Skeletal Muscle Myosin Light Chain Research Articles

Properties of a monoclonal antibody directed to the calmodulin-binding domain of rabbit skeletal muscle myosin light chain kinase

A synthetic peptide representing the calmodulin-binding domain of rabbit skeletal muscle myosin light chain kinase (K-R-R-W-K-K-N-F-I-A-V-S-A-A-N-R-F-K-K-I-S-S-S-G-A-L) was used as an antigen to produce a monoclonal antibody. The antibody (designated MAb RSkCBP1, of the IgM class) reacted with similar affinity (KD approximately 20 nM) by competitive enzyme-linked immunoassay (ELISA) with the antigen peptide and intact rabbit skeletal muscle myosin light chain kinase. MAb RSkCBP1 inhibited rabbit skeletal muscle myosin light chain kinase activity competitively with respect to calmodulin (Ki = 20 nM). The antibody also inhibited myosin light chain kinase activity in extracts of skeletal muscle from several mammalian species (rabbit, sheep, and bovine) and an avian species (chicken). The concentration of MAb RSKCBP1 required for 50% inhibition of enzyme activity was similar for the mammalian species (80 nM) but was significantly higher for the avian species (1.2 microM). A competitive ELISA protocol was used to analyze weak cross-reactivity to other calmodulin-binding peptides and proteins. This assay demonstrated no cross-reactivity with the venom peptides melittin or mastoparan; smooth muscle myosin light chain kinases from hog carotid, bovine trachea, or chicken gizzard; bovine brain calmodulin-dependent calcineurin; or rabbit skeletal muscle troponin I. These data support the contention that the synthetic peptide used as the antigen represents the calmodulin-binding domain of rabbit skeletal muscle myosin light chain kinase and that the calmodulin-binding domains of different calmodulin-regulated proteins may have distinct primary and/or higher order structures.

Rabbit skeletal muscle myosin light chain kinase. The calmodulin binding domain as a potential active site-directed inhibitory domain.

A synthetic peptide modeled after the calmodulin (CaM)-binding domain of rabbit skeletal muscle myosin light chain kinase, Lys-Arg-Arg-Trp-Lys5-Lys-Asn-Phe-Ile-Ala10-Val-Ser-Ala-Ala-+ ++Asn15-Arg-Phe-Glycyl amide (M5), inhibited the CaM-independent chymotryptic fragment of the enzyme, C35 (Edelman, A. M., Takio, K., Blumenthal, D. K., Hansen, R. S., Walsh, K. A., Titani, K., and Krebs, E. G. (1985) J. Biol. Chem. 260, 11275-11285), with a Ki of 3.2 +/- 2.1 microM. Inhibition was competitive with respect to the peptide substrate Lys-Lys-Arg-Ala-Ala5-Arg-Ala-Thr-Ser-Asn10-Val-Phe-Ala and was of the noncompetitive linear mixed type with respect to ATP. M5 and homologues with a serine residue substituted at positions 9, 13, or 14 were phosphorylated with the following order of preference: M5(Ser9) greater than M5(Ser13) much greater than M5(Ser14) greater than M5. The order of preference observed agreed with that predicted by comparison of the sequence of these peptides with the phosphorylation sites of myosin P-light chains. Both inhibition of C35 by M5 and phosphorylation of M5 and its serine-substituted homologues were severely curtailed by the addition of a stoichiometric excess of CaM over peptide. Thus, synthetic peptides modeled after the CaM-binding domain of skeletal muscle myosin light chain kinase can function as calmodulin-regulated active site-directed inhibitors of the enzyme.

Structural studies of rabbit skeletal muscle myosin light chain kinase with monoclonal antibodies.

Monoclonal antibodies directed against rabbit skeletal muscle myosin light chain kinase have been used to study the domains of this kinase. Specificity of nine monoclonal antibodies against rabbit skeletal muscle myosin light chain kinase was demonstrated by immunoblot analysis and immunoadsorption of kinase activity. None of the antibodies reacted by immunoblot analysis with either chicken skeletal or rabbit smooth muscle myosin light chain kinases. Epitope mapping of trypsin-digested rabbit skeletal muscle myosin light chain kinase showed that antibodies 2a, 9a, 9b, 12a, 12b, 16a, and 16b are directed against the 40-kDa catalytic domain. In addition, these seven antibodies reacted with sites that are clustered within a 14-kDa fragment of the kinase generated by Staphylococcus aureus V8 protease digestion. Two monoclonal antibodies, 14a and 19a, reacted with two distinct epitopes located within the inactive, asymmetric trypsin fragment. Six of nine monoclonal antibodies (2a, 9a, 9b, 12a, 12b, and 14a) inhibited kinase activity. Kinetic analyses demonstrated that antibodies 2a, 12a, and 14a inhibited kinase activity competitively with respect to myosin phosphorylatable light chain; 2a, 12a, and 14a exhibit noncompetitive inhibition with respect to calmodulin. These data suggest that monoclonal antibodies 2a, 12a, and 14a bind at or adjacent to the active site of the kinase.

The calmodulin binding domain of chicken smooth muscle myosin light chain kinase contains a pseudosubstrate sequence.

Smooth muscle myosin light chain kinase contains a 64 residue sequence that binds calmodulin in a Ca2+-dependent manner (Guerriero, V., Jr., Russo, M. A., and Means, A. R. (1987) Biochemistry, in press). Within this region is a sequence with homology to the corresponding sequence reported for the calmodulin binding region of skeletal muscle myosin light chain kinase (Blumenthal, D. K., Takio, K., Edelman, A. M., Charbonneau, H., Titani, L., Walsh, K. A., and Krebs, E. G. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 3187-3191). Inspection of these sequences reveals that they both share a similar number and spatial arrangement of basic residues with those present in the myosin light chain substrate. We have synthesized a 22-residue peptide corresponding to residues 480-501 (determined from the cDNA) of the smooth muscle myosin light chain kinase. This peptide, Ala-Lys-Lys-Leu-Ser-Lys-Asp-Arg-Met-Lys-Lys-Tyr-Met-Ala-Arg-Arg-Lys-Trp- Gln-Lys-Thr-Gly, inhibited calmodulin-dependent activation of the smooth muscle myosin light chain kinase with an IC50 of 46 nM. At saturating concentrations of calmodulin, the 22-residue peptide inhibited myosin light chain and synthetic peptide substrate phosphorylation competitively with IC50 values of 2.7 and 0.9 microM, respectively. An 11-residue synthetic peptide analog, corresponding to part of the calmodulin-binding sequence in skeletal muscle myosin light chain kinase, Lys-Arg-Arg-Trp-Lys-Lys-Asn-Phe-Ile-Ala-Val, also competitively inhibited synthetic peptide substrate phosphorylation with a Ki of 1 microM. The competitive inhibitory activity of the calmodulin binding regions is similar to the apparent Km of 2.7 microM for phosphorylation of the 23-residue peptide analog of the smooth muscle myosin light chain and raises the possibility that the calmodulin binding region of the myosin light chain kinase may act as a pseudosubstrate inhibitor of the enzyme.

10] Preparation and properties of the calmodulin-binding domain of skeletal muscle myosin light chain kinase

Peptides corresponding to the calmodulin-binding domain of MLCK and other target enzymes promise to be useful tools in the study of calmodulin action. Not only will they help to elucidate the molecular interactions of calmodulin with specific target enzymes, but they should also prove to be useful calmodulin antagonists because of their high affinity and high specificity. Given the lack of sequence similarity in the peptides presently known to avidly bind calmodulin, it seems as likely as not that there will be little sequence homology found between calmodulin-binding domains from different calmodulin-dependent enzymes. If this is the case, it may be difficult to identify the calmodulin-binding domain of a target enzyme by simple inspection of the enzyme's sequence. However, the techniques described in this chapter require only nominal amounts of target enzyme and should, therefore, prove useful in a situation where the sequence of a target enzyme is known from DNA cloning work, but only a small quantity of pure enzyme is available for structure-function studies.

37] Recognition and characterization of calmodulin-binding sequences in peptides and proteins

Publisher Summary This chapter provides methods for the recognition and analysis of basic, amphiphilic calmodulin-binding regions in peptides and proteins. The first calmodulin-binding peptides were discovered through screening of peptide hormones for calmodulin-binding activity. Calmodulin also contains a hydrophobic patch that becomes more exposed upon calcium binding. Recent evidence has given credence to the premise that amphiphilic α helical peptides are good models for the calmodulin-binding site of target enzymes. A 27-residue peptide has been isolated from a CNBr digest of skeletal muscle myosin light chain kinase (MLCK), which appears to comprise this enzyme's calmodulin-binding domain. The helical wheel is a widely used qualitative method for locating potential calmodulin-binding sites within a protein sequence. A rapid method of quantitating the information presented on a helical wheel involves calculation of the average helical hydrophobic moment (μH), which is defined as the mean vector sum of the hydrophobicities of the residues in the helix. Binding of peptides to calmodulin can be determined in a number of ways that rely on monitoring changes in the physical and spectroscopic properties of calmodulin and/or peptides that accompany complex formation. Fluorescence spectroscopy is an excellent method for evaluating. It is also shown that peptides become more helical when they bind calmodulin and that this increase in helicity is readily measured by far-ultraviolet circular dichroism spectroscopy (CD).

Amino acid sequence of rabbit skeletal muscle myosin light chain kinase.

The amino acid sequence of the amino-terminal, 235-residue segment of rabbit skeletal muscle myosin light chain kinase has been determined. Together with the carboxyl-terminal segment previously described [Takio, K., Blumenthal, D. K., Edelman, A. M., Walsh, K. A., Krebs, E. G., & Titani, K. (1985) Biochemistry 24, 6028], the present work completes the 603-residue sequence of this protein. The amino-terminal segment that has been analyzed herein corresponds to a domain reported to be of highly asymmetrical shape and as yet unknown function. Secondary structure calculations failed to provide any evidence of alpha-helix or beta-structures, but polyproline II like helical structure is possible. Sequence analysis indicates the presence of approximately equal quantities of two isoforms differing in a single amino acid replacement. Unexpected difficulties were encountered in the present sequence analysis due to the presence of acid-labile Asp-Pro bonds and to five separable variants of a blocked 21-residue amino-terminal peptide, arising from rearrangement at an Asn-Gly bond.

Differential recognition of calmodulin-enzyme complexes by a conformation-specific anti-calmodulin monoclonal antibody.

An anti-calmodulin monoclonal antibody having an absolute requirement for Ca2+ has been produced from mice immunized with a mixture of calmodulin and calmodulin-binding proteins. Radioimmune assays were developed for the determination of its specificity. the epitope for this antibody resides on the COOH-terminal half of the mammalian protein. Plant calmodulin or troponin C had little reactivity. The apparent affinity of the antibody for calmodulin was increased approximately 60-fold in the presence of heart calmodulin-dependent phosphodiesterase. The presence of heart phosphodiesterase in the radioimmune assay greatly enhanced the sensitivity for calmodulin. The intrinsic calmodulin subunit of phosphorylase kinase and calmodulin which was bound to brain phosphodiesterases was also recognized with high affinity by the antibody. The antibody reacted poorly with calmodulin which was bound to heart or brain calcineurin, skeletal muscle myosin light chain kinase, or other calmodulin-binding proteins. In direct binding experiments, most of the calmodulin-binding proteins studied were unreactive with the antibody. This selectivity allowed purification of heart and two brain calmodulin-dependent cyclic nucleotide phosphodiesterase isozymes on immobilized antibody affinity columns. Phosphodiesterase activity was adsorbed directly from crude samples and specifically eluted with EGTA. Isozyme separation was accomplished using a previously described anti-heart phosphodiesterase monoclonal antibody affinity support. The brain isozymes differed not only in reactivity with the anti-phosphodiesterase antibody, but also in apparent subunit molecular weight, and relative specificity for cAMP and cGMP as substrates. The calmodulin activation constants for the brain enzymes were 10-20-fold greater than for the heart enzyme. The data suggest that the binding of ligands to Ca2+/calmodulin induce conformation changes in calmodulin which alter reactivity with the anti-calmodulin monoclonal antibody. The differential antibody reactivity toward calmodulin-enzyme complexes indicates that target proteins either induce very different conformations in calmodulin and/or interact with different geometries relative to the antibody binding site. The anti-calmodulin monoclonal antibody should be useful for the purification of other calmodulin-dependent phosphodiesterases as well as isozymes of phosphorylase kinase.

Phosphorylation of synthetic peptides by skeletal muscle myosin light chain kinases.

Substrate determinants for rabbit and chicken skeletal muscle myosin light chain kinases were examined with synthetic peptides. Both skeletal muscle myosin light chain kinases had similar phosphorylation kinetics with synthetic peptide substrates. Average kinetic constants for skeletal muscle myosin light chain heptadecapeptide, (formula; see text) where S(P) is phosphoserine, were Km, 2.3 microM and Vmax, 0.9 mumol/min/mg of enzyme. Km values were 122 and 162 microM for skeletal muscle peptides containing A-A for basic residues at positions 2-3 and 6-7, respectively. Average kinetic constants for smooth muscle myosin light chain peptide, (formula; see text), were Km, 1.4 microM and Vmax 27 mumol/min/mg of enzyme. Average Km values for the smooth muscle peptide, residues 11-23, were 10 microM which increased 6- and 11-fold with substitutions of alanine at residues 12 and 13, respectively. Vmax values decreased and Km values increased markedly by substitution of residue 16 with glutamate in the 11-23 smooth muscle tridecapeptide. Basic residues located 3 and 6-7 residues toward the NH2 terminus from phosphoserine in smooth muscle myosin light chain and 6-8 and 10-11 residues toward the NH2 terminus from phosphoserine in skeletal muscle myosin light chain appear to be important substrate determinants for skeletal muscle myosin light chain kinases. These properties are different from myosin light chain kinase from smooth muscle.

Characterization of myosin light-chain kinase from bovine adrenal medulla

Partially purified bovine adrenal medullary myosin light-chain kinase (MLCK) possesses a Stoke's radius of 79 Å and a sedimentation coefficient of 3.95 ± 0.45 S, yielding a native molecular weight of 150,000 ± 17,000 g/mol and a frictional ratio of 2.24. It catalyzes the phosphorylation of the isolated light chain of skeletal muscle myosin and the light chain of intact adrenal medullary myosin, but not phosphorylase b or histone. The activation of MLCK by calmodulin is specific and dose dependent, yielding a K 0.5 value of 9.0 n m; the dose response curve with respect to free Ca 2+ is biphasic, exhibiting a stimulatory phase at low free Ca 2+ concentrations ( K 0.5 = 0.17 μM) and an inhibitory phase at higher free Ca 2+ concentrations (400–3000 μ m). Michaelis-Menten kinetics are observed for ATP, yielding a K m for ATP of 25 μ m and a V max of 23.2 nmol/min/mg. However, positive cooperative kinetics are observed for the skeletal muscle myosin light chain, yielding a Hill coefficient of 3.57, a K 0.5 for light chain of 27 μ m and a V max of 16.6 nmol/min/mg. A stoichiometry of phosphorylation of approximately 1 mol of phosphate/ mol of skeletal muscle myosin light chain was observed. Therefore, adrenal medullary MLCK is similar in most, but not all, of its physical and kinetics properties to MLCKs isolated from other sources and may serve to regulate actin-myosin contractile activity in the adrenal medulla.

Steady‐state kinetics of skeletal muscle myosin light chain kinase indicate a strong down regulation by products

European Journal of BiochemistryVolume 153, Issue 2 p. 333-334 Free Access Steady-state kinetics of skeletal muscle myosin light chain kinase indicate a strong down regulation by products Ursula GEUSS, Ursula GEUSSSearch for more papers by this authorGeorg W. MAYR, Georg W. MAYRSearch for more papers by this authorLudwig M. G. HEILMEYER Jr., Ludwig M. G. HEILMEYER Jr.Search for more papers by this author Ursula GEUSS, Ursula GEUSSSearch for more papers by this authorGeorg W. MAYR, Georg W. MAYRSearch for more papers by this authorLudwig M. G. HEILMEYER Jr., Ludwig M. G. HEILMEYER Jr.Search for more papers by this author First published: December 1985 https://doi.org/10.1111/j.1432-1033.1985.tb09306.xCitations: 1AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article.Citing Literature Volume153, Issue2December 1985Pages 333-334 RelatedInformation

Steady-state kinetics of skeletal muscle myosin light chain kinase indicate a strong down regulation by products.

The kinetic behaviour of myosin light chain kinase isolated from skeletal muscle was studied under steady-state conditions using highly purified phosphorylatable light chains 2 (LC2). Forward reaction, product inhibition, and reverse reaction data indicate a sequential mechanism which can be interpreted best by a rapid-equilibrium random bi-bi reaction model. The forward reaction parameters are KATP = 150 microM, KLC2 = 5.3 microM, and Ki LC2 = 7.6 microM. The enzyme forms a dead-end complex with ADP and light chain 2; Kd, ADP of this complex is 50 microM. The forward reaction is also strongly inhibited by the phosphorylated light chain 2, Ki, LC2P is 1.5 microM. An equilibrium constant Keq of about 70 can be calculated from the kinetic parameters which agrees with the directly measured value of about 60. The role of the two inhibitory mechanisms in the regulation of the enzyme and of the high energy of the light chain phosphate bond as deducible from Keq are discussed.

Interaction of calmodulin and a calmodulin-binding peptide from myosin light chain kinase: major spectral changes in both occur as the result of complex formation.

Many different enzymes are activated by direct interaction with calmodulin; this interaction is thought to occur through a distinct calmodulin-binding domain in each of these enzymes. We have recently reported the sequence of a 27-residue peptide (denoted M13), derived from skeletal muscle myosin light chain kinase (MLCK), that exhibits the properties expected of a calmodulin-binding domain [Blumenthal, D. K., Takio, K., Edelman, A. M., Charbonneau, H., Titani, K., Walsh, K. A., & Krebs, E. G. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 3187-3191]. The interaction between chemically synthesized M13 peptide and calmodulin has been studied by circular dichroism (CD) and proton nuclear magnetic resonance (NMR) spectroscopy. In the presence of Ca2+, the observed ellipticity of an equimolar mixture of M13 and calmodulin is much greater than the sum of the ellipticities of the two isolated proteins. In the absence of Ca2+, the measured ellipticity of the mixture is approximately the sum of the two components. Addition of the peptide to calmodulin causes dramatic changes in the proton NMR spectrum; at a 1:1 molar ratio, no evidence of either free peptide or free calmodulin is observed. Moreover, these data demonstrate that a unique species of the M13-calmodulin complex is formed, indicating that the peptide binds to calmodulin in only one way. The many resonances affected by M13 binding include residues in both halves of the calmodulin molecule. The observed CD and NMR effects suggest that secondary and tertiary conformational changes occur both in M13 and in calmodulin upon complex formation.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium binding to complexes of calmodulin and calmodulin binding proteins.

The free energy of coupling for binding of Ca2+ and the calmodulin-sensitive phosphodiesterase to calmodulin was determined and compared to coupling energies for two other calmodulin binding proteins, troponin I and myosin light chain kinase. Free energies of coupling were determined by quantitating binding of Ca2+ to calmodulin complexed to calmodulin binding proteins with Quin 2 to monitor free Ca2+ concentrations. The geometric means of the dissociation constants (-Kd) for Ca2+ binding to calmodulin in the presence of equimolar rabbit skeletal muscle troponin I, rabbit skeletal muscle myosin light chain kinase, and bovine heart calmodulin sensitive phosphodiesterase were 2.1, 1.1, and 0.55 microM. The free-energy couplings for the binding of four Ca2+ and these proteins to calmodulin were -4.48, -6.00, and -7.64 kcal, respectively. The Ca2+-independent Kd for binding of the phosphodiesterase to calmodulin was estimated at 80 mM, indicating that complexes between calmodulin and this enzyme would not exist within the cell under low Ca2+ conditions. The large free-energy coupling values reflect the increase in Ca2+ affinity of calmodulin when it is complexed to calmodulin binding proteins and define the apparent positive cooperativity for Ca2+ binding expected for each system. These data suggest that in vitro differences in free-energy coupling for various calmodulin-regulated enzymes may lead to differing Ca2+ sensitivities of the enzymes.

Chromosomal distribution of genes coding for fast twitch skeletal muscle myosin light chains.

The mouse fast twitch skeletal muscle myosin light chains are encoded by a multigene family which comprises the gene coding for the myosin light chain 2 (Myl2f), and the gene coding for both myosin light chains 1 and 3 (Myl1f/Myl3f). In addition, a Myl1f/Myl3f-related pseudogene is present in the domestic mouse Mus musculus. The members of this gene family were assigned to chromosomes by molecular hybridization, using DNA extracted from a panel of cloned mouse-Chinese hamster somatic hybrid cells and specific DNA probes. The genes coding for the light chains of the myosin molecule are dispersed on several chromosomes, while genes coding for the heavy chain of myosin are located on a single, different chromosome.

Amino acid sequence of an active fragment of rabbit skeletal muscle myosin light chain kinase.

The amino acid sequence of a 368-residue segment at the carboxyl-terminus of rabbit skeletal muscle myosin light chain kinase (MLCK) has been determined. The sequence was derived primarily from analysis of two complementary sets of fragments obtained by cleavage at methionyl and arginyl bonds in S-carboxymethylated MLCK. The segment included a 360-residue fragment produced by limited tryptic digestion of MLCK. This fragment was both catalytically active and dependent on Ca2+-calmodulin. Unique structural features of MLCK have been identified, and a likely calmodulin interaction site is suggested. Sequence comparisons of MLCK to other protein kinases indicate close structural relationships in spite of marked differences in physicochemical properties, enzymatic characteristics, and regulatory response among these enzymes.

Characterization of the calmodulin-binding and catalytic domains in skeletal muscle myosin light chain kinase.

Limited proteolysis has been utilized to study the structural organization of rabbit skeletal muscle myosin light chain kinase. The enzyme (Mr approximately 89,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) consists of an amino-terminal, protease-susceptible region of unidentified function and a carboxyl-terminal, protease-resistant region of Mr approximately 40,000 containing the catalytic and calmodulin-binding domains. Partial digestion with trypsin produced an intermediate 56,000-dalton fragment and a stable 38,000-dalton fragment, both of which were catalytically active and calmodulin-dependent. Chymotryptic digestion yielded three catalytically active fragments of about 37,000, 36,000, and 35,000 daltons. The Mr = 37,000 fragment was calmodulin-dependent with an apparent affinity equivalent to that of the native enzyme (approximately 1 nM). The 36,000-dalton fragment was also calmodulin-dependent but had a approximately 200-fold lower apparent affinity. The Mr = 35,000 fragment was calmodulin-independent. These three chymotryptic fragments, had identical amino termini. Nineteen residues were missing from the carboxyl terminus of the calmodulin-independent chymotryptic fragment whereas only 8 or 9 carboxyl-terminal residues were missing from the calmodulin-dependent tryptic fragments. These results suggest that the 11-residue sequence (IAVSAANRFKK) in the carboxyl-terminal region of myosin light chain kinase contributes directly to the binding of calmodulin. This conclusion is in accord with data (Blumenthal, D. K., Takio, K., Edelman, A. M., Charbonneau, H., Titani, K., Walsh, K. A., and Krebs, E. G. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 3187-3191) that the carboxyl-terminal, 27-residue CNBr peptide of the native enzyme shows Ca2+-dependent, high affinity binding to calmodulin and that similar calmodulin-binding activity, although detectable in unfractionated CNBr digests of calmodulin-dependent enzyme forms, is much reduced in a CNBr digest of the calmodulin-independent, Mr = 35,000 chymotryptic fragment.

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.

Identification of the calmodulin-binding domain of skeletal muscle myosin light chain kinase.

In the course of determining the primary structure of rabbit skeletal muscle myosin light chain kinase (MLCK; ATP:protein phosphotransferase, EC 2.7.1.37) a peptide fragment was obtained that appears to represent the calmodulin-binding domain of this enzyme. Low concentrations of the peptide inhibited calmodulin activation of MLCK (Ki congruent to 1 nM). The peptide was not associated with a catalytically active, calmodulin-independent form of MLCK that was obtained by limited proteolysis. The peptide is 27 residues in length and represents the carboxyl terminus of MLCK. The sequence of the peptide shows no significant homology with any known protein sequence. The peptide contains one tryptophanyl residue and a high percentage of basic and hydrophobic residues, but no acidic or prolyl residues. Much of the sequence has a high probability of forming alpha helix. A chemically synthesized peptide has been prepared to study the interactions of the peptide and calmodulin in more detail. The intrinsic tryptophan fluorescence of the synthetic peptide shows a significant enhancement (approximately equal to 45%) in the presence of Ca2+ and calmodulin; fluorescence enhancement is maximal at a peptide:calmodulin stoichiometry of 1:1. Calmodulin-Sepharose affinity chromatography in the presence of 2 M urea indicates that the interaction of peptide and calmodulin is Ca2+-dependent. The results of these studies indicate that the catalytic and calmodulin-binding domains of MLCK represent distinct and separable regions of the protein. In addition, the results provide a basis for future studies of the molecular and evolutionary details of calmodulin-dependent enzyme regulation.

Tissue-specific expression of rat myosin light-chain 2 gene in transgenic mice.

One approach to determining how the differential expression of specific genes is regulated in higher organisms is to introduce cloned copies of the genes (or parts of the genes) into the genomes of individual organisms from the very beginning of their development. The way in which the exogenous genetic information behaves during the development of the experimental organisms can then provide a means of defining the DNA sequences that restrict the expression of the gene to specific cell types and times of development. So far, several different genes have been introduced into the genomes of mice, but in only a few cases have the exogenous genes retained the tissue specificity of expression of the equivalent endogenous genes. I report here that in two out of three 'transgenic' mice carrying copies of the rat gene for skeletal muscle myosin light chain 2, the exogenous gene is expressed specifically in skeletal muscle cells. The sequences contained in the cloned copy of the myosin light-chain 2 gene used in these experiments are thus sufficient to confer a tissue-specific pattern of expression.