Proteolytic susceptibility of both isolated and bound light chains from various myosins to myopathic hamster protease

Myopathic hamster protease was incubated with turkey gizzard, scallop adductor, and Loligo mantle retractor myosins in order to establish if the regulatory light chain could be selectively digested. In contrast to cardiac or skeletal muscle myosin in which almost all of the regulatory light chain is degraded, these light chains from smooth and invertebrate muscle myosins were remarkably resistant to proteolysis. In the case of scallop myosin, increasing the protease to myosin ratio resulted in comparable digestions of both the regulatory and essential light chains regardless of the presence of Mg2+. The isolated light chains on the other hand were readily digested into smaller fragments. In addition, it was observed that the myosin heavy chains were extremely sensitive and that it was possible to cleave them quantitatively to produce a new band moving with a mobility on SDS gels corresponding to an Mr of approximately 150,000. This was again at variance with cardiac or skeletal myosin where the breakdown of the heavy chains was shown to be minimal. In spite of the significant extent of heavy chain cleavage, gizzard myosin appears to maintain its tertiary structure as demonstrated by sedimentation velocity and equilibrium ultracentrifugation analysis. Moreover, upon examination by electron microscopy, both intact and cleaved gizzard myosin revealed the characteristic folded structure which had a sedimentation rate of about 10 S when dialyzed into a low salt, Mg X ATP-containing buffer. The effects and implications of such modifications on catalytic activities of gizzard, scallop, and Loligo myosins are discussed in detail.     

Effects of proteolysis on the adenosinetriphosphatase activities of thymus myosin

Limited proteolysis was used to identify regions on the heavy chains of calf thymus myosin which may be involved in ATP and actin binding. Assignments of the various proteolytic fragments to different parts of the myosin heavy chain were based on solubility, gel filtration, electron microscopy, and binding of 32P-labeled regulatory light chains. Chymotrypsin rapidly cleaved within the head of thymus myosin to give a 70,000-dalton N-terminal fragment and a 140,000-dalton C-terminal fragment. These two fragments did not dissociate under nondenaturing conditions. Cleavage within the myosin tail to give heavy meromyosin occurred more slowly. Cleavage at the site 70,000 daltons from the N-terminus of the heavy chain caused about a 30-fold decrease in the actin concentration required to achieve half-maximal stimulation of the magnesium-adenosinetriphosphatase (Mg-ATPase) activity of unphosphorylated thymus myosin. The actin-activated ATPase activity of this digested myosin was only slightly affected by light chain phosphorylation. Actin inhibited the cleavage at this site by chymotrypsin. In the presence of ATP, chymotrypsin rapidly cleaved the thymus myosin heavy chain at an additional site about 4000 daltons from the N-terminus. Cleavage at this site caused a 2-fold increase in the ethylenediaminetetraacetic acid-ATPase activity and 3-fold decreases in the Ca2+- and Mg-ATPase activities of thymus myosin. Thus, cleavage at the N-terminus of thymus myosin was affected by ATP, and this cleavage altered ATPase activity. Papain cleaved the thymus myosin heavy chain about 94,000 daltons from the N-terminus to give subfragment 1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Two smooth muscle myosin heavy chains differ in their light meromyosin fragment

Smooth muscle myosin heavy chains [SM1, approximately 205 kilodaltons (kDa), and SM2, approximately 200 kDa] were separated on sodium dodecyl sulfate (SDS)-polyacrylamide gels. Peptide maps of the two heavy chains showed unique patterns. Limited proteolytic cleavage of purified swine stomach myosin was performed by using a variety of proteases to produce the major myosin fragments which were resolved on SDS gels. A single band was obtained for heavy meromyosin in the soluble fraction following chymotrypsin digestion. However, a variable number of bands were observed for light meromyosin fragments in the insoluble fraction after chymotrypsin digestion. Peptide mapping indicated that the two bands observed after short digestion times with chymotrypsin had relative mobility and solubility properties consistent with approximately 100- and 95-kDa light meromyosin (LMM) fragments. These results indicate that the region of difference between SM1 and SM2 lies in the LMM fragment.     

Changes in myosin isozymes during development of chicken gizzard muscle

The distribution of myosin isozymes in embryonic and adult chicken gizzard muscle were examined by electrophoresis in a non-denaturing gel system (pyrophosphate acrylamide gel electrophoresis), and both light and heavy chains of embryonic and adult myosin isozymes were compared. In pyrophosphate acrylamide gel electrophoresis, there were three isozyme components in embryonic gizzard myosin, but only one isozyme in adult gizzard myosin. The mobility of the fastest migrating embryonic isozyme was similar to that of the adult isozyme. The three embryonic isozymes differ from each other in the light chain distribution. Two of them contain an embryo-specific myosin light chain, which is characterized by its molecular weight and isoelectric point, whereas the other embryonic myosin isozyme contained the same light chains as the adult myosin. The pattern of peptide fragments of embryonic heavy chain produced by digestion with alpha-chymotrypsin in the presence of SDS was not distinguishable from that of adult myosin heavy chain. Thus there are myosin isozymes specific to embryonic gizzard muscle which exhibit embryo-specific light chain compositions, but are similar to adult gizzard myosin in their heavy chain structure.     

Light chains of chicken embryonic gizzard myosin.

1. Myosin from gizzards of 15-day-old chicken embryos was highly purified by ammonium sulfate fractionation in the presence of ATP and MgCl2, ultra-centrifugation and Sepharose 4B chromatography. 2. The myosin composed of heavy and three light chains as determined by sodium dodecyl sulfate (SDS) gel electrophoresis. The molecular weights of the light chains were 23,000 (L23), 20,000 (L20), and 17,000 (L17), respectively. The amount of L23 light chain decreased and disappeared, and the L17 light chain increased steadily in the course of development. The amount of L20 light chain did not change. 3. ATPase activity of the embryonic myosin was essentially the same as that of adult myosin. The change in the light chain pattern in the course of development did not correlate to the ATPase activity. 4. Antigenicity of the heavy chains in the embryonic myosin was the same as that of the adult heavy chains. However, antibodies to light chains were not detected in the antibodies to either the embryonic or adult myosins.     

A new cardiac myosin characterized from the canine atria.

Canine atrial myosin light chains were electrophoretically distinct from myosins of canine ventricles on 5–20% polyacrylamide gradient slab gels (SDS), giving molecular weights of 26,000 and 21,000 as compared to 28,000 and 18,500 for ventricular myosin light chains. While atrial myosin heavy chains were immunologically identical with ventricular myosin heavy chains, in contrast, there was 8.0% relative cross-reactivity of atrial myosin light chains with left ventricular myosin light chains by radioimunoassay. According to charge separation on two-dimensional polyacrylamide urea gels, atrial myosin light chains were different from those of ventricular myosins. Variances in ATPase activities between atrial and ventricular myosins were strongly demonstrated. There was a lower K⁺ activated ATPase activity in atrial myosin, however the Ca²⁺ activated ATPase activity, at ATP saturation levels, was higher in atrial myosin as compared to ventricular myosins.     

Separation and identification of Drosophila myosin light chains

Myosin was extracted from the larvae and adult flies of Drosophila melanogaster, and purified by column chromatography in the presence of KI. Myosin light chains were separated from heavy chains by column chromatography after treatment of the myosin with urea, and they were identified by 2D-gel electrophoresis. Tubular muscles and fibrillar muscles have different light chains. Lt1 (Mw = 31,000), Lt2 (Mw = 30,000), Lt2' (Mw = 30,000), and Lt3 (Mw = 20,000) exist in the tubular myosin of both larvae and adult flies; Lf1 (Mw = 34,000), Lf2 (Mw = 30,000), Lf2' (Mw = 30,000), and Lf3 (Mw = 20,000) exist in the fibrillar myosin. Polyacrylamide gel electrophoresis of myosin under nondissociating conditions revealed that there was one major myosin isozyme in each type of adult muscle, and the re-electrophoresis of each isozyme on SDS gel confirmed our identification of the light chains.     

Conformationally altered aortic myosin light chains

Aorta smooth myosin contains two types of light chain, LC20 and LC17, which fold together with the N-terminal region of each heavy chain to form the globular head region of myosin. We demonstrate an altered conformation of LC20 after its separation from heavy chain by high concentrations of urea, on the basis of the following evidende: 1) A polyclonal antibody against LC20 was not able to recognize this conformationally altered form; 2) Myosin reconstituted from heavy chains and urea-dissociated light chains exhibited extremely low ATPase activity. Circular dichroism unfolding profiles showed that light chains dissociated from heavy chains by SDS appeared to be more stable than those generated by urea dissociation.     

Comparison of myosin isoenzymes present in skeletal and cardiac muscles of the Arctic charr Salvelinus alpinus (L.). Sequential expression of different myosin heavy chains during development of the fast white skeletal muscle.

The expression of myosin isoforms and their subunit composition in the white skeletal body musculature of Arctic charr (Salvelinus alpinus) of different ages (from 77-day embryos until about 5 years old) was studied at the protein level by means of electrophoretic techniques. Myosin from the white muscle displayed three types of light chain during all the developmental stages examined: two myosin light chains type 1 (LC1F) differing in both apparent molecular mass and pI, one myosin light chain type 2 (LC2F) and one myosin light chain type 3 (LC3F). The fastest-migrating form of LC1F seemed to be predominant during the embryonic and eleutheroembryonic periods. The slowest-migrating form of LC1F was predominant in the 5-year-old fish. Between 1 year and 4 years, both types of LC1F were present in similar amounts. Cardiac as well as red muscle myosin from 3-year-old fish had two types of light chain. The myosin light chains from atria and ventriculi were indistinguishable by two-dimensional electrophoresis, but were different from the myosin light chains from red muscle. Neither the light chains from cardiac nor red muscle were coexpressed with the myosin light chains of white muscle at any of the developmental stages examined. Two myosin heavy chain bands were resolved by SDS/glycerol/polyacrylamide gel electrophoresis of the extract from embryos. One of the bands was present in minor amounts. The other, and most abundant, band comigrated with the only band found in the extracts of white muscle myosin from older fish. One-dimensional Staphylococcus aureus V8 protease peptide mapping of these bands revealed some differences during development of the white muscle tentatively interpreted as follows. The myosin heavy chain band present in minor amounts in the embryos may represent an early embryonic form that is replaced by a late embryonic or foetal form in the eleutheroembryos. The foetal myosin heavy chain appears to be present until the resorption of the yolk sack and beginning of the free-swimming stage. A new form of myosin heavy chain, termed neonatal and probably expressed around hatching, is present until about 1 year of age.(ABSTRACT TRUNCATED AT 400 WORDS)     

Regenerating adult chicken skeletal muscle and satellite cell cultures express embryonic patterns of myosin and tropomyosin isoforms

Regenerating areas of adult chicken fast muscle (pectoralis major) and slow muscle (anterior latissimus dorsi) were examined in order to determine synthesis patterns of myosin light chains, heavy chains and tropomyosin. In addition, these patterns were also examined in muscle cultures derived from satellite cells of adult fast and slow muscle. One week after cold-injury the regenerating fast muscle showed a pattern of synthesis that was predominately embryonic. These muscles synthesized the embryonic myosin heavy chain, beta-tropomyosin and reduced amounts of myosin fast light chain-3 which are characteristic of embryonic fast muscle but synthesized very little myosin slow light chains. The regenerating slow muscle, however, showed a nearly complete array of embryonic peptides including embryonic myosin heavy chain, fast and slow myosin light chains and both alpha-fast and slow tropomyosins. Peptide map analysis of the embryonic myosin heavy chains synthesized by regenerating fast and slow muscles showed them to be identical. Thus, in both muscles there is a return to embryonic patterns during regeneration but this return appears to be incomplete in the pectoralis major. By 4 weeks postinjury both regenerating fast and slow muscles had stopped synthesizing embryonic isoforms of myosin and tropomyosin and had returned to a normal adult pattern of synthesis. Adult fast and slow muscles yielded a satellite cell population that formed muscle fibers in culture. Fibers derived from either population synthesized the embryonic myosin heavy chain in addition to alpha-fast and beta-tropomyosin. Thus, muscle fibers derived in culture from satellite cells of fast and slow muscles synthesized a predominately embryonic pattern of myosin heavy chains and tropomyosin. In addition, however, the satellite cell-derived myotubes from fast muscle synthesized only fast myosin light chains while the myotubes derived from slow muscle satellite cells synthesized both fast and slow myosin light chains. Thus, while both kinds of satellite cells produced embryonic type myotubes in culture the overall patterns were not identical. Satellite cells of fast and slow muscle appear therefore to have diverged from each other in their commitment during maturation in vivo.     

Purification and characterization of myosin from the clonal rat glial cell strain C-6.

A myosin was isolated from the clonal rat glial cell strain C-6 and compared with rat skeletal muscle myosin. After cell extracts were subjected to gel filtration chromatography in the presence of KI and magnesium pyrophosphate the C-6 myosin was rapidly purified by a procedure similar to that used for skeletal muscle myosin. The C-6 myosin resembles muscle myosin both physically and enzymatically. It contains heavy chains of 200,000 daltons and two classes of light chains of 17,000 and 19,000 daltons in approximately equal molar ratios. This myosin forms bipolar thick filaments in 0.1 M KCl and binds reversibly to skeletal muscle F-actin, the binding being inhibited by MgATP. Skeletal muscle F-actin stimulates the C-6 myosin adenosine triphosphatase 2- to 3-fold in the presence of KCl and Mg2+. The action activation of muscle myosin ATPase at low ionic strength is 10-fold greater than that of C-6 myosin. Ca2+ and EDTA stimulated the ATPase activities of both enzymes. When assayed in the presence of 0.6 M KCl and 1 mM EDTA the skeletal muscle myocin ATPase demonstrates substrate saturation while the C-6 myosin enzyme activity is stimulated by ATP concentrations above 2.5 mM.     

Turnover of myosin heavy and light chains in cultured embryonic chick cardiac and skeletal muscle

The relative rates of synthesis and breakdown of myosin heavy and light chains were studied in primary cell cultures of embryonic chick cardiac and skeletal muscle. Measurements were made after 4 days in culture, at which time both skeletal and cardiac cultures were differentiated and contracted spontaneously. Following a 4-hr pulse of radioactive leucine, myosin and its heavy and light chains were extracted to 90% or greater purity and the specific activities of the proteins were determined. In cardiac muscle, myosin heavy chains were synthesized approximately 1.6 times the rate of myosin light chains, and in skeletal muscle, heavy chains were synthesized at approximately 1.4 times the rate of light chains. Relative rates of degradation of muscle proteins were determined using a dual-isotope technique. In general, the soluble and myofibrillar proteins of both types of muscle had decay rates proportional to their molecular weights (larger proteins generally had higher decay rates) based on analyses utilizing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A notable exception to this general rule was myosin heavy chains, which had decay rates only slightly higher than the myosin light chains. Direct measurements on purified proteins indicated that the heavy chains of myosin were turning over at a slightly greater rate (approximately 20%) than the myosin light chains in both cardiac and skeletal muscle. The reasons for the apparent discrepancy between these measurements of myosin heavy and light chain synthesis and degradation are discussed.     

Effect of monoclonal antibodies on the properties of smooth muscle myosin

Monoclonal antibodies were generated against turkey gizzard myosin, and their effects on some of the properties of myosin were assayed. Ca2+- and Mg2+-ATPase activities of myosin were enhanced by the anti-subfragment 2 antibodies at low ionic strength (i.e., with 10S myosin). Tryptic fragments of an anti-S2 IgM also activated these activities. Antibodies directed against subfragment 1 and light meromyosin had no effect. The Mg2+-ATPase activity of heavy meromyosin also was activated by an anti-S2 antibody. Actin-activated ATPase activity of phosphorylated myosin was enhanced by the anti-S2 IgM fragments at low MgCl2 concentrations. This increase was reflected by a 5-fold increase in Vmax and a slight decrease in the apparent dissociation constant for actin. The actin-activated ATPase of dephosphorylated myosin was not affected by intact anti-S2 antibody or its fragments. The rates of phosphorylation and dephosphorylation of the 20,000-dalton light chains were increased by interaction of myosin with anti-S2 antibody. Limited proteolysis of myosin was used as a conformational probe. Interaction of anti-S2 antibody with 10S myosin increased the extent of cleavage at the S1-S2 junction. Proteolysis of 6S myosin was rapid and was not influenced by anti-S2 antibody. Our interpretation of these results is that interaction of the anti-S2 antibodies with myosin alters the conformation in the S2 region and this in turn modifies some of the properties of myosin. This is consistent with the hypothesis that the S2 region of smooth muscle myosin is a determinant of its biological properties.     

Formation and characterization of myosin hybrids containing essential light chains and heavy chains from different muscle myosins

Light chain exchange in 4.7 M NH4Cl was used to hybridize the essential light chain of cardiac myosin with the heavy chain of fast muscle myosin subfragment 1, S-1. The actin-activated ATPase properties of this hybrid were compared to those of the two fast S-1 isoenzymes, S-1(A1), fast muscle subfragment 1 which contains only the alkali-1 light chain, and S-1(A2), fast muscle myosin subfragment 1 which contains only the alkali-2 light chain. This hybrid S-1 behaved like S-1(A1)., At low ionic strength in the presence of actin, this hybrid had a maximal rate of ATP hydrolysis about the same as that of S-1(A1) and about one-half that of S-1(A2), while at higher ionic strengths the actin-activated ATPases of these three S-2 species were all similar. Light chain exchange in NH4Cl was also used to hybridize the essential light chains of fast muscle myosin with the heavy chains of cardiac myosin and to hybridize the essential light chains of cardiac myosin with the heavy chains of fast muscle myosin. In 60 and 100 mM KCl, the actin-activated ATPases of these two hybrid myosins were very different from those of the control myosins with the same essential light chains but were very similar to those of the control myosins with the same heavy chains, differing at most by one-third.     

Dinitrophenylation of chicken gizzard myosin: reactivity of the 17 000-dalton light chain

Chicken gizzard myosin rapidly incorporated 3 mol of 1-fluoro-2,4-dinitrobenzene per 4.7 x 10(5) g of protein with little change in the ATPase (ATP phosphohydrolase, EC 3.6.1.3) activity. During an interval when 2 additional mol of the reagent were bound the K+-ATPase activity in the presence of EDTA was inhibited and the Ca2+-ATPase activity was altered to a lesser extent. Cysteine residues were modified in the dinitrophenylated gizzard myosin. The dinitrophenyl group was located mainly in the active proteolytic fragment, subfragment 1. Dinitrophenylation of the heavy and light chains was observed but major changes in the ATPase activity occurred when the 17 000-dalton light chain and some heavy chains were modified as judged by dissociation experiments in sodium dodecyl sulfate. Thiolysis of the dinitrophenylated gizzard myosin with 2-mercaptoethanol restored the ATPase activity and approx. 2 mol of the dinitrophenyl group were removed. The restoration of the enzymic activity, however, occurred when 1 mol of the label was thiolytically cleaved from cysteine residues of the 17 000-dalton light chain. Substrate Mg-ATP(2-) or MgADP did not protect the ATPase activity of modified gizzard myosin. In the presence of nucleotide there was an increase in the incorporation of the reagent, and a change in its distribution into the light and heavy chains. Calcium had no effect on the dinitrophenylation of this myosin. these results indicate that the reagent, 1-fluoro-2,4-dinitrobenzene, could detect chemical differences in smooth muscle myosin when compared to the reactivity of other myosins. Thiol groups of the 17 000-light chain (and some heavy chains) are probably located peripheral to the active site region of gizzard myosin and they are involved in maintaining the enzymic activity of this protein.     

Isolation and characterization of myosin from amoebae of Physarum polycephalum

Myosin was isolated from amoebae of Physarum polycephalum and compared with myosin from plasmodia, another motile stage in the Physarum life cycle. Amoebal myosin contained heavy chains (Mr approximately 220,000), phosphorylatable light chains (Mr 18,000), and Ca2+-binding light chains (Mr 14,000) and possessed a two-headed long-tailed shape in electron micrographs after rotary shadow casting. In the presence of high salt concentrations, myosin ATPase activity increased in the following order: Mg-ATPase activity less than K-EDTA-ATPase activity less than Ca-ATPase activity. In the presence of low salt concentrations, Mg-ATPase activity was activated approximately 9-fold by skeletal muscle actin. This actin-activated ATPase activity was inhibited by micromolar levels of Ca2+. Amoebal myosin was indistinguishable from plasmodial myosin in ATPase activities and molecular shape. However, the heavy chain and phosphorylatable light chains of amoebal myosin could be distinguished from those of plasmodial myosin in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, peptide mapping, and immunological studies, suggesting that these are different gene products. Ca2+-binding light chains of amoebal and plasmodial myosins were found to be identical using similar criteria, supporting our hypothesis that the Ca2+-binding light chain plays a key role in the inhibition of actin-activated ATPase activity in Physarum myosins by micromolar levels of Ca2+.     

Vanadate-mediated photocleavage of rabbit skeletal myosin

The heavy chain of myosin from rabbit skeletal muscle can be cleaved at three sites by irradiation with near-ultraviolet light in the presence of 0.1-1.0 mM vanadate. The sigmoidal dependence upon vanadate concentration, with half-maximal rate occurring at about 0.5 mM vanadate and a sigmoidicity of 2.7, is consistent with the chromophore responsible for cleavage being oligomeric vanadate. Cleavage occurs at two sites located within the head region of the molecule, 23 kDa and 75 kDa from the NH2-terminus; these sites are cleaved equally well in heavy meromyosin and subfragment 1. In the presence of 1 mM vanadate, the half-times for cleavage of the 23-kDa and 75-kDa sites are about 15 and 10 min, respectively. The rate of cleavage at both these sites is retarded 2-3-fold by the presence of greater than 10 microM MgATP. The third photocleavage site is located about 5-10 kDa from the COOH terminus of the intact heavy chain, and cleaves equally well in the isolated rod and in light meromyosin. Cleavage at this site occurs with a half-time of 138 min, and its rate is unaffected by the presence of MgATP. The vanadate-mediated cleavage of the heavy chains is accompanied by characteristic changes in the myosin ATPase properties, with the Ca2+, Mg2+ and actin-activated Mg2+ ATPases becoming elevated, whereas the K+/EDTA ATPase becomes inactivated. The sites of photocleavage in the myosin heavy chain might be associated with sites of phosphate binding.     

Localization and topography of antigenic domains within the heavy chain of smooth muscle myosin

We have produced and characterized monoclonal antibodies that label antigenic determinants distributed among three distinct, nonoverlapping peptide domains of the 200-kD heavy chain of avian smooth muscle myosin. Mice were immunized with a partially phosphorylated chymotryptic digest of adult turkey gizzard myosin. Hybridoma antibody specificities were determined by solid-phase indirect radioimmunoassay and immunoreplica techniques. Electron microscopy of rotary-shadowed samples was used to directly visualize the topography of individual [antibody.antigen] complexes. Antibody TGM-1 bound to a 50-kD peptide of subfragment-1 (S-1) previously found to be associated with actin binding and was localized by immunoelectron microscopy to the distal aspect of the myosin head. However, there was no antibody-dependent inhibition of the actin-activated heavy meromyosin ATPase, nor was antibody TGM-1 binding to actin-S-1 complexes inhibited. Antibody TGM-2 detected an epitope of the subfragment-2 (S-2) domain of heavy meromyosin but not the S-2 domain of intact myosin or rod, consistent with recognition of a site exposed by chymotryptic cleavage of the S-2:light meromyosin junction. Localization of TGM-2 to the carboxy-terminus of S-2 was substantiated by immunoelectron microscopy. Antibody TGM-3 recognized an epitope found in the light meromyosin portion of myosin. All three antibodies were specific for avian smooth muscle myosin. Of particular interest is that antibody TGM-1, unlike TGM-3, bound poorly to homogenates of 19-d embryonic smooth muscles. This indicates the expression of different myosin heavy chain epitopes during smooth muscle development.     

Contraction kinetics and myosin isoform composition in smooth muscle from hypertrophied rat urinary bladder

Mechanical properties and isoform composition of myosin heavy and light chains were studied in hypertrophying rat urinary bladders. Growth of the bladder was induced by partial ligation of the urethra. Preparations were obtained after 10 days. In maximally activated skinned preparations from the hypertrophying tissue, the maximal shortening velocity and the rate of force development following photolytic release of ATP were reduced by about 20 and 25%, respectively. Stiffness was unchanged. The relative content of the basic isoform of the essential 17 kDa myosin light chain was doubled in the hypertrophied tissue. The expression of myosin heavy chain with a 7 amino acid insert at the 25K/50K region was determined using a peptide-derived antibody against the insert sequence. The relative amount of heavy chain with insert was decreased to 50%, in the hypertrophic tissue. The kinetics of the cross-bridge turn-over in the newly formed myosin in the hypertrophic smooth muscle is reduced, which might be related to altered expression of myosin heavy or light chain isoforms. © 1996 Wiley-Liss, Inc.     

The Two Myosin Isoenzymes of Chicken Anterior Latissimus dorsi Muscle Contain Different Myosin Heavy Chains Encoded by Separate mRNAs

Abstract. The two myosin isozymes (SM1 and SM2) of the anterior latissimus dorsi muscle of the chicken change in relative concentration during development. As SM1 decreases from 13 days of embryonic growth through 1 year of adult maturation, SM2 increases. In the adult muscle SM2 accounts for over 95% of the total myosin. The myosin heavy chains of the two isozymes are distinctly different and may be separated from each other by 5% SDS polyacrylamide gel electrophoresis. The faster migrating myosin heavy chain is identified as originating from SM1 and the slower migrating myosin heavy chain from SM2 myosin isozymes. The myosin heavy chains change in relative concentration during development exactly parallel with changes in SM1 and SM2 isozyme levels. Peptide map analysis also reveals that SM1 myosin heavy chains and SM2 myosin heavy chains are distinctly different. When RNA from the ALD muscle is added to reticulocyte lysate protein synthesizing systems the translation products are shown to include both SM1 and SM2 myosin heavy chains. These comigrate exactly on 5% SDS polyacrylamide gels with authentic counterparts from ALD muscle. Finally, when peptide maps of SM1 and SM2 myosin heavy chains synthesized in the reticulocyte lysate are compared they are again found to be distinctly different and each is identical to a peptide map of respective authentic SM1 and SM2 myosin heavy chains. It is concluded that the myosin heavy chains of SM1 and SM2 myosin isozymes of ALD muscle have different primary structures and that they are encoded by two distinctly different mRNAs.