The primary structure of skeletal muscle myosin heavy chain: I. Sequence of the amino-terminal 23 kDa fragment

Subfragment-1 was prepared from adult chicken pectoralis myosin by limited digestion with alpha-chymotrypsin, and an amino-terminal 23 kDa fragment of the heavy chain was obtained by digesting the subfragment-1 with trypsin. The 205-residue sequence of the fragment was determined by sequencing its cyanogen bromide, tryptic, and chymotryptic peptides. The amino-terminal alpha-amino group of the fragment was acetylated, and two methylated lysines; epsilon-N-monomethyllysine and epsilon-N-trimethyllysine were recognized at the 35th and 130th positions, respectively, as in rabbit skeletal myosin. Comparing the 205-residue sequence of the skeletal myosin with those of cardiac, and gizzard myosins from chicken, considerable differences are recognized, especially in the amino-terminal region, but strong homologies are observed around the reactive lysine residue, around the epsilon-N-trimethyllysine residue, and around the consensus sequence of GXXGXGKT for nucleotide-binding proteins. On the other hand, only 12 amino acid substitutions are recognized between adult and embryonic skeletal myosins, allowing for the post-translational methylation.     

The primary structure of skeletal muscle myosin heavy chain: II. Sequence of the 50 kDa fragment of subfragment-1

The complete amino acid sequence of the 50 kDa fragment of subfragment-1 from adult chicken pectoralis muscle myosin was determined. It contained 431 residues including an epsilon-N-trimethyllysine at position 346. The 431-residue sequence corresponds to the sequence of residues 206 to 639 of chicken embryonic breast muscle myosin heavy chain which was predicted from the nucleotide sequence of the cDNA by Molina et al. [Molina, M. I., Kropp, K.E., Gulick, J., & Robbins, J. (1987) J. Biol. Chem. 262, 6478-6488]. Comparing the two sequences, 23 amino acid substitutions and three deletions/insertions are recognized.     

The primary structure of skeletal muscle myosin heavy chain: IV. Sequence of the rod, and the complete 1,938-residue sequence of the heavy chain

In the preceding paper [Maita, T., Miyanishi, T., Matsuzono, K., Tanioka, Y., & Matsuda, G. (1991) J. Biochem. 110, 68-74], we reported the amino-terminal 837-residue sequence of the heavy chain of adult chicken pectoralis muscle myosin. This paper describes the carboxyl terminal 1,097-residue sequence and the linkage of the two sequences. Rod obtained by digesting myosin filaments with alpha-chymotrypsin was redigested with the protease at high KCl concentration, and two fragments, subfragment-2 and light meromyosin, were isolated and sequenced by conventional methods. The linkage of the two fragments was deduced from the sequence of an overlapping peptide obtained by cleaving the rod with cyanogen bromide. The rod contained 1,039 amino acid residues, but lacked the carboxyl-terminal 58 residues of the heavy chain. A carboxyl-terminal 63-residue peptide obtained by cleaving the whole heavy chain with cyanogen bromide was sequenced. Thus, the carboxyl terminal 1,097-residue sequence of the heavy chain was completed. The linkage of subfragment-1 and the rod was deduced from the sequence of an overlapping peptide between the two which was obtained by cleaving heavy meromyosin with cyanogen bromide. Comparing the sequence of the adult myosin thus determined with that of chicken embryonic myosin reported by Molina et al. [Molina, M.I., Kropp, K.E., Gulick, J., & Robbins, J. (1987) J. Biol. Chem. 262, 6478-6488], we found that the sequence homology is 94%.     

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.     

Electron microscopic study on the location of 23 kDa and 50 kDa fragments in skeletal myosin head

The functional activities of myosin head are located in a 95 kilodalton (kDa) heavy chain which can be divided into three fragments of 23 kDa, 50 kDa, and 20 kDa. ATP hydrolysis sites were suggested to be located in the 23 kDa and 50 kDa fragments, and actin binding sites were in the 50 kDa and 20 kDa fragments. In this study, we obtained electron microscopic images of the myosin molecule bound with antibodies directed to the 23 kDa and 50 kDa fragments. We determined that the antigenic sites for 23 kDa fragment are located at 140-180 A from the head-rod junction of myosin, and those for 50 kDa fragment at 160 A from the junction and at the tip of the head itself. The relationship between the spatial locations and the primary structures is discussed.     

Effects of exercise on plasma myosin heavy chain fragments and MRI of skeletal muscle.

The effects of a single series of high-force eccentric contractions involving the quadriceps muscle group (single leg) on plasma concentrations of muscle proteins were examined as a function of time, in the context of measurements of torque production and magnetic resonance imaging (MRI) of the involved muscle groups. Plasma concentrations of slow-twitch skeletal (cardiac beta-type) myosin heavy chain (MHC) fragments, myoglobin, creatine kinase (CK), and cardiac troponin T were measured in blood samples of six healthy male volunteers before and 2 h after 70 eccentric contractions of the quadriceps femoris muscle. Screenings were conducted 1, 2, 3, 6, 9, and 13 days later. To visualize muscle injury, MRI of the loaded and unloaded thighs was performed 3, 6, and 9 days after the eccentric exercise bout. Force generation of the knee extensors was monitored on a dynamometer (Cybex II+) parallel to blood sampling. Exercise resulted in a biphasic myoglobin release profile, delayed CK and MHC peaks. Increased MHC fragment concentrations of slow skeletal muscle myosin occurred in late samples of all participants, which indicated a degradation of slow skeletal muscle myosin. Because cardiac troponin T was within the normal range in all samples, which excluded a protein release from the heart (cardiac beta-type MHC), this finding provides evidence for an injury of slow-twitch skeletal muscle fibers in response to eccentric contractions. Muscle action revealed delayed reversible increases in MRI signal intensities on T2-weighted images of the loaded vastus intermedius and deep parts of the vastus lateralis. We attributed MRI signal changes due to edema in part to slow skeletal muscle fiber injury.(ABSTRACT TRUNCATED AT 250 WORDS)     

Exercise-induced alterations in skeletal muscle myosin heavy chain phenotype: dose-response relationship.

This study investigated the effects of exercise training duration on the myosin heavy chain (MHC) isoform distribution in rat locomotor muscles. Female Sprague-Dawley rats (120 days old) were assigned to either a sedentary control group or to one of three endurance exercise training groups. Trained animals ran on a treadmill at approximately 75% maximal O2 uptake for 10 wk (4-5 days/wk) at one of three different exercise durations (30, 60, or 90 min/day). Training resulted in increases (P < 0.05) in citrate synthase activity in the soleus and extensor digitorum longus in both the 60 and 90 min/day duration groups and in the plantaris (Pla) in all three exercise groups. All durations of training resulted in a reduction (P < 0.05) in the percentage of MHCIIb and an increase (P < 0.05) in the percentage of MHCIIa in the Pla. The magnitude of change in the percentage of MHCIIb in the Pla increased as a function of the training duration. In the extensor digitorum longus, 90 min of daily exercise promoted a decrease (P < 0.05) in percentage of MHCIIb and increases (P < 0.05) in the percentages of MHCI, MHCIIa, and MHCIId/x. Finally, training durations >/=60 min resulted in an increase (P < 0.05) in the percentage of MHCI and a concomitant decrease (P < 0.05) in the percentage of MHCIIa in the soleus. These results demonstrate that increasing the training duration elevates the magnitude of the fast-to-slow shift in MHC phenotype in rat hindlimb muscles.     

Metabolism of myosin heavy chain in steady-state chick skeletal muscle cultures.

Synthesis, accumulation and breakdown of the 200000-mol.wt. heavy subunit of myosin were analysed over an 11 day period in muscle cell cultures isolated from the leg muscle of 12-day chick embryos. Muscle cells accumulated myosin heavy chain rapidly from days 2 to 5 and maintained a maximum, constant myosin-heavy-chain concentration between days 7 and 11. Myosin-heavy-chain content and breakdown rate were compared in steady-state muscle cultures grown either in the presence of an optimum batch of horse serum (control) or in the presence of horse serum that had been pre-selected for its ability to inhibit several-fold the rate of synthesis of myosin heavy chain (inhibitory). The quantity of myosin heavy chain in the inhibited cultures was decreased in direct proportion to the decrease in the rate of synthesis of myosin heavy chain; however, the half-lives of myosin heavy chain (control, 17.7h; inhibitory, 17.0h) were virtually identical. In contrast, the absolute rate of breakdown of myosin heavy chain, expressed as molecules/min per nucleus, was approx. 5-fold lower in the inhibited cultures (4.3 X 10(3) molecules/min per nucleus) than in the control cultures (21.7 X 10(3) molecules/min per nucleus). Thus, inhibition of myosin-heavy-chain synthesis in this case was accompanied by diminished myosin-heavy-chain concentration and absolute breakdown rate at the altered steady state, but relative myosin-heavy-chain breakdown rates were unchanged.     

Characterization of the isolated 20 kDa and 50 kDa fragments of the myosin head

We have isolated two proteolytic fragments of subfragment 1 (S-1) of myosin from rabbit skeletal muscle. These fragments, identified by their molecular weights of 20 and 50 kDa, may be functional domains that, when isolated, retain their specific function. We have studied several structural and functional features of the 20 and 50 kDa fragments. Considerable secondary structure in both fragments has been observed in CD spectrum studies. Previously CD spectra showed 64% ordered structure for the 20 kDa fragment (Muhlrad and Morales, M.F. (1984) Proc. Natl. Acad. Sci. 81, 1003) and here we show 71% ordered structures for the 50 kDa fragment. Fluorescence lifetime studies of tryptophan residues in the 50 kDa fragment and 1,5-IAEDANS-labeled SH-1 in the 20 kDa fragment are used to investigate the tertiary structure of the fragments. We find the tertiary structure relating to this measurement of both fragments to be intact; however, the reaction of 1,5-IAEDANS with SH-1 on the isolated 20 kDa fragment is less specific than with S-1. Furthermore, the fragments showed a tendency to aggregate. The domain concept of S-1 was supported by the characteristic biochemical function of the isolated fragments. Both of the fragments were effective in competing with S-1 for binding to actin in acto-S-1 ATPase measurements. From these studies and in direct binding measurement the 20 kDa fragment proved to bind with higher affinity to actin than did the 50 kDa fragment.     

Primary structure of myosin heavy chain from fast skeletal muscle of Chum salmon Oncorhynchus keta

The nucleotide sequence of the cDNA encoding myosin heavy chain of chum salmon Oncorhynchus keta fast skeletal muscle was determined. The sequence consists of 5,994 bp, including 5,814 bp of translated region deducing an amino acid sequence of 1,937 residues. The deduced sequence showed 79% homology to that of rabbit fast skeletal myosin and 84-87% homology to those of fast skeletal myosins from walleye pollack, white croaker and carp. The putative binding-sites for ATP, actin and regulatory light-chains in the subfragment-1 region of the salmon myosin showed high homology with the fish myosins (78-100% homology). However, the Loop-1 and Loop-2 showed considerably low homology (31-60%). On the other hand, the deduced sequences of subfragment-2 (533 residues) and light meromyosin (564 residues) showed 88-93% homology to the corresponding regions of the fish myosins. It becomes obvious that several specific residues of the rabbit LMM are substituted to Gly in the salmon LMM as well as the other fish LMMs. This may be involved in the structural instability of the fish myosin tail region.     

The effect of glucocorticoids on the myosin heavy chain isoforms' turnover in skeletal muscle

The purpose of this study was to find the effect of dexamethasone on the myosin heavy chain (MyHC) isoforms' composition in different skeletal muscles and glycolytic (G) fibres in relation with their synthesis rate and degradation of MyHC isoforms by alkaline proteinases. Eighteen-week-old male rats of the Wistar strain were treated with dexamethasone (100 microg/100 g bwt) during 10 days. The forelimb strength decreased from 9.52 to 6.19 N (P<0.001) and hindlimb strength from 15.54 to 8.55 N (P<0.001). Daily motor activity decreased (total activity from 933 to 559 and ambulatory activity from 482 to 226 movements/h, P<0.001). The degradation rate of muscle contractile proteins increased from 2.0 to 5.9% per day (P<0.001), as well as the myosin heavy chain IIB isoform degradation with alkaline proteinase in fast-twitch (F-T) muscles (12 +/- 0.9%; P<0.05) and glycolytic muscle fibres (15 +/- 1.1%; P<0.001). The synthesis rate of MyHC type II isoforms decreased in Pla muscles (P<0.05) and MyHC IIA (P<0.05) and IIB in EDL muscle and G fibres (P<0.001). The relative content of MyHC IIB isoform decreased in F-T muscles (P<0.001) and in G fibres (P<0.01), and the relative content of IIA and IID isoforms increased simultaneously. Dexamethasone decreased the MyHC IIB isoform synthesis rate and increased the sensibility of MyHC IIB isoform to alkaline proteinase, which in its turn led to the decrease of MyHC IIB isoform relative content in F-T muscles with low oxidative potential and G muscle fibres.     

The genes coding for the muscle contractile proteins, myosin heavy chain, myosin light chain 2, and skeletal muscle actin are located on three different mouse chromosomes.

The chromosomal distribution of murine genes expressed during differentiation of skeletal muscle cells was determined by Southern blot analysis of DNA from mouse-Chinese hamster hybrid cell lines containing incomplete subsets of mouse chromosomes. All detectable myosin heavy chain genes are located on chromosome 11. The gene for the myosin light chain 2 is located on chromosome 7. The skeletal muscle alpha-actin gene and several other actin genes, or pseudogenes, are located on chromosome 3. Additional actin DNA sequences are distributed on other mouse chromosomes.     

The genes coding for the muscle contractile proteins, myosin heavy chain, myosin light chain 2, and skeletal muscle actin are located on three different chromosomes

[This corrects the article on p. 1299 in vol. 1.].     

Isolation and mapping of two porcine skeletal muscle myosin heavy chain isoforms

The present paper describes the isolation and linkage mapping of two isoforms of skeletal muscle myosin heavy chain in pig. Two partial cDNAs (pAZMY4 and pAZMY7), coding for the porcine myosin heavy chain-2B and -β respectively, have been isolated from a pig skeletal muscle cDNA library. Four RFLPs were detected with the putative porcine skeletal myosin heavy chain-2B probe (pAZMY4) and one RFLP was identified with the putative myosin heavy chain-β probe (pAZMY7). Two myosin heavy chain loci were mapped by linkage analysis performed with the five RFLPs against the PiGMaP linkage consortium ResPig database: the MYH1 locus, which identifies the fast skeletal muscle myosin heavy chain gene cluster, was located at the end of the map of porcine chromosome 12, while the MYH7 locus, which identifies the myosin heavy chain-α/-β gene cluster, was assigned to the long arm of porcine chromosome 7.     

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).     

The correlation between the synthesis of skeletal muscle actin,myosin heavy chain,and myosin light chain and the accumulation of corresponding mRNA sequences during myogenesis

Cloned cDNA probes were used to measure the accumulation of myosin heavy chain, myosin light chain 2, and actin mRNA during differentiation of rat skeletal muscle cell cultures. This was compared with the changes in the rate of synthesis of the corresponding proteins. Accumulation of those mRNA sequences was detectable a few hours before the onset of the phase of cell fusion; however, the main increase in hybridizable RNA occurred during the phase of rapid cell fusion. A close correlation was found between the amounts of mRNAs coding for these proteins and the rate of synthesis of the proteins. The results suggest that the activation of stored mRNA is not a major mechanism for controlling the time at which these proteins are synthesized.     

Complete primary structure of a scallop striated muscle myosin heavy chain. Sequence comparison with other heavy chains reveals regions that might be critical for regulation

We have determined the primary structure of the myosin heavy chain (MHC) of the striated adductor muscle of the scallop Aequipecten irradians by cloning and sequencing its cDNA. It is the first heavy chain sequence obtained in a directly Ca(2+)-regulated myosin. The 1938-amino acid sequence has an overall structure similar to other MHCs. The subfragment-1 region of the scallop MHC has a 59-62% sequence identity with sarcomeric and a 52-53% identity with nonsarcomeric (smooth and metazoan nonmuscle) MHCs. The heavy chain component of the regulatory domain (Kwon, H., Goodwin, E. B., Nyitray, L., Berliner, E., O'Neall-Hennessey, E., Melandri, F. D., and Szent-Gy?rgyi, A. G. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 4771-4775) starts at either Leu-755 or Val-760. Ca(2+)-sensitive Trp residues (Wells, C., Warriner, K. E., and Bagshaw, C. R. (1985) Biochem. J. 231, 31-38) are located near the C-terminal end of this segment (residues 818-827). More detailed sequence comparison with other MHCs reveals that the 50-kDa domain and the N-terminal two-thirds of the 20-kDa domain differ substantially between sarcomeric and nonsarcomeric myosins. In contrast, in the light chain binding region of the regulatory domain (residues 784-844) the scallop sequence shows greater homology with regulated myosins (smooth muscle, nonmuscle, and invertebrate striated muscles) than with unregulated ones (vertebrate skeletal and heart muscles). The N-terminal 25-kDa domain also contains several residues which are preserved only in regulated myosins. These results indicate that certain heavy chain sites might be critical for regulation. The rod has features typical of sarcomeric myosins. It is 52-60% and 30-33% homologous with sarcomeric and nonsarcomeric MHCs, respectively. A Ser-rich tailpiece (residues 1918-1938) is apparently nonhelical.     

A monoclonal antibody to the embryonic myosin heavy chain of rat skeletal muscle

A monoclonal antibody, 2B6, has been prepared against the embryonic myosin heavy chain of rat skeletal muscle. On solid phase radioimmunoassay, 2B6 shows specificity to myosin isozymes known to contain the embryonic myosin heavy chain and on immunoblots of denatured contractile proteins and on competitive radioimmunoassay, it reacts only with the myosin heavy chain of embryonic myosin and not with the myosin heavy chain of neonatal or adult fast and slow myosin isozymes or with other contractile or noncontractile proteins. This specificity is maintained with cat, dog, guinea pig, and human myosins, but not with chicken myosins. 2B6 was used to define which isozymes in the developing animal contained the embryonic myosin heavy chain and to characterize the changes in embryonic myosin heavy chain in fast versus slow muscles during development. Finally, 2B6 was used to demonstrate that thyroid hormone hastens the disappearance of embryonic myosin heavy chain during development, while hypothyroidism retards its decrease. This confirmed our previous conclusion that thyroid hormones orchestrate changes in isozymes during development.     

Dimerization specificity of adult and neonatal chicken skeletal muscle myosin heavy chain rods

The dimerization specificity of the recombinantly expressed and purified rod domain of adult and neonatal chicken myosin heavy chain was analyzed using metal chelation chromatography. Our results indicate that full-length adult and neonatal rods preferentially formed homodimers when renatured from an equimolar mixture of the two isoforms denatured in guanidine hydrochloride. The contribution made toward the dimerization specificity by subdomains of the rod has been addressed by making a chimeric protein consisting of the subfragment 2 (S2) region of the adult isoform and the light meromyosin region of the neonatal isoform. The proportion of heterodimers formed in exchange experiments between the chimera and the neonatal and adult rods rose with increase in the sequence homology between the two exchanging proteins. This suggests that multiple regions of the rod domain of chicken MyHC including S2 can contribute toward dimerization specificity.     

Fluorescence polarization of skeletal muscle fibers labeled with rhodamine isomers on the myosin heavy chain.

Fluorescence polarization was used to examine orientational changes of Rhodamine probes in single, skinned muscle fibers from rabbit psoas muscle following either photolysis of caged nucleotides or rapid length changes. Fibers were extensively and predominantly labeled at SH1 (Cys-707) of the myosin heavy chain with either the 5- or the 6-isomer of iodoacetamidotetramethylrhodamine. Results from spectroscopic experiments utilizing the two Rhodamine isomers were quite similar. Following photolysis of either caged ATP or caged ADP, probes promptly reoriented toward the muscle fiber axis. Changes in the fluorescence polarization signals with transients elicited by the photolysis of caged ATP in the presence of saturating Ca2+ greatly preceded active force generation. Photolysis of caged ADP caused only a small, rapid decrease in force but elicited changes in the fluorescence polarization signals with time course and amplitude similar to those following photolysis of caged ATP. Fluorescence polarization signals were virtually unchanged by rapid length steps in both rigor and active muscle fibers. These results indicate that structural changes monitored by Rhodamine probes at SH1 are not associated directly with the force-generating event of muscle contraction. However, the fluorescence polarization transients were slightly faster than the estimated rate of cross-bridge detachment following photolysis of caged ATP, suggesting that the observed structural changes at SH1 may be involved in the communication pathway between the nucleotide- and actin-binding sites of myosin.