Phosphorylated and dephosphorylated myosin light chains of Drosophila fly and larva

1. The present study confirmed that light chains of Drosophila adult fibrillar (flight) muscle myosin consist of Lf1, Lf2, Lf2' and Lf3, and tubular muscle myosin light chains contain Lt1, Lt2, Lt2' and Lt3, as revealed by two-dimensional (isoelectric focusing and SDS-gel electrophoresis) gel electrophoresis. 2. Larva myosin light chains were of all the tubular type. However, it was found that Lt1 and Lt2' are produced by phosphorylation of Lt2, and Lf1 is produced by phosphorylation of Lf2'. 3. Injection of radioactive phosphate into Drosophila fly resulted in phosphorylations of Lf1 and Lt1. When larva or late pupa myosin was incubated with myosin light chain kinase from chicken gizzard or adult flies, phosphorylation of Lt1, Lf2' and Lt2' occurred. Drosophila myosin light chain kinase phosphorylated Lf1 in addition to Lt1 and L2' (Lf2' + Lt2') of adult myosin. 4. Dephosphorylation of adult myosin by potato acid and calf intestine alkaline phosphatases led to the shift of Lf1 (34,000), Lt1 (31,000) and L2' (Lf2' + Lt2') (30,000) to L2 (Lf2 + Lt2) positions (30,000). 5. Peptide mapping analyses revealed that larva Lt1, Lt2', Lt2 and adult Lt1 were all the same; therefore, it is thought that a single species of Lt2 specific to the tubular type of myosin and its phosphorylated isoforms (Lt1, Lt2') exist. 6. The peptide map of Lf1 was slightly different from that of Lt1, but very similar to that of L2' in adult myosin. L2 and L2' of adult myosin showed very similar peptide maps, but there were several different peptide fragments.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Selective purification of the 20,000-Da light chains of smooth muscle myosin

The 20,000-Da light chains of gizzard smooth muscle myosin have been purified to homogeneity. Actomyosin, prepared by MgATP extraction of myofibrils, was denatured in 8 M urea, 1 M guanidine HCl, and 0.05% sodium dodecyl sulfate. Myosin heavy chains were precipitated with ethanol and the light chain enriched fraction was dialyzed and subjected to chromatography on DEAE-Sephacel. Fractions containing the 20,000-Da light chains were further purified by hydrophobic chromatography on phenyl-Sepharose. The 20,000-Da light chains eluted at low ionic strength from the phenyl-Sepharose column were judged to be greater than 95% pure by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and contained only 0.04 mol of phosphate/mol of light chain. The yield of light chains was calculated to be 219 +/- 17 mg/kg of starting gizzard smooth muscle. This method may be useful for preparation of homogeneous 20,000-Da smooth muscle myosin light chains in the quantities necessary for study of contractile systems.     

Coexistence of cardiac-type and fast skeletal-type myosin light chains in embryonic chicken cardiac muscle

Myosin from embryonic chicken ventricle contained a light chain component which comigrated with fast skeletal myosin light chain 1 (Lf1) on two dimensional electrophoresis in addition to cardiac type light chains (Lc1 and Lc2). Immunoblot analysis showed that this minor light chain band reacted with anti-Lf1 antibody. Antigens binding with anti-Lc1 and anti-Lf1 antibodies were located on myofibrils in embryonic cardiac muscle cells in vivo and in vitro. From these observations, we conclude that a small amount of Lf1 exists in embryonic chicken cardiac muscle.     

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.     

Myosin light chains of guinea-pig striated muscles. Similarities and differences with rat myosin light chains

1. Myosin light chains of guinea-pig striated muscles have been screened by two-dimensional gel electrophoresis and compared to rat myosin light chains. 2. The fast type light chains 1F and 3F, slow type light chains 1S and 2S, and embryonic type light chain 1E are shown to differ in the two rodents; only the fast type light chains 2F co-electrophorese on the gel. 3. In guinea-pig, as in rat, ventricle muscle light chains appear the same as the 1S and 2S light chains and atrial light chain type 1 the same as the 1E light chain. We show that this embryonic light chain of guinea-pig myosin is difficult to identify and may be confused with the adult 1F light chain.     

Changes in myosin isozymes during development of chicken breast muscle

The patterns of myosin isozymes in embryonic and adult chicken pectoralis muscle were examined by electrophoresis in a non-denaturing gel system (pyrophosphate acrylamide gel electrophoresis), and both light chains and heavy chains of embryonic and adult myosin isozymes were compared. In pyrophosphate acrylamide gel electrophoresis, the predominant isozyme component in embryonic pectoralis myosin could be clearly distinguished from adult myosin isozymes. SDS-polyacrylamide gel electrophoresis indicated that the light chain composition of embryonic myosin was also different from that of adult myosin. The pattern of peptide fragments produced by myosin digestion with a-chymotrypsin differed significantly between embryonic and adult skeletal myosin. These results suggest that myosin in the embryonic pectoralis muscle is different in both light and heavy chain composition from myosin in the same adult tissue.     

Purification and some properties of rabbit stomach myosin.

Myosin from rabbit stomach was highly purified by ammonium sulfate fractionation in the presence of ATP and MgCl2, ultracentrifugation and Sepharose 4B chromatography. The myosin composed of one heavy and two light chains as determined by SDS-gel electrophoresis. The molecular weights of the light chains were the same as those of gizzard myosin, about 20,000 and 17,000, respectively. The pH-activity curve and the KCl concentration dependency of Ca-ATPase of the stomach myosin were similar to those of other smooth muscle myosins. The stomach myosin was more resistant to pepsin digestion than skeletal myosin. Other proteolytic enzymes, trypsin, chymotrypsin, papain, and nagarse, digested the myosin in the same way as skeletal myosin.     

Characterization of cardiac myosin from rabbit embryos and adult rabbits.

1. Ca2+-ATPase of myosin and electrophoretic pattern of light chains of myosin were investigated in cardiac muscles of 22-day-old rabbit embryos, new-born and adult rabbits. 2. Ca2+-ATPase activity was found to decrease during development and in contrast to that of adult rabbit, cardiac myosin prepared from 22-day-old embryos, is stable on exposure to pH 9.5. 3. Myosin from the cardiac muscle of rabbit embryos reveals light chains of both fast and slow types, that from adult animals, however, reveals light chains of the slow type only. 4. These studies suggest that unlike the cardiac muscle of adult rabbit, cardiac muscle of rabbit embryos contains both fast and slow types of myosin.     

An electrophoretic study of native myosin isozymes and of their subunit content.

Myosin polymorphism in muscles has been studied by a variety of electrophoretic techniques, in non-dissociating and in dissociating conditions. The analysis of myosin isozymes in the native state was achieved in pyrophosphate buffer and required only minute amounts of protein; identical results were obtained with purified or crudely extracted myosin. The determination of the subunit content of each isozyme was done in the presence of sodium dodecyl sulphate or urea for light chain, and in a phenol, acetic acid and urea system for heavy chain screening. Electrophoresis in non-dissociating conditions has led to the separation of up to a dozen of myosin isozymes, differing in mobilities by as much as 30%. Muscle specificity of myosin was clearly established. Apart from a few exceptions, all the muscles tested were shown to contain more than one myosin species; fast-twitch muscles for instance all contained the same three isozymes, but in variable ratios. Class specificity of myosin appeared related to the relative proportions of isozymes in a given muscle. A second electrophoresis in dissociating solvents of the myosin bands first resolved in pyrophosphate buffer has then allowed a further characterization of the various isozymes. The differences in mobilities observed in the native state were shown to come either from the light chains, or from the heavy chains, or from both. The first case was illustrated by the three species present in fast muscles, which were shown to correspond to three alkali light-chain isozymes, the heterodimer representing in some instances up to 40% of the total. Next to light-chain muscle type specificity, electrophoresis in the phenol, acetic acid, urea system has led to the detection of differences in the heavy chains of fast, slow and cardiac myosins. The application of these various electrophoretic techniques to the analysis of the modification of myosin isozymes during development or in pathology studies can be considered.     

The relation between ATPASE activity and light chains of myosin in developing, adult and denervated muscles of several animals species.

Ca2+ATPase activity and light chains of myosin, fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, in developing, adult and denervated fast, slow and cardiac muscles of the rat, guinea-pig, cat, rabbit and chick were studied. It has been shown that in normal adult muscles the electrophoretic pattern of light chains of myosin reflects the myosin ATPase activity only when muscles from the same animal species are compared. In homologous muscles from adult animals differing in size, the size-dependent difference in myosin ATPase activity is not revealed in the electrophoretic pattern. Both in developing and in denervated muscle, changes in myosin ATPase activity are either connected with changes in the pattern of light chains of myosin or this pattern does not change. This relation is different in fast and slow muscles and also differs in chick and rabbit muscles. There are several possibilities of explaining the relation between ATPase activity of myosin and the pattern of light chains of myosin. The observation that myosin from the soleus muscle of 1-month-old rabbit contains light chains corresponding to both fast and slow type of myosin, indicates that the change in myosin ATPase activity during development is due to changes in the ratio between the fast and slow type of myosin.     

Characterization of the myosin-phosphorylating system in normal murine astrocytes and derivative sv40 wild-type and A-mutant transformant

Myosin and myosin light-chain kinase have been isolated and characterized from small quantities of normal and SV40-transformed, murine astrocytic neuroglial cells in culture and from intact normal mouse brain. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of the astrocyte myosins revealed a heavy chain of 200,000 daltons and two light chains of 20,000 and 15,000 daltons. These myosins are similar to other cytyplasmic myosins. The astrocyte 20,000-dalton light chain can be phosphorylated by an endogenous myosin light-chain kinase which has properties similar to those of the myosin light-chain kinase found in human platelets. No differences were detected in either the astrocyte myosins or myosin light-chain kinases between (a) the normal and transformed cells, (b) the transformed cells grown at the permissive and nonpermissive temperatures, or (c) the SV40 wild-type and A-mutant transformants.     

Evidence for myosin heterogeneity inDrosophila melanogaster

Summary Electrophoresis of myosin extracts from larvae and adult tissues ofDrosophila melanogaster under non-dissociating conditions indicate that two of the bands seen are myosins. They stain for Ca²⁺ ATPase activity and when cut and re-run under dissociating conditions are found to contain a myosin heavy chain that co-migrates with rabbit skeletal muscle myosin heavy chain. One of the forms of myosin seen is found primarily in extracts from the leg. The other is common to the adult fibrillar flight muscles and the larval body wall muscles.The electrophoretic evidence for two myosin types is strengthened by the histochemical demonstration of two myofibrillar ATPases on the basis of their lability to acid or alkali preincubation. The myofibrillar ATPase in the leg and the Tergal Depressor of the Trochanter (TDT) are shown to be relatively acid labile and alkali stable. The larval body wall muscles and the adult fibrillar flight muscles have an ATPase which is acid stable and alkali labile. This distribution of the two myofibrillar ATPase coincides with that predicted by electrophoresis of extracts from whole tissue and also locates the two myosins to specific muscle types.     

Synthesis of myosin heavy and light chains in muscle cultures

The weight ratio of myosin/actin, the myosin heavy chain content as the percentage of total protein (wt/wt), and the kinds of myosin light chains were determined in (a) standard muscle cultures, (b) pure myotube cultures, and (c) fibroblast cultures. Cells for these cultures were obtained from the breast of 11-day chick embryos. Standard cultures contain, in addition to myotubes, large numbers of replicating mononucleated cells. By killing these replicating cells with cytosine arabinoside, pure myotube cultures were obtained. The myosin/actin ratio (wt/wt) for pure myotube, standard muscle, and fibroblast cultures average 3.1, 1.9, and 1.1 respectively. By day 7, myosin in myotube cultures represents a minimum of 7% of the total protein, but about 3% in standard cultures and less than 1.5% in fibroblasts cultures. Myosin from standard cultures contains light chain LC1, LC2, and LC3, with a relative stoichiometry of the molarity of 1.0:1.9:0.5 and mol wt of 25,000, 18,000 and 16,000 daltons, identical to those in adult fast muscle. Myosin from pure myotubes exhibits light chains LC1 and LC2, with a molar ratio of 1.5:1.6. Myosin from fibroblast cultures possesses two light chains with a stoichiometry of 1.8:1.8 and mol wt of 20,000 and 16,000 daltons. Clearly, the faster migrating light chain, LC3, found in standard cultures is synthesized not by the myotubes but ty the mononucleated cells. In myotubes, both the assembly of the sarcomeres and the interaction between thick and thin filaments required for spontaneous contraction occur in the absence of light chain LC3. One set of structural genes for the myosin light and heavy chains appears to be active in mononucleated cells, whereas another set appears to be active in multinucleated myotubes.     

Myosin isoforms of the developing skeletal muscles in the loach

Changes in the myosin isozyme spectrum were studied in the loach developing skeletal muscle. It was shown using disk-electrophoresis in polyacrylamide gel and peptide mapping that light and heavy myosin chains from the larval muscles, as well as from the red and white muscle of adult fish differ from each other. Forms of myosin light and heavy chains were found which were characteristic of the larval muscle only.     

Volume occupation, environment, and accessibility in proteins. Environment and molecular area of RNase-S

The myosin light chains of cultured muscle cells and embryonic muscle tissue have been examined by two-dimensional gel electrophoresis. Myosin purified from primary cultures of rat muscle cells or the myogenic cell line L6 contain not only the light chains corresponding to those of fast twitch muscle but also another protein, differing slightly in molecular weight and isoelectric point from the adult LC1 protein. By a number of criteria this additional protein is shown to be a myosin light chain: (1) it is found in highly purified myosin preparations; (2) in L6 myosin it replaces the other LC1-type light chains in stoichiometric amounts; (3) it is part of the subfragment-1 complex of myosin produced by chymotrypsin. as expected for an LC1-type light chain. Total extracts of fused cultured muscle cells, when analyzed by two-dimensional electrophoresis, contain substantial amounts of this additional LC1-type protein, strongly suggesting that it is not a proteolytic fragment produced during myosin isolation. Unfused cultures do not synthesize detectable amounts of the adult light chains or the additional LC1-type light chain. This additional LC1 protein can be detected in embryonic or newborn muscle tissue but it is not present in adult myosin or myofibrils. These results indicate that a novel form of myosin light chain, referred to as an embryonic LC1 or LC1_emb, is characteristic of the early stages of muscle development.     

Myosin transitions in developing fast and slow muscles of the rat hindlimb

Abstract. Myosin isozymes from the slow soleus and fast EDL muscles of the rat hindlimb were analyzed by pyrophosphate gel electrophoresis, by peptide mapping of heavy chains, and by antibody staining. At the earliest stage examined, 20 days gestation, distinctions between the developing fast and slow muscles were seen by all these criteria; all fibers in the distal hindlimb reacted strongly with antibody to adult fast myosin. Some fibers also reacted with antibody to adult slow myosin; these fibers had a precise, axial distribution in the hindlimb. This pattern of staining which includes the entire soleus, foreshadows the adult distribution of slow fibers and may indicate that the specific pattern of innervation of the limb is already determined. In the early developing soleus there are four fetal and neonatal isozymes plus two isozymes present in equal proportions in the 'slow' area of the pyrophosphate gel. The mobility of these two slow isozymes decreases with maturity and the slowest moving isozyme gradually becomes the dominant species. Thus early diversity between the soleus and EDL is expressed by myosins which are distinct from the mature isozymes. The relative proportion of slow isozymes significantly increases with development and as this occurs the fetal and neonatal isozymes are progressively eliminated. Transiently at least one mature fast isozyme appears in the soleus. This is present at 15 days postpartum and probably correlates with the population of fast, type II fibers, which comprise 50% of this muscle cell population at 15 days. The EDL contained three fetal and neonatal isozymes and only one slow isozyme which does not change in mobility with age. Slow isozymes in the soleus and EDL are thus not identical. Each muscle underwent a unique series of changes until the adult pattern of isozymes and heavy chains was reached about one month postpartum.     

Ontogenic differentiation of pig atrial and ventricular myosin

Myosin was isolated from pig atrial and ventricular myocardium during postnatal development and Ca2+-ATPase was determined and myosin light chains were analysed by electrophoresis in sodium dodecylsulfate polyacrylamide gel. During ontogenesis ATPase activity of ventricular myosin remains virtually unchanged, whereas that of atrial myosin increases. The patterns of myosin light chains of atrial and ventricular myosin differ from each other, but the individual pattern remains unchanged during the development.     

Myosin isoenzymes and their subunits in urodelan amphibian fast skeletal muscle. Coexistence of larval and adult heavy chains in neotenic individuals

The distributions of native myosin isoforms were examined by electrophoresis under non-dissociating conditions, in the fast twitch dorsal skeletal muscle of young larvae, neotenic adults and metamorphosed adults of urodelan amphibians. Both heavy and light chains of myosin isoenzymes were analysed. In pyrophosphate acrylamide gel electrophoresis three isoenzymes were demonstrated in larval myosin; other isoforms of lower electrophoretic mobility were observed in metamorphosed adults myosin. Larval and adult isoenzymes were shown to coexist in myosin from neotenic adults. Analysis of heavy chains in denaturing conditions and proteolytic digestion revealed the sequential occurrence during development of two types of heavy chains, one larval and one adult, that coexist in the myosin of neotenic adults only. Analysis of light chain patterns under denaturing conditions revealed the existence of three fast light chains which displayed no modification during the course of development. The neotenic urodelan amphibian species model represents actually the only model in which the coexistence of larval (or neonatal) and adult heavy chains is maintained throughout life in adults.     

Calcium regulation of muscle contraction.

Calcium triggers contraction by reaction with regulatory proteins that in the absence of calcium prevent interaction of actin and myosin. Two different regulatory systems are found in different muscles. In actin-linked regulation troponin and tropomyosin regulate actin by blocking sites on actin required for complex formation with myosin; in myosin-linked regulation sites on myosin are blocked in the absence of calcium. The major features of actin control are as follows: there is a requirement for tropomyosin and for a troponin complex having three different subunits with different functions; the actin displays a cooperative behavior; and a movement of tropomyosin occurs controlled by the calcium binding on troponin. Myosin regulation is controlled by a regulatory subunit that can be dissociated in scallop myosin reversibly by removing divalent cations with EDTA. Myosin control can function with pure actin in the absence of tropomyosin. Calcium binding and regulation of molluscan myosins depend on the presence of regulatory light chains. It is proposed that the light chains function by sterically blocking myosin sites in the absence of calcium, and that the "off" state of myosin requires cooperation between the two myosin heads. Both myosin control and actin control are widely distributed in different organisms. Many invertebrates have muscles with both types of regulation. Actin control is absent in the muscles of molluscs and in several minor phyla that lack troponin. Myosin control is not found in striated vertebrate muscles and in the fast muscles of crustacean decapods, although regulatory light chains are present. While in vivo myosin control may not be excluded from vertebrate striated muscles, myosin control may be absent as a result of mutations of the myosin heavy chain.