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 cardiac muscle actin, the skeletal muscle actin and the cytoplasmic beta-actin are located on three different mouse chromosomes.

The actins are a group of highly conserved proteins encoded by a multigene family. We have previously reported that the skeletal muscle actin gene is located on mouse chromosome 3, together with several other unidentified actin DNA sequences. We show here that the gene coding for the cardiac muscle actin, which is closely related to the skeletal muscle actin (1.1% amino acid replacements), is located on mouse chromosome 17. The gene coding for the cytoplasmic beta-actin is located on mouse chromosome 5. Thus, these three actin genes are located on three different chromosomes.     

Effect of skeletal muscle myosin light chain 2 on the Ca2+-sensitive interaction of myosin and heavy meromyosin with regulated actin

A low-speed centrifugation assay has been used to examine the binding of myosin filaments to F-action and to regulated actin in the presence of MgATP. While the cross-linking of F-actin by myosin was Ca2+ insensitive, much less regulated actin was cross-linked by myosin in the absence of Ca2+ than in its presence. Removal of the 19000-dalton, phosphorylatable light chain from myosin resulted in the loss of this Ca2+ sensitivity. Readdition of this light chain partially restored the Ca2+-sensitive cross-linking of regulated actin by myosin. Urea gel electrophoresis has been used to distinguish that fraction of heavy meromyosin which contains intact phosphorylatable light chain from that which contains a 17000-dalton fragment of this light chain. In the absence of Ca2+, heavy meromyosin which contained digested light chain bound to regulated actin in MgATP about 10-fold more tightly than did heavy meromyosin which contained intact light chain. The regulated actin-activated ATPases of heavy meromyosin also showed that cleavage of this light chain causes a substantial increase in the affinity of heavy meromyosin for regulated actin in the absence of Ca2+. Thus, the binding of both myosin and heavy meromyosin to regulated actin is Ca2+ sensitive, and this sensitivity is dependent on the phosphorylatable light chain.     

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.     

Characterization of cDNA coding for the complete light meromyosin portion of a rabbit fast skeletal muscle myosin heavy chain

Myosin-heavy-chain-specific cDNA clones have been isolated from a cDNA library prepared from hind leg muscle of a 14-day-old rabbit. According to restriction enzyme analysis these can be grouped into at least two, probably three different classes. RNA dot-blot hybridization shows that all of these clones correspond to mRNAs expressed in fast skeletal muscle. The clones of the most abundant form, class I, can be aligned to cover the complete light meromyosin portion of myosin heavy chain. The sequence of the coding and the 3'-untranslated region, together comprising 2143 base pairs, has been determined. The class I clone detects a multigene family of 8-12 members on a Southern blot of rabbit genomic DNA.     

The genes for human gastrin and cholecystokinin are located on different chromosomes

Summary The polypeptide hormones gastrin and cholecystokinin are structurally related, having the identical pentapeptide GWMDF located at their C-terminus. The precursors to these two hormones also show amino acid homology, suggesting that they may have a common ancestral origin. Recombinant DNA clones corresponding to gene fragments encoding human gastrin and cholecystokinin were used to determine their respective chromosomal localization by analyzing human-rodent cell lines. We have assigned the cholecystokinin gene to human chromosome 3q12-3pter and the gastrin gene to chromosome 17q.     

The human desmin and vimentin genes are located on different chromosomes

We have used somatic cell hybrids of Chinese hamster X man and mouse X man to localize the genes (des and vim) encoding the intermediate filaments desmin and vimentin in the human genome. Southern blots of DNA prepared from each cell line were screened with hamster cDNA probes specific for des and vim genes, respectively. The single-copy human des gene is located on chromosome 2, and the single-copy human vim gene is assigned to chromosome 10. Partial restriction maps of the two human genomic loci are presented. A possible correlation of the des locus with several reported hereditary myopathies is discussed.     

Effect of glucocorticoids on the turnover rate of actin and myosin heavy and light chains on different types of skeletal muscle fibres

The catabolic action of glucocorticoids on the molecular level of the two main muscular proteins, myosin and actin, was found to depend on the type of muscle fibres. The synthesis rate of actin and myosin heavy chain was decreased in all types of muscle fibres, and in myosin light chain only in the slow-twitch red fibres. The turnover rate of actin and myosin heavy chain was also found decreased in all types of muscle fibres. The myosin light chains turned over more rapidly in dexamethasone-treated than in the control rats in all types of muscle fibres except in the case of the slow-twitch red ones as was shown by single and double isotope methods. Dexamethasone treatment enhanced the urinary 3-methylhistidine excretion in rats by 60%.     

Analysis of myosin light and heavy chain types in single human skeletal muscle fibers

In this study, myosin types in human skeletal muscle fibers were investigated with electrophoretic techniques. Single fibers were dissected out of lyophilized surgical biopsies and typed by staining for myofibrillar ATPase after preincubation in acid or alkaline buffers. After 14C-labelling of the fiber proteins in vitro by reductive methylation, the myosin light chain pattern was analysed on two-dimensional gels and the myosin heavy chains were investigated by one-dimensional peptide mapping. Surprisingly, human type I fibers, which contained only the slow heavy chain, were found to contain variable amounts of fast myosin light chains in addition to the two slow light chains LC1s and LC2s. The majority of the type I fibers in normal human muscle showed the pattern LC1s, LC2s and LC1f. Further evidence for the existence in human muscle of a hybrid myosin composed of a slow heavy chain with fast and slow light chains comes from the analysis of purified human myosin in the native state by pyrophosphate gel electrophoresis. With this method, a single band corresponding to slow myosin was obtained; this slow myosin had the light chain composition LC1s, LC2s and LC1f. Type IIA and IIB fibers, on the other hand, revealed identical light chain patterns consisting of only the fast light chains LC1f, LC2f and LC3f but were found to have different myosin havy chains. On the basis of the results presented, we suggest that the histochemical ATPase normally used for fibre typing is determined by the myosin heavy chain type (and not by the light chains). Thus, in normal human muscle a number of 'hybrid' myosins were found to occur, namely two extreme forms of fast myosins which have the same light chains but different heavy chains (IIA and IIB) and a continuum of slow forms consisting of the same heavy chain and slow light chains with a variable fast light chain composition. This is consistent with the different physiological roles these fibers are thought to have in muscle contraction.     

Homologous calcium-binding proteins in the activation of skeletal, cardiac, and smooth muscle myosin light chain kinases

In order to identify the physiological regulator of calcium dependent myosin light chain kinases of cardiac, skeletal, and smooth muscles, the effects of the three homologous calciproteins, calmodulin, troponin C, and parvalbumin, on the kinases isolated from bovine myocardium, rabbit skeletal muscle, and turkey gizzard were examined. Only calmodulin was effective in stimulating the cardiac, skeletal, or smooth muscle kinase; troponin C and parvalbumin exhibited no activation of any of the three kinases, even when examined at concentrations as high as 10-(5) M. It is concluded that calmodulin is the specific regulator of myosin light chain kinase in cardiac, skeletal, and smooth muscle.     

Myosin light chain kinase and the role of myosin light chain phosphorylation in skeletal muscle

Skeletal muscle myosin light chain kinase (skMLCK) is a dedicated Ca²⁺/calmodulin-dependent serine–threonine protein kinase that phosphorylates the regulatory light chain (RLC) of sarcomeric myosin. It is expressed from the MYLK2 gene specifically in skeletal muscle fibers with most abundance in fast contracting muscles. Biochemically, activation occurs with Ca²⁺ binding to calmodulin forming a (Ca²⁺)₄•calmodulin complex sufficient for activation with a diffusion limited, stoichiometric binding and displacement of a regulatory segment from skMLCK catalytic core. The N-terminal sequence of RLC then extends through the exposed catalytic cleft for Ser15 phosphorylation. Removal of Ca²⁺ results in the slow dissociation of calmodulin and inactivation of skMLCK. Combined biochemical properties provide unique features for the physiological responsiveness of RLC phosphorylation, including (1) rapid activation of MLCK by Ca²⁺/calmodulin, (2) limiting kinase activity so phosphorylation is slower than contraction, (3) slow MLCK inactivation after relaxation and (4) much greater kinase activity relative to myosin light chain phosphatase (MLCP). SkMLCK phosphorylation of myosin RLC modulates mechanical aspects of vertebrate skeletal muscle function. In permeabilized skeletal muscle fibers, phosphorylation-mediated alterations in myosin structure increase the rate of force-generation by myosin cross bridges to increase Ca²⁺-sensitivity of the contractile apparatus. Stimulation-induced increases in RLC phosphorylation in intact muscle produces isometric and concentric force potentiation to enhance dynamic aspects of muscle work and power in unfatigued or fatigued muscle. Moreover, RLC phosphorylation-mediated enhancements may interact with neural strategies for human skeletal muscle activation to ameliorate either central or peripheral aspects of fatigue.     

Acidic residues comprise part of the myosin light chain-binding site on skeletal muscle myosin light chain kinase

Myosin light chain kinase is a Ca2+/calmodulin-dependent protein kinase which exhibits a very high degree of protein substrate specificity. The regulatory light chain of myosin is the only known physiological substrate of the enzyme. Based upon epitope mapping of monoclonal antibodies which inhibit kinase activity competitively with respect to the light chain substrate, residues 235-319 of the rabbit skeletal muscle kinase have been proposed to contain a light chain-binding site (Herring, B. P., Stull, J. T., and Gallagher, P. J. (1990) J. Biol. Chem. 265, 1724-1730). With the expression of a truncated kinase, we have further localized this putative binding site to residues 235-294. Mutation of acidic residues at positions 269 and 270 of the kinase resulted in a 10-fold increase in the Km value for the myosin light chain, with no significant change in the Vmax value. In contrast, altering a cluster of acidic amino acids at positions 261-263 had little effect on the Km value for the myosin light chain. These results suggest that residues 269 and 270 may be involved in protein-substrate binding. Interestingly, these residues, located amino-terminal of the homologous catalytic core (positions 302-539), are in a region which is highly conserved among myosin light chain kinases, but not other protein kinases. It is probable that the homologous catalytic core contains structural elements required for phosphotransferase activity. The catalytic domain of myosin light chain kinase would therefore include these conserved elements together with additional specific substrate-binding residues.     

Phosphorylation of myosin light chain modulates the in vitro movement of fibrils composed of actin and myosin filaments from skeletal muscle

In vitro movement of fibrils composed of actin and myosin filaments purified from skeletal muscle was observed by dark field microscopy during superprecipitation at low ionic strengths at room temperature. The movement was activated by phosphorylation of light chain (LC2) of myosin. The activity of the movement was evaluated in terms of the spreading of the area where the fibrils were moving. Adenosine triphosphatase activity of actomyosin was also enhanced by phosphorylation of LC2 and was correlated with the activity of the in vitro movement.     

Myosin light chain phosphorylation and contractile performance of human skeletal muscle

Twitch tension and maximal unloaded velocity of human knee extensor muscles were studied under conditions of low phosphate content of the phosphorylatable light chains (P-light chains) of myosin and elevated phosphate content, following a 10-s maximal voluntary isometric contraction (MVC). After the MVC, twitch tension was significantly potentiated, with greater potentiation observed at a shorter muscle length (p less than 0.05). The MVC was associated with at least a twofold increase in phosphate content of the fast (LC2F) and two slow (LC2S and LC2S') P-light chains, but this increase was unrelated to muscle length. No significant differences in knee extension velocity were observed between conditions where P-light chains had low or elevated phosphate content. Positive but nonsignificant correlations were noted between the extent of twitch potentiation and phosphate content of individual P-light chains as well as the percentage of type II muscle fibres in vastus lateralis muscle. No significant relationships were determined for myosin light chain kinase activity and either P-light chain phosphorylation or type II fibre percentage. These data suggest that, unlike other mammalian fast muscles, P-light chain phosphorylation of mixed human muscles is not strongly associated with altered contractile performance.     

A comparison of the alkali light chain subunits of vertebrate skeletal muscle myosin in free and heavy chain associated states

The conformations of the alkali light-chain subunits A1 and A2 of vertebrate fast-twitch muscle myosin have been compared for these chains both in their free state and their heavy-chain-associated states by examining the fluorescence parameters of the extrinsic probe 2-(4′-maleimidylanilino)naphthalene-6-sulfonic acid attached covalently to the two light chains. The effect of temperature, salt concentration, and ligands such as Mg²⁺ ions, MgADP, and MgATP has also been investigated. In spite of the extensive sequence homology between the two light chains the data indicate that in their free states the fluorophore in the A2 chain resides in a considerably higher hydrophobic environment. It was also found that the presence of the bulky fluorophore on these light chains does not adversely affect their ability to hybridize with Subfragment 1 heavy chains to form ATPase active hybrids. This association to the heavy chains is accompanied by significant changes in the quantum yields of the 2-(4′-maleimidylanilino)naphthalene-6-sulfonic acid label indicating that conformational changes do occur during this transition. Mg²⁺ ions were found to cause either an enhancement or a decrease in fluorescence intensity depending on whether the alkali light chains were free or combined to the heavy chains, respectively. Fluorescence perturbation by nucleotide was only observed for the heavy-chain-associated state.     

Disulfide formation within the regulatory light chain of skeletal muscle myosin

Thiol-disulfide exchange reactions between myosin and 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) lead to the formation of 5-thio-2-nitrobenzoic acid (TNB)-mixed disulfides as well as to protein disulfide bonds. After incubation with DTNB, myosin was treated with an excess of N-ethylmaleimide (NEM) before electrophoretic analysis of the protein subunits in sodium dodecyl sulfate (SDS) without prior reduction by dithiothreitol (DTT). Without NEM treatment, thiol-disulfide rearrangement reactions occurred in the presence of SDS between the residual free thiols and DTNB. In the absence of divalent metal ions at 25 degrees C, DTNB was shown to induce an intrachain disulfide bond between Cys-127 and Cys-156 of the RLC. This intrachain cross-link restricts partially the unfolding of the RLC in SDS and can be followed as a faster migrating species, RLC'. Densitometric evaluation of the electrophoretic gel patterns indicated that the stoichiometric relation of the light chains (including RLC and RLC') remained unchanged. The two cysteine residues of the fast migrating RLC' were no more available for reaction with [14C]NEM, but upon reduction with DTT, the electrophoretic mobility of the RLC' reverted to that of unmodified RLC and of the RLC modified with two TNB groups. Ca2+ or Mg2+ was able to prevent this disulfide formation in the RLC of myosin by 50% at a free ion concentration of 1.1 X 10(-8) and 4.0 X 10(-7) M, respectively, at 25 degrees C and pH 7.6. Intrachain disulfide formation of RLC never occurred in myosin at 0 degree C.(ABSTRACT TRUNCATED AT 250 WORDS)