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At least 19 recordsLinked to original sources

PubMed1989

Immunochemical study of connectin (titin) in neuromuscular diseases using a monoclonal antibody: connectin is degraded extensively in Duchenne muscular dystrophy.

Connectin (also called titin) is a myofibrillar elastic filament which links a thick filament to a neighbouring Z line in a sarcomere and thus contributes significantly to the elastic property of myofibrils. In the present study, the degradation state of connectin in biopsied skeletal muscles from various neuromuscular diseases was investigated by Western blot analysis using a monoclonal antibody which reacts extensively with the degradation products of connectin. In Duchenne muscular dystrophy (DMD), connectin was degraded progressively and relentlessly after 5 years of age. In Becker muscular dystrophy, degradation of connectin was much less than in DMD. Connectin was well preserved in normal controls, and was only minimally degraded in Charcot-Marie-Tooth disease, amyotrophic lateral sclerosis, limb girdle muscular dystrophy and myotonic dystrophy, even when the biopsied muscles showed a similar degree of weakness as those of DMD. The degradation of connectin, even though secondary, is presumed to play an important role in the pathogenesis of myofibrillar degeneration in DMD.

Adolescent↗

PubMed1992

Calcium-induced splitting of connectin filaments into beta-connectin and a 1,200-kDa subfragment.

When rabbit skeletal muscle myofibrils were treated with a solution containing 0.1 mM Ca2+ and 30 micrograms of leupeptin/ml, alpha-connectin, which forms very thin filaments in myofibrils, was split into beta-connectin and a 1,200-kDa subfragment. A part of beta-connectin located near the junction between beta-connectin and the subfragment seems to have an affinity for calcium ions and to be susceptible to the binding of large amounts of calcium ions. The calcium-binding site on beta-connectin is localized near the N2 line in the I band, and the subfragment is localized adjacent to the Z disk. It is possible that connectin filaments change their elasticity during the contraction-relaxation cycle of skeletal muscle at the physiological concentration of calcium ions. Because postmortem skeletal muscles lose their elasticity and become plastic in association with the calcium-specific splitting of connectin filaments, the splitting is considered to be a factor in meat tenderization during postrigor ageing.

Animals↗

PubMed1992

Characterization and localization of alpha-connectin (titin 1): an elastic protein isolated from rabbit skeletal muscle.

A simplified procedure to isolate alpha-connectin (titin 1, TI), a gigantic elastic protein, from rabbit skeletal muscle is described. A rapid column chromatography step to concentrate alpha-connectin is introduced. Separation of alpha-connectin from beta-connectin is introduced. Separation of alpha-connectin from beta-connectin (titin 2, TII) in the presence of 4 M urea at pH 7.0 did not cause any change in the secondary structure of alpha-connectin as judged by circular dichroic spectra. Ultraviolet absorption spectra and the amino acid composition of alpha-connectin (MW, approximately 3 x 10(6)) were similar to those of its proteolytic product, beta-connectin (MW, approximately 2 x 10(6)). Circular dichroic spectra suggested that both alpha- and beta-connectin consist of 60% beta-sheet and 30% beta-turn. It thus appears that the whole elastic filament of connectin has a folded beta-strand structure. Proteolysis of alpha-connectin by calpain resulted in formation of beta-connectin and smaller peptides. The alpha-connectin interacted with both myosin and actin filaments similarly to beta-connectin. Polyclonal antibodies raised against 1200 kDa peptides obtained from aged rabbit skeletal myofibrils reacted with alpha-connectin (titin 1, TI) but only weakly with beta-connectin (titin 2, TII) in rabbit skeletal muscle. Immunoelectron microscopy and indirect immunofluorescence microscopy revealed that the antibodies bound at the Z-line and at the epitope regions in the I-band near the binding site of a monoclonal antibody SM1 whose position depends on sarcomere length. It thus appears that beta-connectin extends from the edge of M-line to the above epitope region in the I-band.

Actins↗

PubMed1986

Native connectin from porcine cardiac muscle.

Native connectin was isolated from porcine cardiac muscle using the method developed for the preparation of native connectin from chicken breast muscle (Kimura et al. (1984) J. Biochem. 96, 1947-1950). It was not necessary to keep cardiac muscle at 0 degrees C before preparation: the proteolysis of alpha-connectin to beta-connectin proceeded during the preparation of myofibrils. Cardiac connectin showed almost the same properties as those of skeletal muscle connectin: mobility in SDS gel electrophoresis, filamentous structure under an electron microscope, circular dichroism spectra, UV absorption spectra, and amino acid composition. Porcine cardiac connectin cross-reacted with antiserum against chicken breast muscle connectin as revealed by an immunoblot method. Immunoelectron microscopical observations revealed an abundance of connectin antigenic sites around the A-I junction area of cardiac myofibrils. Cardiac connectin also interacted with myosin and actin filaments at low ionic strengths to form aggregates. The extent of interaction was somewhat weaker in the case of cardiac connectin than skeletal muscle connectin, regardless of the origin of myosin and actin (porcine cardiac and rabbit skeletal muscles). In conclusion, cardiac connectin is very similar, but not identical to skeletal muscle connectin.

Amino Acids↗

PubMed1990

Assembly of connectin (titin) in relation to myosin and alpha-actinin in cultured cardiac myocytes.

By using polyclonal and monoclonal antibodies against connectin (titin) which stain the A-I junctional area and the A-band domain (polyclonal anti-connectin and monoclonal 4C9) and the I-band domain (monoclonal SM1), the developmental relationship of this elastic protein with sarcomeric proteins, especially and alpha-actin, was examined in embryonic chick cardiac myocytes in vitro under fluorescence microscopy. During premyofibril stages, I-Z-I proteins were detected first (alpha-actinin dots and diffuse actin [phalloidin and anti-troponin C] staining), and later in these areas connectin and myosin dots appeared with nearly identical distribution. Somewhat later, phalloidin-positive nonstriated fibrils were observed in a straight course. They were always reactive with antibodies against alpha-actinin and troponin C, but unreactive or only weakly reactive with anticonnectin and anti-myosin. Initially, alpha-actinin dots were aligned along these fibrils but did not form striations. As they aggregated to form Z-bands, connectin and myosin started to exhibit typical striation ('doublets' and A-bands, respectively). No difference in the staining pattern was observed with two kinds of monoclonal antibodies against different domains of connectin filaments (4C9 and SM1) at early phases. As myosin staining began to show clear A-bands, connectin epitopes became arranged in polarized positions. We conclude that primitive I-Z-I complexes appear prior to the assembly of connectin and myosin filaments and then connectin filaments, developing intimately and coordinately with myosin, become associated with the alpha-actinin lines. Thus it appears that the putative elastic protein connectin plays some role in integrating myosin filaments with the preexisting I-Z-I brushes. The occasional absence of connectin and A-bands between two Z-bands, beyond both of which clear sarcomeres have been formed, indicates that connectin is not a preformed scaffold of myofibrils on which sarcomeric proteins accumulate.

Actinin↗

PubMed1992

Chicken leg muscle alpha-connectin as studied by a monoclonal antibody to the 1200 kDa fragment.

Chicken leg gracilis muscle contained only alpha-connectin (ca 3000 kDa) without beta-connectin. When myofibrils were kept standing for 20 hr at 4 degrees C, alpha-connectin was degraded to beta-connectin (ca 2000 kDa) and 1200 kDa peptide. The latter was prepared from myofibrils and purified by gel filtration in the presence of SDS. A monoclonal antibody, alpha 7, to this 1200 kDa fragment was prepared. The antibody reacted with the 1200 kDa fragment and its mother molecule alpha-connectin, but not with beta-connectin. Immunoelectron microscopy using alpha 7, as well as other antibodies to chicken breast muscle beta-connectin, revealed that the 1200 kDa peptide covered the portion of alpha-connectin from the Z line to the N2 line region in the I band of chicken leg gracilis muscle sarcomeres. The results were in good agreement with those observed in rabbit skeletal muscle.

Animals↗

PubMed1987

Binding of actin filaments to connectin.

The binding of actin filaments to connectin, a muscle elastic protein, was investigated by means of turbidity and sedimentation measurements and electron microscopy. In the presence of less than 0.12 M KCl at pH 7.0, actin filaments bound to connectin. Long actin filaments formed bundles. Short actin filaments also aggregated into irregular bundles or a meshwork, and were frequently attached perpendicularly to long bundles. The binding of F-actin to connectin was saturated at an equal weight ratio (molar ratio, 50 : 1), as determined by a cosedimentation assay. Larger amounts of sonicated short actin filaments appeared to bind to connectin than intact F-actin. Myosin S1-decorated actin filaments did not bind to connectin. The addition of S1 to connectin-induced actin bundles resulted in partial disaggregation. Thus, connectin does not appear to interfere with actin-myosin interactions, since myosin S1 binds to actin more strongly than connectin.

Actins↗

PubMed1990

Projectin is an invertebrate connectin (titin): isolation from crayfish claw muscle and localization in crayfish claw muscle and insect flight muscle.

A filamentous protein was isolated from crayfish claw muscle. This protein had physiochemical properties very similar to vertebrate skeletal muscle connectin (titin), although its apparent molecular mass (approximately 1200 kDa) was considerably lower than that of connectin (approximately 3000 kDa). Polyclonal as well as monoclonal antibodies against chicken skeletal muscle connectin reacted with the 1200 kDa protein from crayfish claw muscle. Conversely, polyclonal antibodies against crayfish 1200 kDa protein cross-reacted with chicken connectin. Circular dichroic spectra indicated the abundance of beta-sheet structure (approximately 60%). Low-angle shadowed images showed filamentous structures (0.2-0.5 microns) by electron microscopy. Proteolysis of the 1200 kDa protein by alpha-chymotrypsin or V8 protease rapidly resulted in formation of 1000 kDa or 1100 and 800 kDa peptides. The amino acid composition was very similar to those of vertebrate connectins and of honeybee flight muscle projectin. Based on the molecular weight and amino acid composition, the 1200 kDa protein is regarded to be crayfish projectin. Immunofluorescence and immunoelectron microscopy revealed that crayfish projectin was localized in the A/I junction area and A-band except for its centre region in crayfish claw muscles. Polyclonal antibodies against crayfish claw muscle projectin reacted with 1200 kDa projectin of honeybee and beetle flight muscle. A monoclonal antibody against chicken skeletal muscle connectin also reacted with honeybee and beetle projectin. Immunoelectron microscopic observations revealed that anti-crayfish projectin antibodies bound the connecting filaments linking the Z-line and the thick filaments up to the M-line of honeybee muscle sarcomere. Anti-crayfish projectin antibodies bound the I-band region near the Z-line of beetle flight muscle. It is concluded that the 1200 kDa projectin from crayfish claw muscle is an invertebrate connectin (titin). Recent work with locust flight muscle mini-titin (Nave & Weber, 1990) is in good agreement with the present study, except that the isolated mini-titin estimated as 600 kDa appears to be a proteolytic product (approximately 1100 kDa) of the parent molecule (approximately 1200 kDa).

Actin Cytoskeleton↗

PubMed1992

Localization and elasticity of connectin (titin) filaments in skinned frog muscle fibres subjected to partial depolymerization of thick filaments.

The localization and elasticity of connectin (titin) filaments in skinned fibres of frog skeletal muscle were examined for changes in the localization of connectin and in resting tension during partial depolymerization of thick filaments with a relaxing solution containing increased KCl concentrations. Immunoelectron microscopic studies revealed that deposites of antibodies against connectin at a sarcomere length of 3.0 microns remained at about 0.8 microns from the M-line, until the thick filament was depolymerized to the length of approximately 0.4 microns. On further depolymerization, the bound antibodies were found to move towards the Z-line and, on complete depolymerization, were observed to be within 0.3 microns of the Z-line; a marked decrease in resting tension accompanied this further depolymerization. These results suggest that connectin filament starts from the Z-line, extends to the M-line, and contributes to resting tension. After partial depolymerization of thick filaments, the distances between the anti-connectin deposits and the Z-line and between anti-connectin deposits and the M-line increased with sarcomere length, suggesting that connectin filaments are elastic along their entire length.

Actin Cytoskeleton↗

PubMed1990

Degradation of connectin (titin) in Fukuyama type congenital muscular dystrophy: immunochemical study with monoclonal antibodies.

Connectin (also called titin) is a myofibrillar elastic filament which links a thick filament to a neighbouring Z line in a sarcomere and thus contributes significantly to the elasticity of myofibrils. In a previous study, we demonstrated by Western blot analysis of the biopsied skeletal muscles using an anti-connectin monoclonal antibody that connectin was degraded extensively after 5 years of age in Duchenne muscular dystrophy (DMD), while it was degraded mildly in Becker muscular dystrophy and only minimally in myotonic dystrophy, limb girdle dystrophy, amyotrophic lateral sclerosis and Charcot-Marie-Tooth disease. In the present study, we investigated the degradation state of connectin in Fukuyama type congenital muscular dystrophy (FCMD) by a similar method using 2 distinct anti-connectin monoclonal antibodies. In FCMD, connectin degradation began much earlier than in DMD: Definite degradation was already observed in 5-8-month-old patients. It was presumed that connectin degradation would play an important role in the myofibrillar degeneration in the early stage of FCMD.

Antibodies, Monoclonal↗

PubMed1989

Isolation of alpha-connectin, an elastic protein, from rabbit skeletal muscle.

alpha-Connectin (also called titin 1) has been isolated from rabbit back muscle. Myofibrils were well washed with 5 mM NaHCO3 and then extracted with 0.2 M sodium phosphate, pH 7.0. The extract was dialyzed against 0.1 M potassium phosphate, pH 7.0, to sediment myosin. The supernatant, adjusted to 0.18 M potassium phosphate, pH 7.0, and 4 M urea, was subjected to DEAE Toyopearl column chromatography. beta-Connectin was eluted in the flow-through fraction and alpha-connectin was eluted at around 0.1 M NaCl, when a 0 to 0.25 M NaCl gradient was applied. The separated alpha-connectin was dialyzed against 0.2 M potassium phosphate, pH 7.0. The resultant alpha-connectin showed the same mobility as that in an SDS extract of rabbit back muscle on SDS gel electrophoresis using 1.8% polyacrylamide gels. A monoclonal antibody against chicken breast muscle beta-connectin reacted with the alpha-connectin isolated from rabbit back muscle.

Animals↗

PubMed1985

Studies on exercise and an elastic protein "connectin" in hindlimb muscle of growing rat.

We examined the effect of physical training on the properties of an elastic protein, connectin, in the hindlimb muscles of 6 growing rats running on a treadmill, in comparison with its properties in 6 growing non-running rats. The experimental results are as follows. 1) The body weight of exercised (R-) and control (C-) rats gradually increased during housing for 31 days, from an average of 46.2 g to 275 g. The average amount of running in the 6 R-rats was 38,160 +/- 12,122 (S.D.) g X m/hr. 2) The SDS-insoluble connectin in R-rat muscle was lower in quantity than that in C-rat muscle, but the SDS-soluble connectin of R-rat muscle was greater in quantity. 3) Amino acid compositions of SDS-soluble connectin in R- and C-rats, calculated from SDS-insoluble connectin and soleus ghost, were significantly different. 4) The soleus ghost preparation consisted mainly of connectin as shown by SDS polyacrylamide gel electrophoresis and by immunohistological staining. 5) The maximum passive tension of the soleus ghost, calculated from the factors of T/phi and (dT/dx)/phi, was higher in R-rats than in C-rats. The above results suggest that physical training might alter the biosynthetic pattern of connectin to suit the developing muscle, just as physical training alters that of myosin isoenzymes.

Amino Acids↗

PubMed1989

Elastic behavior of connectin filaments during thick filament movement in activated skeletal muscle.

Connectin (also called titin) is a huge, striated muscle protein that binds to thick filaments and links them to the Z-disc. Using an mAb that binds to connectin in the I-band region of the molecule, we studied the behavior of connectin in both relaxed and activated skinned rabbit psoas fibers by immunoelectron microscopy. In relaxed fibers, antibody binding is visualized as two extra striations per sarcomere arranged symmetrically about the M-line. These striations move away from both the nearest Z-disc and the thick filaments when the sarcomere is stretched, confirming the elastic behavior of connectin within the I-band of relaxed sarcomeres as previously observed by several investigators. When the fiber is activated, thick filaments in sarcomeres shorter than 2.8 microns tend to move from the center to the side of the sarcomere. This translocation of thick filaments within the sarcomere is accompanied by movement of the antibody label in the same direction. In that half-sarcomere in which the thick filaments move away from the Z-disc, the spacings between the Z-disc and the antibody and between the antibody and the thick filaments both increase. Conversely, on the side of the sarcomere in which the thick filaments move nearer to the Z-line, these spacings decrease. Regardless of whether I-band spacing is varied by stretch of a relaxed sarcomere or by active sliding of thick filaments within a sarcomere of constant length, the spacings between the Z-line and the antibody and between the antibody and the thick filaments increase with I-band length identically. These results indicate that the connectin filaments remain bound to the thick filaments in active fibers, and that the elastic properties of connectin are unaltered by calcium ions and cross-bridge activity.

Actin Cytoskeleton↗

PubMed1977

Connectin, an elastic protein of muscle. Comparative Biochemistry.

Connectin, an elastic protein, was isolated from both skeletal and cardiac muscles of various species of vertebrates, and also from smooth muscles (gizzard) of the chicken. The amino acid compositions of these preparations were very similar. Connectin was also obtained from claw and tail muscles of the crayfish, but preparations from clam adductor muscles and insect thoracic muscles were heavily contaminated with collagen and resilin, respectively. Connectin-like protein was obtained from cell membranes of erythrocytes and fluorescent anti-connectin staining suggested that it is located on the cytoplasmic surface of the membrane. An attempt to isolate an elastic protein from insoluble residues of amoebae of the slime mold and those of bacterial cell body (Salmonella) was inconclusive. The present comparative bio-chemical study has shown that connectin or connectin-like protein is widely distributed in various types of muscles and in some nonmuscle cells.

Amino Acids↗

PubMed1992

Connectin: a homophilic cell adhesion molecule expressed on a subset of muscles and the motoneurons that innervate them in Drosophila.

Each abdominal hemisegment in the Drosophila embryo contains a stereotyped array of 30 muscles, each specifically innervated by one or a few motoneurons. We screened 11,000 enhancer trap lines, isolated several expressing beta-galactosidase in small subsets of muscle fibers prior to innervation, and identified two of these as inserts in connectin and Toll, members of the leucine-rich repeat gene family. Connectin contains a signal sequence, ten leucine-rich repeats, and a putative phosphatidylinositol membrane linkage; in S2 cells, connectin can mediate homophilic cell adhesion. Connectin is expressed on the surface of eight muscles, the motoneurons that innervate them, and several glial cells along the pathways leading to them. During synapse formation, the protein localizes to synaptic sites; afterward, it largely disappears. Thus, connectin is a novel cell adhesion molecule whose expression suggests a role in target recognition.

Amino Acid Sequence↗

PubMed1989

Binding of connectin to myosin filaments.

Binding of native connectin (2,100 kDa fragment of alpha-connectin) to myosin filaments was investigated using a sedimentation technique and densitometric estimations of the separated proteins. In the presence of 60 mM KCl and 5 mM phosphate buffer, pH 7.0, as much as 1.5 mol of connectin was bound to 1 mol of myosin, suggesting that some 150 connectin filaments bound to a single myosin filament of approximately 0.5 micron in length. This value was much more than the ratio found in muscle (12:1). It appeared that C protein did not affect the binding of connectin to myosin filaments.

Animals↗

PubMed1986

Sodium dodecyl sulfate gel electrophoresis studies of connectin-like high molecular weight proteins of various types of vertebrate and invertebrate muscles.

Using an SDS gel electrophoresis method, connectin, very high molecular weight (approximately 10(6) dalton) protein, was detected in an SDS extract of whole tissues of various types of muscles of vertebrates and invertebrates. Connectin bands were clearly recognized in all the types of striated muscles (skeletal and cardiac) of the vertebrates examined: rabbit, chicken, turtle, snake, newt, frog, and fish. This was also the case with skeletal muscle of prochordate, Amphioxus. In invertebrates, the situation was much complicated. Connectin-like protein bands were detected in C. elegans (nematode), but not in earthworm (annelid). Smaller sizes of proteins (approximately 10(6)) were faintly found in molluscan adductor muscles. In arthropods, connectin-like proteins were clearly detected in some muscles (e.g., claw muscles of crab and crayfish; leg muscles of several insects) but not at all in other muscles (e.g., tail muscles of crayfish and shrimp; thoracic muscles of some insects). These peculiar observations might be related to the presence of such specific elastic proteins as projectin in honeybee flight muscle. The present study has revealed that connectin is an elastic protein of vertebrate striated muscle, skeletal and cardiac muscles.

Animals↗

PubMed1979

Post-mortem changes in skeletal muscle connectin.

Changes in connectin and elasticity of skeletal muscle were determined during post-mortem ageing. The amount of connectin decreased with increasing time of post-mortem storage whereas the rate of the decrease depended on the source of muscles. The loss in elasticity of muscle coincided well with the decrease in connectin contents. Electron microscopically, a network structure between the Z discs vanished when the amount of connectin fell to zero. We have concluded that the continuous net structure of connectin is responsible for about 30% of the total elasticity of living skeletal muscle and its degradtaion is responsible for post-mortem tenderization of meat.

Animals↗