that has high actin-activated Mg2+-ATPase activity when 1 serine residue is phosphorylated. The heavy chain contains two F-actin-binding sites, one associated with the catalytic site and a second which allows myosin IB to cross-link actin filaments but has no direct effect on catalytic activity. Tryptic digestion of the heavy chain initially produces an NH2-terminal 62-kDa peptide that contains the ATP-binding site and the regulatory phosphorylation site, and a COOH-terminal 68-kDa peptide. F-actin, in the absence of ATP, protects this site and tryptic cleavage then produces an NH2-terminal 80-kDa peptide. Both the 62- and the 80-kDa peptides retain the (NH+4,EDTA)-ATPase activity of native myosin IB and both bind to F-actin in an ATP-sensitive manner. However, only the 80-kDa peptide retains a major portion of the actin-activated Mg2+-ATPase activity. This activity requires phosphorylation of the 80-kDa peptide by myosin I heavy chain kinase but, in contrast to the activity of intact myosin IB, it has a simple, hyperbolic dependence on the concentration of F-actin. Also unlike myosin IB, the 80-kDa peptide cannot cross-link F-actin filaments indicating the presence of only a single actin-binding site. These results allow the assignment of the actin-binding site involved in catalytic activity to the region near, and possibly on both sides of, the tryptic cleavage site 62 kDa from the NH2 terminus, and the second actin-binding site to the COOH-terminal 45-kDa domain. Thus, the NH2-terminal 80 kDa of the myosin IB heavy chain is functionally similar to the 93-kDa subfragment 1 of muscle myosin and most likely has a similar organization of functional domains.     

Molecular charge dominates the inhibition of actomyosin in skinned muscle fibers by SH1 peptides.

It is not definitively known whether the highly conserved region of myosin heavy chain around SH1 (Cys 707) is part of the actin-binding site. We tested this possibility by assaying for competitive inhibition of maximum Ca-activated force production of skinned muscle fibers by synthetic peptides which had sequences derived from the SH1 region of myosin. Force was inhibited by a heptapeptide (IRICRKG) with an apparent K0.5 of about 4 mM. Unloaded shortening velocity of fibers, determined by the slack test, and maximum Ca-activated myofibrillar MgATPase activity were also inhibited by this peptide, but both required higher concentrations. We found that other cationic peptides also inhibited force in a manner that depended on the charge of the peptide; increasing the net positive charge of the peptide increased its efficacy. The inhibition was not significantly affected by altering solution ionic strength (100-200 mM). Disulfide bond formation was not involved in the inhibitory mechanism because a peptide with Thr substituted for Cys was inhibitory in the presence or absence of DTT. Our data demonstrate that the net charge was the predominant molecular characteristic correlated with the ability of peptides from this region of myosin heavy chain to inhibit force production. Thus, the hypothesis that the SH1 region of myosin is an essential part of the force-producing interaction with actin during the cross-bridge cycle (Eto, M., R. Suzuki, F. Morita, H. Kuwayama, N. Nishi, and S. Tokura., 1990, J. Biochem. 108:499-504; Keane et al., 1990, Nature (Lond.). 344:265-268) is not supported.     

A new, smaller actin-activatable myosin subfragment 1 which lacks the 20-kDa, SH1 and SH2 peptide

The actin-dependent ATPase activity of myosin is retained in the separated heads (S1) which contain the NH2-terminal 95-kDa heavy chain fragment and one or two light chains. The S1 heavy chain can be degraded further by limited trypsin treatment into characteristic 25-, 50-, and 20-kDa peptides, in this order from the NH2-terminal end. The 20-kDa peptide contains an actin-binding site and SH1 and SH2, two thiols whose modification dramatically affects ATPase activity. By treating myosin filaments with trypsin at 4 degrees C in the presence of 2 mM MgCl2, we have now obtained preferential cleavage at the 50-20-kDa heavy chain site without any cleavage at the head-rod junction and hinge region in the rod. Incubation of these trypsinized filaments at 37 degrees C in the presence of MgATP released a new S1 fraction which lacked the COOH-terminal 20-kDa heavy chain peptide region. This fraction, termed S1'(75K), has more than 50% of the actin-activated Mg2+-ATPase activity of S1 and the characteristic Ca2+-ATPase and K+-EDTA ATPase activities of myosin. These results show that SH1 and SH2 are not essential for ATPase activity and that binding of actin to the 20-kDa region is not essential for the enhancement of the Mg2+-ATPase activity.     

Twitchin can regulate the ATPase cycle of actomyosin in a phosphorylation-dependent manner in skinned mammalian skeletal muscle fibres

The effect of twitchin, a thick filament protein of molluscan muscles, on the actin-myosin interaction at several mimicked sequential steps of the ATPase cycle was investigated using the polarized fluorescence of 1.5-IAEDANS bound to myosin heads, FITC-phalloidin attached to actin and acrylodan bound to twitchin in the glycerol-skinned skeletal muscle fibres of mammalian. The phosphorylation-dependent multi-step changes in mobility and spatial arrangement of myosin SH1 helix, actin subunit and twitchin during the ATPase cycle have been revealed. It was shown that nonphosphorylated twitchin inhibited the movements of SH1 helix of the myosin heads and actin subunits and decreased the affinity of myosin to actin by freezing the position and mobility of twitchin in the muscle fibres. The phosphorylation of twitchin reverses this effect by changing the spatial arrangement and mobility of the actin-binding portions of twitchin. In this case, enhanced movements of SH1 helix of the myosin heads and actin subunits are observed. The data imply a novel property of twitchin incorporated into organized contractile system: its ability to regulate the ATPase cycle in a phosphorylation-dependent fashion by changing the affinity and spatial arrangement of the actin-binding portions of twitchin.     

Inhibition of actomyosin subfragment 1 ATPase activity by analog peptides of the actin-binding site around the Cys(SH1) of myosin heavy chain

The synthetic heptapeptide, Ile-Arg-Ile-Cys-Arg-Lsy-Gly-ethoxy, an analog of one of the actin binding sites on myosin head (S-site) (Suzuki, R., Nishi, N., Tokura, S., and Morita, F. (1987) J. Biol. Chem. 262, 11410-11412) was found to completely inhibit the acto-S-1 (myosin subfragment 1) ATPase activity. The effect of the heptapeptide on the binding ability of S-1 for F-actin was determined by an ultracentrifugal separation. Results indicated that the heptapeptide scarcely dissociated the acto-S-1 complex during the ATPase reaction. Consistent results were obtained from the acto-S-1 ATPase activities determined as a function of S-1 concentrations in the absence or presence of the heptapeptide at a fixed F-actin concentration. The heptapeptide reduced the maximum acto-S-1 ATPase activity without affecting the apparent dissociation constant of the acto-S-1 complex. The heptapeptide bound by a site on actin complementary to the S-site probably inhibits the activation of S-1 ATPase by F-actin. These results suggest that S-1 ATPase is necessary to rebind transiently with F-actin at the S-site in order to be activated by F-actin. This is consistent with the activation mechanism proposed assuming the two actin-binding sites on S-1 ATPase (Katoh, T., and Morita F. (1984) J. Biochem. (Tokyo) 96, 1223-1230).     

Caspase-mediated cleavage of actin-binding and SH3-domain-containing proteins cortactin, HS1, and HIP-55 during apoptosis.

Reorganization of the actin cytoskeleton occurs during apoptosis. We found that actin-binding and Src homology 3 (SH3)-domain-containing proteins cortactin, hematopoietic-specific protein 1 (HS1), and hematopoietic progenitor kinase 1-interacting protein of 55 kDa (HIP-55, also called SH3P7 and Abp1) were degraded in a caspase-dependent manner during apoptosis. Cortactin, HS1, and HIP-55 were direct substrates of caspase 3. Cortactin and HS1 have two clusters of potential caspase cleavage sites; one is in their actin-binding domains, and the other is close to their carboxy-terminal SH3 domains. HIP-55 has one caspase recognition site, EHID(361). The HIP-55 (D361A) mutant was resistant to caspase cleavage. Cleavage of HIP-55 by caspases dissociated its actin-binding domain from its SH3 domain. The cleavage of these actin-binding and SH3 domain-containing proteins may affect cell signaling to and from the actin cytoskeleton and may be involved in the morphological change of cells during apoptosis.     

The isolated heavy chain of an Acanthamoeba myosin contains full enzymatic activity.

Acanthamoeba myosin IB is a single-headed enzyme containing one heavy chain of 125,000 daltons, one light chain of 27,000 daltons, and one light chain of 14,000 daltons. The 125,000- and 27,000-dalton polypeptides are consistently found in a molar ratio of 1:1. The content of the 14,000-dalton peptide is usually only 0.1 to 0.2, and always less than 0.5, relative to the other two chains and might be a contaminant or a degradation product of one of the other chains. The specific activities of the Ca2+-ATPase, (K+, EDTA)-ATPase, and (after phosphorylation of its heavy chain by a specific kinase) actin-activated Mg2+-ATPase of Acanthamoeba myosin IB are similar to those of rabbit skeletal muscle myosin. After treatment of the enzyme with 2 M LiCl, the 125,000-dalton heavy chain of Acanthamoeba myosin Ib can be obtained, by chromatography on Sephadex G-200, essentially free of the 14,000-dalton peptide and more than 90% free of the 27,000-dalton peptide. This isolated heavy chain has the same specific ATPase activities as the original enzyme. Therefore, the heavy chain of Acanthamoeba myosin IB contains the ATPase catalytic site, the actin-binding site, and the phosphorylation site and is fully active enzymatically in the absence of light chains.     

The myosin SH2-50-kilodalton fragment cross-link: location and consequences

Some of us recently described a new interthiol cross-link which occurs in the skeletal myosin subfragment 1-MgADP complex between the reactive sulfhydryl group "SH2" (Cys-697) and a thiol (named SH chi) of the 50-kilodalton (kDa) central domain of the heavy chain; this link leads to the entrapment of the nucleotide at the active site [Chaussepied, P., Mornet, D., & Kassab, R. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 2037-2041]. In the present study, we identify SH chi as Cys-540 of the 50-kDa fragment. The portion of the heavy chain including this residue and also extending to Cys-522 that is cross-linkable to the "SH1" thiol [Ue, K. (1987) Biochemistry 26, 1889-1894] is near the SH2-SH1 region. Furthermore, various spectral and enzymatic properties of the (Cys697-Cys540)-N,N'-p-phenylenedimaleimide (pPDM)-cross-linked myosin chymotryptic subfragment 1 (S-1) were established and compared to those for the well-known (SH1-SH2)-pPDM-cross-linked S-1. The circular dichroism spectra of the new derivative were similar to those of native S-1 complexed to MgADP. At 15 mM ionic strength, (Cys697-Cys540)-S-1 binds very strongly to unregulated actin (Ka = 7 X 10(6) M-1), and the actin binding is very weakly affected by ionic strength. Joining actin with the (Cys697-Cys540)-S-1 heavy chain, using 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide, produces different species than does joining unmodified S-1 with actin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Functional analysis of tail domains of Acanthamoeba myosin IC by characterization of truncation and deletion mutants

Acanthamoeba myosin IC has a single 129-kDa heavy chain and a single 17-kDa light chain. The heavy chain comprises a 75-kDa catalytic head domain with an ATP-sensitive F-actin-binding site, a 3-kDa neck domain, which binds a single 17-kDa light chain, and a 50-kDa tail domain, which binds F-actin in the presence or absence of ATP. The actin-activated MgATPase activity of myosin IC exhibits triphasic actin dependence, apparently as a consequence of the two actin-binding sites, and is regulated by phosphorylation of Ser-329 in the head. The 50-kDa tail consists of a basic domain, a glycine/proline/alanine-rich (GPA) domain, and a Src homology 3 (SH3) domain, often referred to as tail homology (TH)-1, -2, and -3 domains, respectively. The SH3 domain divides the TH-3 domain into GPA-1 and GPA-2. To define the functions of the tail domains more precisely, we determined the properties of expressed wild type and six mutant myosins, an SH3 deletion mutant and five mutants truncated at the C terminus of the SH3, GPA-2, TH-1, neck and head domains, respectively. We found that both the TH-1 and GPA-2 domains bind F-actin in the presence of ATP. Only the mutants that retained an actin-binding site in the tail exhibited triphasic actin-dependent MgATPase activity, in agreement with the F-actin-cross-linking model, but truncation reduced the MgATPase activity at both low and high actin concentrations. Deletion of the SH3 domain had no effect. Also, none of the tail domains, including the SH3 domain, affected either the K(m) or V(max) for the phosphorylation of Ser-329 by myosin I heavy chain kinase.     

Regulation of the actin-activated ATPase activity of Acanthamoeba myosin II by copolymerization with phosphorylated and dephosphorylated peptides derived from the carboxyl-terminal end of the heavy chain

Myosin II from Acanthamoeba castellanii is a conventional myosin composed of two heavy chains and two pairs of light chains. The amino-terminal approximately 90 kDa of each heavy chain form a globular head that contains the ATPase site and an ATP-sensitive actin-binding site. The carboxyl-terminal approximately 80 kDa of both heavy chains interact to form a coiled coil, helical rod (through which the molecules self-associate into bipolar filaments) ending in a short nonhelical tailpiece. Phosphorylation of 3 serine residues at the tip of the tail (at positions 11, 16, and 21 from the carboxyl terminus) inactivates the actin-activated Mg2(+)-ATPase activity of myosin II filaments. Previous work had indicated that the activity of each myosin II molecule in a filament reflects the global state of phosphorylation of the filament rather than the phosphorylation state of the molecule itself. We have now purified the approximately 28-kDa carboxyl-terminal region of the heavy chain lacking the last two phosphorylation sites, and we have shown that this peptide copolymerizes with and regulates the actin-activated Mg2(+)-ATPase activities of native dephosphorylated and phosphorylated myosin II. It can be concluded from these studies that the biologically relevant enzymatic activity of myosin II is regulated by a phosphorylation-dependent conformational change in the myosin filaments.     

Limited tryptic digestion of Acanthamoeba myosin IA abolishes regulation of actin-activated ATPase activity by heavy chain phosphorylation

Acanthamoeba myosin IA is a globular protein composed of a 140-kDa heavy chain and a 17-kDa light chain. It expresses high actin-activated Mg2+-ATPase activity when one serine on the heavy chain is phosphorylated. We previously showed that chymotrypsin cleaves the heavy chain into a COOH-terminal 27-kDa peptide that can bind to F-actin but has no ATPase activity and a complex containing the NH2-terminal 112-kDa peptide and the light chain. The complex also binds F-actin and has full actin-activated Mg2+-ATPase activity when the regulatory site is phosphorylated. We have now localized the ATP binding site to within 27 kDa of the NH2 terminus and the regulatory phosphorylatable serine to a 20-kDa region between 38 and 58 kDa of the NH2 terminus. Under controlled conditions, trypsin cleaves the heavy chain at two sites, 38 and 112 kDa from the NH2 terminus, producing a COOH-terminal 27-kDa peptide similar to that produced by chymotrypsin and a complex consisting of an NH2-terminal kDa peptide, a central 74-kDa peptide, and the light chain. This complex is similar to the chymotryptic complex but for the cleavage which separates the 38- and 74-kDa peptides. The tryptic complex has full (K+, EDTA)-ATPase activity (the catalytic site is functional) and normal ATP-sensitive actin-binding properties. However, the actin-activated Mg2+-ATPase activity and the F-actin-binding characteristics of the tryptic complex are no longer sensitive to phosphorylation of the regulatory serine. Therefore, cleavage between the phosphorylation site and the ATP-binding site inhibits the effects of phosphorylation on actin binding and actin-activated Mg2+-ATPase activity without abolishing the interactions between the ATP- and actin-binding sites.     

Roles of the amino acid side chains in the actin-binding S-site of myosin heavy chain

The heptapeptide Ile-Arg-Ile-Cys-Arg-Lys-Gly-OEt is the analog of the S-site, one of the actin-binding sites in myosin [Suzuki et al. (1987) J. Biol. Chem. 262, 11410-11412]. Various substituted heptapeptides were synthesized, and the dissociation constants of each acto-heptapeptide complex was measured. Comparison of the dissociation constants indicated that the hydrophobic side chain of Ile-1 was critical for the binding with F-actin, but not that of Ile-3. The positive charge and the side chain length of Arg-2 were also important. The presence of a sulfur atom in the Cys-4 was also necessary. The affinity of the N-terminal Ile-Arg-Ile part for F-actin was influenced by the kind of residues in the C-terminal tetrapeptide part. Based on these results, the side chains of Ile(702), Arg(703), and Cys(SH1)(705) in myosin subfragment-1 heavy chain were assigned to be critical for the binding with F-actin. The amino acid sequence of S-1 heavy chain containing these critical residues for the S-site from residue number 700 to 717 can be predicted as an analogue of the segment B of the ATP-binding site [Walker et al. (1982) EMBO J. 1, 945-951]. The actin-binding S-site possibly shares a part of the ATP-binding site in myosin. We discuss the possibility that the S-site is an inhibitory site of myosin ATPase and the so-called actin-activation of myosin ATPase is a deinhibition induced by transient binding of F-actin to the S-site.     

Studies of ligand-induced conformational perturbations in myosin subfragment 1. An examination of the environment about the SH2 and SH1 thiols using a photoprobe

The effect of ligand binding on the environment near the SH2 and SH1 thiols in myosin subfragment 1 has been investigated by photocross-linking after specific labeling of these thiols individually with 4-(N-maleimido)benzophenone (MBP). On photolysis, cross-linking occurred from SH2-MBP to the middle 50-kDa segment, and subsequent immunopeptide mapping revealed that the cross-link was made to a peptide stretch 31-32 kDa from the N terminus in the absence of MgADP, whereas in its presence the cross-link occurred at about 60-61 kDa from the N terminus. Photolysis of SH1-MBP in the absence of MgADP resulted in a major cross-link to the 27-kDa N-terminal segment and minor cross-links to the 50-kDa middle segment. In the presence of MgADP, no new cross-link occurred but the amount of cross-linking to the 50-kDa segment increased at the expense of the other. Immunopeptide mapping indicated that the regions in the 27- and 50-kDa peptides that were cross-linked to SH1-MBP are at about 14-16 and 55-56 kDa from the N terminus respectively. These results indicate that when nucleotide binds to S1, SH2 is displaced relative to the 50-kDa segment, whereas the local environment around SH1 does not change significantly because photolysis in the presence of MgADP resulted in a change at the site of cross-linking for SH2-MBP but caused only a redistribution of the relative amounts of the cross-links formed from SH1-MBP.     

Characterization of the myosin light chain kinase from smooth muscle as an actin-binding protein that assembles actin filaments in vitro

In addition to its kinase activity, myosin light chain kinase has an actin-binding activity, which results in bundling of actin filaments [Hayakawa et al., Biochem. Biophys. Res. Commun. 199, 786-791, 1994]. There are two actin-binding sites on the kinase: calcium- and calmodulin-sensitive and insensitive sites [Ye et al., J. Biol. Chem. 272, 32182-32189, 1997]. The calcium/calmodulin-sensitive, actin-binding site is located at Asp2-Pro41 and the insensitive site is at Ser138-Met213. The cyanogen bromide fragment, consisting of Asp2-Met213, is furnished with both sites and is the actin-binding core of myosin light chain kinase. Cross-linking between the two sites assembles actin filaments into bundles. Breaking of actin-binding at the calcium/calmodulin-sensitive site by calcium/calmodulin disassembles the bundles.     

Isolation of Sugar Compounds of Asparagine,Phenylalanine, Tyrosine and Valine from Cured Tobacco Leaves

Scanning electron microscopy and rigidity characterization were used in this study of myosin gelation in the presence of actin. The heat-induced gel formation of myosin was aided by the addition of actin to myosin at a molar ratio of 1: 2.7. However, this actin effect was not observed when the actin-binding site(s) of myosin was blocked by p-chloromercuri-benzoate treatment or denaturation, or when the myosin-binding site(s) of actin was blocked by trinitrophenylation.

The effect also disappeared with an aging myosin-actin mixture, with ATP or pyrophosphate as dissociating agent prior to thermal treatment of the system.

The present findings indicate the indispensability of myosin binding to actin for enhancing the thermal gelation of myosin. The gelation process appears to involve a large electrostatic contribution, as well as such chemical reactions as oxidation of SH groups.     

The interaction and photolabeling of myosin subfragment 1 with 3'(2')-O-(4-benzoyl)benzoyladenosine 5'-triphosphate

The photoprobe 3'(2')-O-(4-benzoyl)benzoyladenosine 5'-triphosphate (Bz2ATP) was used to characterize the nucleotide-binding site of myosin subfragment 1 (SF1). Improved synthesis and purification of Bz2ATP are reported. 1H NMR and ultraviolet spectroscopy show that Bz2ATP is a 60:40 mixture of the 3'(2')-ribose isomers and that the epsilon M261 is 41,000 M-1 cm-1. Bz2ATP is hydrolyzed by SF1 comparably to ATP in the presence of actin or K+, NH4+, or Mg2+ ions; and the product, Bz2ADP, has a single binding site on SF1 (K'a = 3.0 X 10(5) M-1). [3H]Bz2ATP was photoincorporated into SF1 with concomitant loss of K+-EDTA-ATPase activity. Analysis of photolabeled SF1 showed that the three major tryptic peptides (23, 50, and 20 kDa) of the heavy chain fragment and the alkali light chains were labeled. The presence of ATP during irradiation protected only the 50-kDa peptide, indicating that the other peptides were nonspecifically labeled. If Bz2ATP was first trapped on SF1 by cross-linking the reactive thiols, SH1 and SH2, with p-phenylenedimaleimide, only the 50-kDa tryptic peptide was labeled. These results confirm and extend previous observations that [3H]Bz2ATP trapped on SF1 by cobalt(III) phenanthroline photolabeled the same 50-kDa peptide (Mahmood, R., and Yount, R.G. (1984) J. Biol. Chem. 259, 12956-12959). Thus, the 50-kDa peptide is labeled with or without thiol cross-linking, indicating that the relative position of SH1 and SH2 does not affect the labeling pattern.     

Location of a contact site between actin and myosin in the three-dimensional structure of the acto-S1 complex

Using fluorescence resonance energy transfer (FRET), we measured distances from chromophores located at or near the actin-binding stretch of amino acids 633-642 of myosin subfragment 1 (S1), to five points in the acto-S1 complex. Specific labeling of this site was achieved by first attaching the desired chromophore to an "antipeptide" that by means of its charge complementarity specifically binds to this segment of S1 [Chaussepied & Morales (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 7471] and then cross-linking the fluorescent peptide to the protein. According to this technique, antipeptides containing three different labels, viz., N-dansylaziridine, (iodoacetamido)fluorescein, and monobromobimane, were purified and covalently bound to S1. A second chromophoric group, required for FRET measurements, was selected in such a way as to provide a good spectral overlap with the corresponding peptide chromophore. Cys-707 (SH1) and Cys-697 (SH2) on S1 were modified by using iodoacetamido and maleimido derivatives of rhodamine, 1,N6-ethenoadenosine 5'-diphosphate was trapped at the S1 active site with orthovanadate, Cys-374 on actin was modified with either N-[4-[4-(dimethylamino)phenyl]azo]phenyl]maleimide or N-[(iodoacetyl)-amino]ethyl]-5-naphthylamine-1-sulfonate, and ADP bound to F-actin was exchanged with the fluorescent etheno analogue. By use of excited-state lifetime fluorometry, the following distances from the stretch 633-642 of S1 to other points on S1 or actin have been measured: Cys-707 (S1), 50.3 A; Cys-697 (S1), 49.4 A; active site of S1, greater than or equal to 44 A; nucleotide binding site (actin), 41.1 A; and Cys-374 (actin), approximately 53 A.(ABSTRACT TRUNCATED AT 250 WORDS)     

Embryonic chicken skeletal, cardiac, and smooth muscles express a common embryo-specific myosin light chain

It has been demonstrated that embryonic chicken gizzard smooth muscle contains a unique embryonic myosin light chain of 23,000 mol wt, called L23 (Katoh, N., and S. Kubo, 1978, Biochem. Biophys. Acta, 535:401-411; Takano-Ohmuro, H., T. Obinata, T. Mikawa, and T. Masaki, 1983, J. Biochem. (Tokyo), 93:903-908). When we examined myosins in developing chicken ventricular and pectoralis muscles by two-dimensional gel electrophoresis, the myosin light chain (Le) that completely comigrates with L23 was detected in both striated muscles at early developmental stages. Two monoclonal antibodies, MT-53f and MT-185d, were applied to characterize the embryonic light chain Le of striated muscles. Both monoclonal antibodies were raised to fast skeletal muscle myosin light chains; the former antibody is specific to fast muscle myosin light chains 1 and 3, whereas the latter recognizes not only fast muscle myosin light chains but also the embryonic smooth muscle light chain L23. The immunoblots combined with both one- and two-dimensional gel electrophoresis showed that Le reacts with MT-185d but not with MT-53f. These results strongly indicate that Le is identical to L23 and that embryonic chicken skeletal, cardiac, and smooth muscles express a common embryo-specific myosin light chain.