Amino acid sequence of the amino-terminal 24 kDa fragment of the heavy chain of chicken gizzard myosin

Chicken gizzard myosin was modified with N-iodoacetyl-N'-(5-sulfo-1-naphthyl)-ethylenediamine (IAEDANS) in the presence of ATP and in 0.15 M KCl, where the myosin assumed 10S conformation. From the tryptic digest of the modified myosin, a fluorescent fragment (24 kilodaltons) was isolated by gel filtration on a Sephadex G-100 column followed by chromatography on a CM 52 column. The amino acid sequence of the fragment was analyzed by conventional methods, and was: (S,Z)K-P-L-S-D-D-E-K-F-L-F-V-D-K-N-F-V-N-N-P-L-A-Q-A-D-W-S-A-K-K- L-V-W-V-P-S-E-K-H-G-F-E-A-A-S-I-K-E-E-K-G-D-E-V-T-V-E-L-Q-E-N-G-K-K- V-T-L-S-K-D-D-I-Q-K-M-N-P-P-K-F-S-K-V-E-D-M-A-E-L-T-C-L-N-E-A-S-V-L- H-N-L-R-E-R-Y-F-S-G-L-I-Y-T-Y-S-G-L-F-C-V-V-I-N-P-Y-K-Q-L-P-I-Y-S-E-K-I- I-D-M-Y-K-G-K-K-R-H-E-M-P-P-H-I-Y-A-I-A-D-T-A-Y-R-S-M-L-Q-D-R-E-D-Q- S-I-L-C-T-G-E-S-G-A-G-K-T-E-N-T-K-K-V-I-Q-Y-L-A-V-V-A-S-S-H-K-G-K. The amino-terminus was blocked, and the fragment was assigned as an amino-terminal part of the heavy chain of gizzard myosin. Position 127 was occupied by epsilon-N-trimethyllysine. Trp-130 of rabbit skeletal myosin heavy chain, which was reported to cross-link to an azide derivative of ATP by Okamoto and Yount (Proc. Natl. Acad. Sci. U.S. 82, 1575-1579 (1985], was replaced by glutamine in gizzard myosin. Cys-93 of the fragment is the amino acid residue whose reaction with IAEDANS alters the ATPase activity of gizzard myosin (Onishi, H. (1985) J. Biochem. 98, 81-86).     

Amino acid sequence of chicken gizzard gamma-tropomyosin

Chicken gizzard muscle tropomyosin has been fractionated into its two major components, beta and gamma and the amino acid sequence of the gamma component established by the isolation and sequence analysis of fragments derived from cyanogen bromide cleavage and tryptic digestions. Despite its much slower mobility on sodium dodecyl sulfate-polyacrylamide electrophoretic gels, it has the same polypeptide chain length (284 residues) as the alpha and beta components of rabbit skeletal muscle. Evidence for microheterogeneity of the chicken gizzard component was detected both on electrophoretic gels and in the sequence analysis. The gamma component is more closely related to rabbit skeletal alpha-tropomyosin than to the beta component. While the protein is highly homologous to the rabbit skeletal tropomyosins, significant sequence differences are observed in two regions; between residues 42-83 and 258-284. In the latter region (COOH-terminal) the alterations in sequence are very similar to those seen in platelet tropomyosin when compared with the skeletal proteins.     

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.     

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.     

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.     

Amino acid sequence of a segment of the Acanthamoeba myosin II heavy chain containing all three regulatory phosphorylation sites

The actin-activated Mg2+-ATPase activity of myosin II from the soil amoeba Acanthamoeba castellanii is regulated by phosphorylation of 3 serine residues on the myosin II heavy chain. Partial chymotryptic digestion of 32P-labeled myosin II cleaves from the tail end of the myosin II heavy chain a small peptide which contains all three phosphorylation sites. During purification the phosphorylated peptide is resolved into several different species as a result of heterogeneity both in phosphate content and in size (probably due to chymotryptic cleavage at the carboxyl terminus). However, all forms of the peptide have an identical amino terminus. The sequence of the first 58 residues of the peptide is: N-S-A-L-E-S-D-K-Q-I10-L-E-D-E-I-G-D-L-H- E20-K-N-K-Q-L-Q-A-K-I-A30-Q-L-Q-D-E-I-D-G-T- P40-S-S-R-G-G-S-T-R-G-A50-S-A-R-G-A-S-V-R. The phosphorylated serines are at positions 46, 51, and 56. The first 36 residues of the sequence display a repeating 3-4-3-4 pattern of hydrophobic residues suggesting that this section of the peptide forms an alpha-helical coiled-coil structure. A -Gly-Thr-Pro sequence at residues 38-40 disrupts the alpha-helix and, at the same point, the repeating pattern of non-polar residues is lost. It is likely that the residues extending from Gly-38 to the end of the myosin II tail, which include the 3 phosphorylatable serines, form a randomly coiled or small globular structure. This is the first report of the sequence around the regulatory phosphorylation sites on any myosin heavy chain.     

Amino acid sequence of chicken gizzard smooth muscle SM22 alpha

The complete amino acid sequence of SM22 alpha, a novel and abundant 22-kDa protein from chicken gizzard smooth muscle, was determined by a combination of automated and manual Edman degradation methods on fragments produced by suitable chemical and proteolytic cleavages. The protein consists of a single polypeptide chain of 197 residues, has a Mr of 21, 978, and a net charge of +4.5 at neutral pH. The pattern of alternating hydrophilic and hydrophobic regions throughout the length of SM23 alpha is typical of a globular protein. The overall secondary structural analysis, using several algorithms based on the sequence, predicts approximately 31% alpha-helix, 24% beta-sheet, 18% beta-turn, and 27% random coil. A search against the National Biomedical Research Foundation Protein Sequence Databank (Washington) and GenBank (Los Alamos) failed to demonstrate significant similarity with any other protein of known sequence.     

Amino acid sequence of a peptide containing an essential cysteine residue of pig heart aconitase

Pig heart aconitase reacts with one mole of phenacyl bromide per molecule to give complete inactivation due to the alkylation of a cysteine residue at the active site. A tryptic peptide containing this essential residue has been isolated and its amino acid sequence determined as Ile-Gln-Leu-Leu-Cys ¹-Pro-Leu-Leu-Asn-Gln-Phe-Asp-Lys by manual methods and by the use of an automated solid phase sequencer. There is a limited similarity in amino acid sequence between this peptide and other peptides containing the cysteine residues involved in the binding of the iron-sulfur clusters of high-potential iron-sulfur protein of Rhodopseudomonas gelatinosa and rubredoxins from various bacteria.     

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.     

Functional domains of chicken gizzard myosin light chain kinase

The proteolytic susceptibility of chicken gizzard myosin light chain kinase, a calmodulin-dependent enzyme, has been utilized to define the relative location of the catalytic and regulatory domains of the enzyme. Myosin light chain kinase isolated from this source exhibits a Mr of 130,000 and is extremely sensitive to trypsin at 24 degrees C; however, the molecule is divided into susceptible and resistant domains such that proteolysis proceeds rapidly and at multiple sites in the sensitive regions even at 4 degrees C while the rest of the molecule remains relatively resistant to digestion. One of these sensitive areas is the calmodulin-binding domain. On the other hand, Staphylococcus aureus V8 protease digestion generates a calmodulin-binding fragment (Mr = 70,000) that retains Ca2+/calmodulin-dependent enzymatic activity and both of the phosphorylation sites recognized by cAMP-dependent protein kinase. In contrast, treatment with chymotrypsin produces a 95,000 Mr calmodulin-binding fragment that contains only the calmodulin-modulated phosphorylation site. Sequential proteolytic digestion studies demonstrated that the chymotryptic cleavage site responsible for the generation of this 95,000 Mr peptide is within 3,000 Mr of the V8 protease site which produces the 70,000 Mr fragment. Moreover, the non-calmodulin-modulated phosphorylation site must exist in this 3,000 Mr region. A calmodulin-Sepharose affinity adsorption protocol was developed for the digestion and used to isolate both the 70,000 and 95,000 Mr fragments for further study. Taken together, our results are compatible with a model for chicken gizzard myosin light chain kinase in which there is no overlap between the active site, the calmodulin-binding region, and the two sites phosphorylated by cAMP-dependent protein kinase with regard to their relative position in the primary sequence of the molecule.     

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.     

Amino acid sequence of chicken gizzard beta-tropomyosin. Comparison of the chicken gizzard, rabbit skeletal, and equine platelet tropomyosins

Chicken gizzard beta-tropomyosin has the same chain length (284 residues) as other muscle tropomyosins, and is most closely related to the beta component of rabbit skeletal muscle. The majority of the amino acid substitutions are restricted to two regions of the structure, residues 185-216 and 258-284. The altered sequences at the COOH-terminal ends (residue 258-284) of the two gizzard components are very similar to each other and to those in platelet tropomyosin and can be correlated with the reduced affinity of interaction of all three tropomyosins with skeletal troponin T and its T1 fragment. The virtually identical NH2-terminal sequences of all four muscle tropomyosin chains indicates that the gizzard proteins' greater ability to polymerize head-to-tail is due to the sequence changes at its COOH terminus. On the other hand, the weaker head-to-tail aggregation of the platelet protein must be due to its NH2-terminal sequence alterations. Examination of the distribution of amino acids and the frequency of their substitution in the a to g positions of the repeating pseudoheptapeptide for all five tropomyosin sequences (four muscle and one platelet) emphasizes the importance of Glu residues at position e. Examination of those features of the muscle sequences implicated in the stabilization of their coiled-coil structures and in their interactions with F-actin suggest only marginal differences among them, with the possible exception of the chicken gizzard gamma component.     

Composition of the myosin light chain kinase from chicken gizzard.

The Ca²⁺-dependent protein kinase (ATP:myosin light chain phosphotransferase) from chicken gizzard smooth muscle requires two proteins for enzymatic activity. These have approximate molecular weights of 105,000 and 17,000 daltons. The isolation procedure for each component is described. Neither component alone markedly alters either the actin-moderated ATPase activity or the phosphorylation of myosin. Activation of ATPase activity by a combination of the two components occurred only in the presence of Ca²⁺ and was always accompanied by the phosphorylation of myosin. The simultaneous activation of ATPase activity and myosin phosphorylation establishes a direct correlation between the two events.     

Amino acid sequence around the serine phosphorylated by casein kinase II in brain myosin heavy chain

Casein kinase II from bovine brain transfers about one mole of phosphate to a serine residue near the COOH terminus of the heavy chain of myosin isolated from bovine brain. We have purified and characterized a peptide that contains this phosphoserine. The peptide was generated by chymotryptic and thermolytic digestion and was isolated by gel filtration, Fe3+ affinity chromatography, and reverse-phase high pressure liquid chromatography. Its sequence, Leu-Glu-Leu-Ser(PO4)-Asp-Asp-Asp-Asp-Glu-Ser-Lys-Ala-Ser-(Xaa)-Ile-Asn-Glu-Thr- Gln-Pro-Pro-Gln, shows that the Ser(PO4) is in an acidic environment, as is typical for casein kinase II phosphorylation sites. The "hydrophobic repeat" typical of alpha-helical coiled-coils is absent, suggesting that the sequence is part of a non-helical "tail piece" of the heavy chain. A synthetic peptide corresponding to residues 1-9 is shown to be an effective substrate for casein kinase II.     

Amino acid sequence of a peptide containing an essential cysteine residue of Escherichia coli GMP synthetase.

The amino acid sequence of a 51-residue tryptic peptide of citraconylated [1-14C]carboxamidomethyl-labeled Escherichia coli GMP synthetase was determined by sequenator analyses of the intact peptide and fragments obtained by cleavage of the peptide with cyanogen bromide, trypsin, and Staphylcoccus aureus strain V8 protease. The cysteine residue of this peptide fragment is essential for glutamine-dependent GMP synthesis activity and is implicated in formation of a hypothetical covalent glutamyl-enzyme intermediate. There is essentially cysteine-containing regions of two other glutamine amidotransferases, Pseudomonas putida anthranilate synthetase Component II and chicken liver formylglycinamide ribonucleotide amidotransferase. There is, however, a cluster of amino acids with "antipathy" for helix formation and a "nonessential" cysteine of anthranilate synthetase Component II.     

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.     

Interaction of the heavy chain of gizzard myosin heads with skeletal F-actin

To probe the molecular properties of the actin recognition site on the smooth muscle myosin heavy chain, the rigor complexes between skeletal F-actin and chicken gizzard myosin subfragments 1 (S1) were investigated by limited proteolysis and by chemical cross-linking with 1-ethyl-3-[3-(dimethyl-amino)propyl]carbodiimide. Earlier, these approaches were used to analyze the actin site on the skeletal muscle myosin heads [Mornet, D., Bertrand, R., Pantel, P., Audemard, E., & Kassab, R. (1981) Biochemistry 20, 2110-2120; Labbé, J.P., Mornet, D., Roseau, G., & Kassab, R. (1982) Biochemistry 21, 6897-6902]. In contrast to the case of the skeletal S1, the cleavage with trypsin or papain of the sensitive COOH-terminal 50K-26K junction of the head heavy chain had no effect on the actin-stimulated Mg2+-ATPase activity of the smooth S1. Moreover, actin binding had no significant influence on the proteolysis at this site whereas it abolished the scission of the skeletal S1 heavy chain. The COOH-terminal 26K segment of the smooth papain S1 heavy chain was converted by trypsin into a 25K peptide derivative, but it remained intact in the actin-S1 complex. A single actin monomer was cross-linked with the carbodiimide reagent to the intact 97K heavy chain of the smooth papain S1. Experiments performed on the complexes between F-actin and the fragmented S1 indicated that the site of cross-linking resides within the COOH-terminal 25K fragment of the S1 heavy chain. Thus, for both the striated and smooth muscle myosins, this region appears to be in contact with F-actin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Amino acid sequence of the alpha chain of chicken AI hemoglobin.

Adult chicken hemoglobin is heterogeneous and contains two major components, AI and AII (1). The amino acid sequence of the alpha chain of the AI component from white leghorns (small A type) was determined and compared with that of the alpha chain of the AII component, previously determined by the authors (2). An unexpectedly large difference of 65 amino acids was found between these two chains.     

Amino acid sequence of the regulatory light chain of squid mantle muscle myosin

The amino acid sequence of the regulatory light chain of mantle muscle myosin from squid (Todarodes pacificus) was determined by conventional methods. It was: xA-E-E-A-P-R-R-V-K-L-S-Q-R-Q-M-Q-E-L-K-E-A-F-T-M-I-D-Q-D-R-D-G-F-I-G-M- E-D-L-K-D-M-F-S-S-L-G-R-V-P-P-D-D-E-L-N-A-M-L-K-E-C-P-G-Q-L-N-F-T- A-F-L-T-L-F-G-E-K-V-S-G-T-D-P-E-D-A-L-R-N-A-F-S-M-F-D-E-D-G-Q-G-F-I-P- E-D-Y-L-K-D-L-L-E-N-M-G-D-N-F-S-K-E-E-I-K-N-V-W-K-D-A-P-L-K-N-K-Q-F- N-Y-N-K-M-V-D-I-K-G-K-A-E-D-E-D. The alpha-amino group of this light chain was blocked, and a typical calcium-binding structure was recognized at the sequence of residue 26 to residue 37, like those in other myosin regulatory light chains.     

Amino acid sequence of the phosphorylation site of bovine cardiac myosin light chain

Amino acid sequences of peptides containing the phosphorylation site of bovine cardiac myosin light chain (L2) were determined. The site was localized to a serine residue in the tentative amino terminus of the light chain and is homologous to phosphorylation sites in other myosin light chains. Phosphorylation of bovine cardiac light chain by chicken gizzard myosin light chain kinase was Ca2+-calmodulin dependent. Kinetic data gave a Km of 107; microM and a Vmax of 23.6 mumol min-1 mg-1. In contrast to what has been observed with smooth muscle light chains, neither the phosphorylation site fragment of the cardiac light chain nor a synthetic tetradecapeptide containing the phosphorylation site were effectively phosphorylated by the chicken gizzard kinase. Phosphorylation of cardiac myosin light chains by chicken gizzard myosin light chain kinase, therefore, requires other regions of the light chain in addition to a phosphate acceptor site.