scholarly journals Further studies on single fibres of bovine μscles

1982 ◽  
Vol 203 (1) ◽  
pp. 179-184 ◽  
Author(s):  
O A Young
Keyword(s):  
Heavy Chain ◽  
Myosin Light Chain ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Fast Muscle ◽  
Muscle Fibres ◽  
Myofibrillar Proteins ◽  
Single Fibres ◽  
Physiological Demands ◽  
Muscle Myosin

Young & Davey (1981) (Biochem. J. 195, 317-327) identified numbers of polymorphs of myofibrillar proteins by sodium dodecyl sulphate/polyacrylamide gel electrophoresis of single muscle fibres isolated from three bovine muscles. Fibres were classed according to the distribution of polymorphs. The study has now been extended to eight diverse bovine muscles. The previous distinction made between fast and slow fibres is valid without exception in the extended study. Within these classes, variations in myofibrillar expression are examined within and between fibres, muscles and animals. Two slow muscles are contrasted; masseter is homogeneous in fibre type, whereas diaphragm is subtly heterogeneous, possibly arising from greater physiological demands. Of the myofibrillar polymorphs, attention is concentrated on two variants of fast-muscle myosin heavy chain. Both are present in all fast and mixed muscles examined, except sternomandibularis, and each is respectively associated with certain unidentified proteins. Within a muscle the fast-muscle myosin light-chain expression is the same irrespective of the heavy-chain variant. Histochemical techniques demonstrated that the variants are respectively associated with types IIA and IIB as defined by other investigators.

scholarly journals Myosin heavy-chain composition in striated muscle after tenotomy

1992 ◽  
Vol 282 (1) ◽  
pp. 237-242 ◽  
Author(s):  
A Jakubiec-Puka ◽  
C Catani ◽  
U Carraro
Keyword(s):  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Striated Muscle ◽  
Slow Muscle ◽  
Fast Muscle ◽  
Muscle Fibres ◽  
Adult Rats ◽  
Tendon Regeneration ◽  
Adult Muscle ◽  
Gastrocnemius Muscles

The myosin heavy-chain (MHC) isoform pattern was studied by biochemical methods in the slow-twitch (soleus) and fast-twitch (gastrocnemius) muscles of adult rats during atrophy after tenotomy and recovery after tendon regeneration. The tenotomized slow muscle atrophied more than the tenotomized fast muscle. During the 12 days after tenotomy the total MHC content decreased by about 85% in the slow muscle, and only by about 35% in the fast muscle. In the slow muscle the ratio of MHC-1 to MHC-2A(2S) remained almost unchanged, showing that similar diminution of both isoforms occurs. In the fast muscle the MHC-2A/MHC-2B ratio decreased, showing the loss of MHC-2A mainly. After tendon regeneration, the slow muscle recovered earlier than the fast muscle. Full recovery of the muscles was not observed until up to 4 months later. The embryonic MHC, which seems to be expressed in denervated adult muscle fibres, was not detected by immunoblotting in the tenotomized muscles during either atrophy or recovery after tendon regeneration. The influence of tenotomy and denervation on expression of the MHC isoforms is compared. The results show that: (a) MHC-1 and MHC-2A(2S) are very sensitive to tenotomy, whereas MHC-2B is much less sensitive; (b) expression of the embryonic MHC in adult muscle seems to be inhibited by the intact neuromuscular junction.

Expression of the catalytic domain of myosin light chain kinase increases paracellular permeability

1996 ◽  
Vol 271 (5) ◽  
pp. C1678-C1684 ◽  
Author(s):  
G. Hecht ◽  
L. Pestic ◽  
G. Nikcevic ◽  
A. Koutsouris ◽  
J. Tripuraneni ◽  
...  
Keyword(s):  
Tight Junction ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Catalytic Domain ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Epithelial Tight Junction ◽  
Tight Junction Permeability ◽  
Mlc20 Phosphorylation

Contractile events resulting from phosphorylation of the 20-kDa myosin light chain (MLC20) have been implicated in the regulation of epithelial tight junction permeability. To address this question, Madin-Darby canine kidney cells were transfected with a murine leukemia retroviral vector containing DNA encoding either the catalytic domain of myosin light chain kinase (tMK) or the beta-galactosidase gene (beta-gal). Autoradiograms of sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of myosin immunoprecipitated from 32Pi-labeled transfected cells demonstrated that MLC20 phosphorylation was increased 3.1 +/- 0.9-fold in cells expressing tMK compared with cells expressing beta-gal. Phosphopeptide mapping confirmed that myosin light chain kinase was responsible for the increased MLC20 phosphorylation. Transepithelial electrical resistance, a measurement of barrier function, of tMK cell monolayers was consistently < 10% (123 +/- 20 omega.cm2) of that of monolayers comprised of wild-type cells (1,456 +/- 178 omega.cm2) or cells expressing beta-gal (1,452 +/- 174 omega.cm2). Dual 22Na+ and [3H]mannitol flux studies indicated that the decrease in resistance in tMK cells was attributable to increased paracellular flow. These data support the idea that MLC20 phosphorylation by myosin light chain kinase is involved in regulating epithelial tight junction permeability.

scholarly journals Purified mu EBP-E binds to immunoglobulin enhancers and promoters.

1988 ◽  
Vol 8 (11) ◽  
pp. 4972-4980 ◽  
Author(s):  
C L Peterson ◽  
S Eaton ◽  
K Calame
Keyword(s):  
Heavy Chain ◽  
Binding Sites ◽  
Dissociation Constants ◽  
Sequence Similarity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Binding Studies ◽  
Apparent Homogeneity ◽  
Enhancer Binding Protein ◽  
Chemical Nuclease

We describe the purification to apparent homogeneity of the murine immunoglobulin heavy-chain (IgH) enhancer-binding protein mu EBP-E from murine plasmacytoma cells by ion exchange and affinity chromatography. Glycerol gradient sedimentation, UV cross-linking, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis confirm that mu EBP-E is a 45-kilodalton molecular mass protein. Orthophenanthroline-copper chemical nuclease footprinting with purified protein has identified high-affinity binding sites for mu EBP-E within the IgH enhancer at the previously identified site E and at sites within IgH promoters and in the kappa light-chain enhancer. Equilibrium binding studies indicate that the dissociation constants for mu EBP-E binding to site E within the enhancer and to a binding site within the V1 heavy-chain promoter are quite low, about 2 x 10(-11) M. Comparison of four mu EBP-E recognition sequences detects only limited sequence similarity among binding sites.

scholarly journals Degradation of myofibrillar proteins by trypsin-like serine proteinases.

1982 ◽  
Vol 201 (2) ◽  
pp. 279-285 ◽  
Author(s):  
J Kay ◽  
L M Siemankowski ◽  
R F Siemankowski ◽  
J A Greweling ◽  
D E Goll
Keyword(s):  
Skeletal Muscle ◽  
Gel Electrophoresis ◽  
Cysteine Proteinase ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Serine Proteinase ◽  
Myofibrillar Proteins ◽  
Serine Proteinases ◽  
Bovine Trypsin

The effects of the Ca2+-activated cysteine proteinase, the rat trypsin-like serine proteinase and bovine trypsin on myofibrillar proteins from rabbit skeletal muscle are compared. 2. Myofibrils that had been treated at neutral pH with the Ca2+-dependent proteinase and with the rat enzyme were (a) analyzed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and (b) examined in the electron microscope. Treatment with each proteinase resulted in the loss of the Z-discs, but the rat enzyme caused much more extensive disruption of the ultrastructure and degraded more of the myofibrillar proteins. 3. Purified F-actin was almost totally resistant to the proteinases, whereas G-actin was degraded by the rat trypsin-like proteinase at a rate approx. 15 times faster than was obtained with bovine trypsin. 4. Similar results were obtained with alpha-actinin, whereas tropomyosin was degraded more readily by bovine trypsin than by the rat trypsin-like proteinase. 5. The implications of these findings for the non-lysosomal breakdown of myofibrillar proteins in vivo are considered.

scholarly journals The Heavy-chain Stoichiometry of Smooth Muscle Myosin is a Characteristic of Smooth Muscle Tissues

1988 ◽  
Vol 41 (4) ◽  
pp. 409 ◽  
Author(s):  
Mukhallad A Mohammad ◽  
Malcolm P Sparrow
Keyword(s):  
Smooth Muscle ◽  
Heavy Chain ◽  
Relative Proportion ◽  
Sodium Dodecyl ◽  
Smooth Muscle Myosin ◽  
Human Bronchus ◽  
Heavy Chains ◽  
Tissue Extracts ◽  
Muscle Tissues ◽  
Muscle Myosin

The stoichiometry of the two heavy chains of myosin in smooth muscle was determined by electrophoresing extracts of native myosin and of dissociated myosin on sodium dodecyl sulfate (SDS) 4%-polyacrylamide gels. The slower migrating heavy chain was 3�6 times more abundant in toad stomach, 2�3 in rabbit myometrium, 2�0 in rat femoral artery, 1�3 in guinea pig ileum, 0�93 in pig trachea and 0�69 in human bronchus, than the more rapidly migrating chain. Both heavy chains were identified as smooth muscle myosin by immunoblotting using antibodies to smooth muscle and nonmuscle myosin. The unequal proportion of heavy chains suggested the possibility of native isoforms of myosin comprised of heavy-chain homodimers. To test this, native myosin extracts were electrophoresed on non-dissociating (pyrophosphate) gels. When each band was individually analysed on SDS-polyacrylamide gel the slowest was found to be filamin and the other bands were myosin in which the relative proportion of the heavy chains was unchanged from that found in the original tissue extracts. Since this is incompatible with either a heterodimeric or a homodimeric arrangement it suggests that pyrophosphate gel electrophoresis is incapable of separating putative isoforms of native myosin.

scholarly journals Characterization of a factor IX variant with a glycine207 to glutamic acid mutation

Blood ◽  
1994 ◽  
Vol 84 (6) ◽  
pp. 1866-1873 ◽  
Author(s):  
SW Lin ◽  
CN Lin ◽  
N Hamaguchi ◽  
KJ Smith ◽  
MC Shen
Keyword(s):  
Glutamic Acid ◽  
Heavy Chain ◽  
Active Site ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Intrinsic Fluorescence ◽  
Factor Ix ◽  
Active Site Pocket ◽  
Factor Ixa ◽  
Factor Xia

Factor IXTaipei9 is a factor IX variant from a hemophilia B patient with reduced levels of circulating protein molecules (cross-reacting material reduced, CRM). This variant contained a glycine (Gly) to glutamic acid (Glu) substitution at the 207th codon of mature factor IX. The functional consequences of the Gly-->Glu mutation in factor IXTaipei9 (IXG207E) were characterized in this study. Plasma-derived IXG207E exhibited a mobility similar to that of normal factor IX on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Its specific activity was estimated to be 3.5% that of the purified normal factor IX in a one-stage partial thromboplastin time assay (aPTT). Cleavage of factor IXG207E by factor XIa or factor VIIa-tissue factor complex appeared to be normal. When the calcium-dependent conformational change was examined by monitoring quenching of intrinsic fluorescence, both normal factor IX and IXG207E exhibited equivalent intrinsic fluorescence quenching. Activated factor IXG207E (IXaG207E) also binds antithrombin III equally as well as normal factor IXa. However, aberrant binding of the active site probe p-aminobenzamidine was observed for factor XIa-activated factor IXG207E, indicating that the active site pocket of the heavy chain of factor IXaG207E was abnormal. Moreover, the rate of activation of factor X by factor IXaG207E, as measured in a purified system using chromogenic substrates, was estimated to be 1/40 of that of normal factor IXa. A computer-modeled heavy-chain structure of factor IXa predicts a hydrophobic environment surrounding Gly-207 and this Gly forms a hydrogen bound to the active site serine-365. The molecular mechanism of the Gly-->Glu mutation in factor IXTaipei9 might result in the alteration of the microenvironment of the active site pocket which renders the active site serine-365 inaccessible to its substrate.

scholarly journals LMO7-dependent apical constriction requires the binding of the Myosin heavy chain

2021 ◽  
Author(s):  
Miho Matsuda ◽  
Chih-Wen Chu ◽  
Sergei S Sokol
Keyword(s):  
Heavy Chain ◽  
Myosin Light Chain ◽  
Myosin Ii ◽  
Tube Formation ◽  
Epithelial Morphogenesis ◽  
Apical Constriction ◽  
Actomyosin Contractility ◽  
Apical Domain ◽  
Underlying Mechanisms ◽  
Muscle Myosin

The reduction of the apical domain, or apical constriction, is a process that occurs in a single cell or is coordinated in a group of cells in the epithelium. Coordinated apical constriction is particularly important when the epithelium is undergoing dynamic morphogenetic events such as furrow or tube formation. However, the underlying mechanisms remain incompletely understood. Here we show that Lim only protein 7 (Lmo7) is a novel activator of apical constriction in the Xenopus superficial ectoderm, which coordinates actomyosin contractility in a group of cells during epithelial morphogenesis. Like other apical constriction regulators, Lmo7 requires the activation of the Rho-Rock-Myosin II pathway to induce apical constriction. However, instead of increasing the phosphorylation of myosin light chain (MLC), Lmo7 binds muscle myosin II heavy chain A (NMIIA) and increases its association with actomyosin bundles at adherens junctions (AJs). Lmo7 overexpression modulates the subcellular distribution of Wtip, a tension marker at AJs, suggesting that Lmo7 generates mechanical forces at AJs. We propose that Lmo7 increases actomyosin contractility at AJs by promoting the formation of actomyosin bundles.

Contractile properties of the developing diaphragm correlate with myosin heavy chain phenotype

1994 ◽  
Vol 77 (1) ◽  
pp. 481-487 ◽  
Author(s):  
B. D. Johnson ◽  
L. E. Wilson ◽  
W. Z. Zhan ◽  
J. F. Watchko ◽  
M. J. Daood ◽  
...  
Keyword(s):  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Diaphragm Muscle ◽  
Shortening Velocity ◽  
Cross Bridge ◽  
Isoform Expression ◽  
The Relationship ◽  
Laser Densitometry

The objective of this study was to determine the relationship between developmental transitions in myosin heavy chain (MHC) composition and changes in maximum unloaded shortening velocity (Vo) and maximum specific force (Po) of the rat diaphragm muscle. The diaphragm was excised at postnatal days 0, 3, 7, 14, 21, and 28 and in adults. MHC isoform expression was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and laser densitometry. In muscle fiber bundles, Vo was determined at 15 degrees C by use of the “slack” test. Isometric Po was determined at 15 and 26 degrees C. Simple and stepwise regressions were used to evaluate the correlations between Vo, Po, and MHC phenotype transitions and the various developmental ages. The progressive increases in Vo and Po with age were found to be inversely correlated to MHC-neonatal isoform expression (r2 = -0.84 and -0.63, respectively) and positively correlated to MHC-2X (r2 = 0.78 and 0.57) and MHC-2B (r2 = 0.51 and 0.40) isoform expression (P < 0.001). Changes in MHC-neonatal isoform expression contributed to most of the developmental variance in Vo and Po, with changes in MHC-2X and MHC-2B expression also contributing significant increments to total variance. The postnatal increase in Vo most likely relates to differences in the actomyosin adenosinetriphosphatase activity between neonatal and adult fast MHC phenotypes. The increase in Po may reflect inherent differences in myofibrillar density, cross-bridge cycling kinetics, and/or the force produced per cross bridge among fibers composed of the different MHC isoforms.

scholarly journals Characterization of a factor IX variant with a glycine207 to glutamic acid mutation

Blood ◽  
1994 ◽  
Vol 84 (6) ◽  
pp. 1866-1873 ◽  
Author(s):  
SW Lin ◽  
CN Lin ◽  
N Hamaguchi ◽  
KJ Smith ◽  
MC Shen
Keyword(s):  
Glutamic Acid ◽  
Heavy Chain ◽  
Active Site ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Intrinsic Fluorescence ◽  
Factor Ix ◽  
Active Site Pocket ◽  
Factor Ixa ◽  
Factor Xia

Abstract Factor IXTaipei9 is a factor IX variant from a hemophilia B patient with reduced levels of circulating protein molecules (cross-reacting material reduced, CRM). This variant contained a glycine (Gly) to glutamic acid (Glu) substitution at the 207th codon of mature factor IX. The functional consequences of the Gly-->Glu mutation in factor IXTaipei9 (IXG207E) were characterized in this study. Plasma-derived IXG207E exhibited a mobility similar to that of normal factor IX on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Its specific activity was estimated to be 3.5% that of the purified normal factor IX in a one-stage partial thromboplastin time assay (aPTT). Cleavage of factor IXG207E by factor XIa or factor VIIa-tissue factor complex appeared to be normal. When the calcium-dependent conformational change was examined by monitoring quenching of intrinsic fluorescence, both normal factor IX and IXG207E exhibited equivalent intrinsic fluorescence quenching. Activated factor IXG207E (IXaG207E) also binds antithrombin III equally as well as normal factor IXa. However, aberrant binding of the active site probe p-aminobenzamidine was observed for factor XIa-activated factor IXG207E, indicating that the active site pocket of the heavy chain of factor IXaG207E was abnormal. Moreover, the rate of activation of factor X by factor IXaG207E, as measured in a purified system using chromogenic substrates, was estimated to be 1/40 of that of normal factor IXa. A computer-modeled heavy-chain structure of factor IXa predicts a hydrophobic environment surrounding Gly-207 and this Gly forms a hydrogen bound to the active site serine-365. The molecular mechanism of the Gly-->Glu mutation in factor IXTaipei9 might result in the alteration of the microenvironment of the active site pocket which renders the active site serine-365 inaccessible to its substrate.

Age-Related Alterations in Myosin Heavy Chain Isoforms in Rat Intrinsic Laryngeal Muscles

2002 ◽  
Vol 111 (11) ◽  
pp. 962-967 ◽  
Author(s):  
Tatsutoshi Suzuki ◽  
Diane M. Bless ◽  
Nadine P. Connor ◽  
Charles N. Ford ◽  
Kyungah Lee ◽  
...  
Keyword(s):  
Myosin Heavy Chain ◽  
Muscle Fiber ◽  
Heavy Chain ◽  
Fiber Type ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Myosin Heavy Chain Isoforms ◽  
Laryngeal Muscles ◽  
Age Related ◽  
Posterior Cricoarytenoid

Deficits in voice and swallowing are found in the elderly, but the underlying neuromuscular mechanisms are unclear. A potential mechanism may be denervation-induced muscle fiber transformation to a slower-contracting type of muscle fiber. This study examined young, old, and denervated rat laryngeal muscles (lateral thyroarytenoid, lateral cricoarytenoid, and posterior cricoarytenoid) to examine differences in myosin heavy chain (MHC) composition. Results of sodium dodecyl sulfate–polyacrylamide gel electrophoresis analyses indicated that all muscles were composed predominately of type IIB MHC. With aging and denervation, type IIB was reduced and type IIX, a slower-contracting isoform, was increased in the lateral thyroarytenoid and lateral cricoarytenoid muscles. In the posterior cricoarytenoid muscle, the MHC composition was relatively unchanged. These findings suggest that aging may affect laryngeal adductory function by altering muscle fiber type composition to a slower-contracting isoform, in a manner similar to that observed with denervation.

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