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PubMed1999

Interaction of paratropomyosin with beta-connectin and its 400-kilodalton fragment from chicken skeletal muscle as influenced by the calcium ion concentration.

The binding of paratropomyosin to beta-connectin, which has been suggested to interact at the A-I junction of a sarcomere, was confirmed by measuring the changes in turbidity of a mixture with changing NaCl concentration, pH and free calcium ions, and by morphological observation and a coprecipitation assay of the aggregates formed in the mixture. Paratropomyosin also bound to the 400-kDa fragment which is the N-terminal portion of beta-connectin and contains the A-I junction region. Moreover, the interaction of paratropomyosin with the 400-kDa fragment was enhanced by a calcium ion concentration from 10(-7) M to 10(-5) M and markedly suppressed above 10(-4) M calcium ions. We conclude that paratropomyosin probably binds to the 400-kDa fragment of beta-connectin in the A-I junction region in living and pre-rigor skeletal muscle. In postmortem skeletal muscle paratropomyosin may be released from the 400-kDa portion of the connectin filament by increased calcium ion concentration and translocated on to thin filaments to induce meat tenderization.

Animals↗

PubMed2003

The N-terminal sequence of paratropomyosin binding fragments from beta-connectin.

In order to clarify the position where paratropomyosin binds to connectin in the A-I junction region of a sarcomere, chicken beta-connectin was digested by Staphylococcus aureus V8 protease under denaturing conditions and the digested peptides were electrophoretically separated. Five peptides, 150-kDa, 100-kDa, 70-kDa, and 43-kDa fragments, were simultaneously detected by biotinylated paratropomyosin and an anti-connectin monoclonal antibody. The N-terminal sequence of the 43-kDa fragment was found to be YQFRVYAVNK, similar to the sequence of 7556-7565 amino acids in the I51 fibronectin type 3 domain that was located at the A-I junction region of human cardiac titin/connectin. Therefore, we propose that paratropomyosin binds to the 43-kDa fragment from beta-connectin at the A-I junction region in both living muscle and in muscle immediately postmortem, and the N-terminus of the 43-kDa fragment is localized in the I51 domain.

Amino Acid Sequence↗

PubMed2006

Effects of eccentric exercise on joint stiffness and muscle connectin (titin) isoform in the rat hindlimb.

We investigated the effects of repeated eccentric exercise for rat medial gastrocnemius muscle on ankle joint stiffness and muscle connectin (titin) isoform composition (longer form, alpha-connectin; shorter form, beta-connectin). Male Wistar rats were trained on a custom-made, isokinetic dynamometer (eccentric-exercise group, n = 6; sham-operated group, n = 6). The exercise session consisted of 20 eccentric contractions elicited by submaximal electric stimulations under anesthesia. The contracting muscle was forcibly lengthened by an isokinetic dorsi-flexion of the ankle joint (velocity, 30 degrees/s; range of motion, 45 degrees). Rats in the eccentric-exercise group were trained every two days for 20 days (10 sessions in total). The static passive resistive torque (PRT) of 45 degrees at the ankle joint was used as a measure of the joint stiffness, and was determined before and after the experimental period. After 10 sessions of eccentric exercise, the wet weight of medial gastrocnemius muscle significantly increased (P < 0.05), whereas the static PRT significantly decreased (P < 0.05) in the eccentric-exercise group, when compared to the sham-operated group. Myosin-ATPase staining showed a decrease in the number of type IIb/IId fibers (P < 0.001) and an increase in the number of type IIa fibers (P < 0.05). However, no significant difference was seen in the connectin (titin) isoform composition between the eccentric-exercise group and the sham-operated group, suggesting that the reduction in PRT was not due to change in resting mechanical properties of muscle fibers.

Animals↗

PubMed1979

[Biochemical studies on collagen and connectin from human skeletal muscle: age-related changes in the properties of elasticity (author's transl)].

Collagen and connectin, an intracellular elastic protein, play important roles in structural maintenance and mechanical continuity of skeletal muscle. The author has investigated age-related changes in these proteins from human skeletal muscles ranged from 6 lunar month to 79 years of age. The total content of collagen in skeletal muscle at 6 lunar month was 1.7% of wet weight of the tissue. This value was found to decrease to 0.8% by 5 years of age, and no significant change in this value was observed after 5 years of age. Collagen in skeletal muscle tissue was found to consist of 80% of Type I collagen, 19% of Type III collagen and 1% of Type IV collagen. The proportion of these genetically distinct types of collagen was constant throughout the whole life-span. The amount of soluble collagen and formation of reducible cross-links of collagen were found to decrease with age. Although the content of connection in skeletal muscle from human embryo was low, 0.9-1.1% of wet weight of the tissue, it gradually increased to 2.9% at 79 years of age. Following NaB3H4 reduction, connectin was found to contain the reducible cross-links derived from lysine- and hydroxylysine-aldehydes; two of them were identified as lysinonorleucine and histidino-hydroxymerodesmosine, and additional three reducible compounds were eluted between elution positions of dihydroxylysinonorleucine and lysinonorleucine from ion exchange chromatography. These findings strongly indicate that connectin and connective tissue proteins, collagen and elastin, share common features of cross-linking. Furthermore, these reducible cross-links were found to decrease with age. Thus, maturations of both connectin and collagen are apparently due to qualitative changes in their reducible cross-links.

Adolescent↗

PubMed1977

Connectin, an elastic protein of muscle. Its abundance in cardiac myofibrils.

Pure myofibrils were isolated from bovine heart by sucrose layer ultracentrifugation. Cardiac myofibrils thus prepared contained more protein as insoluble stroma than skeletal muscle. The insoluble stroma largely consisted of connectin, an elastic protein of muscle. The connectin content in cardiac myofibrils was about 18% of the total myofibrillar protein and was three times that in skeletal myofibrils. In view of the role of connectin as an elastic component of muscle, the abundance of connectin in cardiac myofibrils may be responsible for keeping myofibrils short at rest. This would account for the more effective tension generation in cardiac muscle on passive stretching due to blood inflow (Stirling's law).

Amino Acids↗

PubMed1993

Dynamics of actin and assembly of connectin (titin) during myofibrillogenesis in embryonic chick cardiac muscle cells in vitro.

Immunogold electron microscopy of cardiac myocytes microinjected with biotin-labeled actin showed that gold labeling was first found around the A band level of myofibrils at their proximal parts. This observation suggests that polymerization of actin and/or the addition of newly formed actin filaments occurs preferentially in association with myosin filaments to increase the myofibrillar girth. At the distal portions of developing myofibrils, their terminal ends were initially labeled, suggesting that continued reorganization and/or de novo formation of myofibrils occurs at these locations. Soon, gold particles were seen along the termini of growing myofibrils. This appears to indicate that actin subunits are added at the membrane-associated ends of preexisting actin filaments to increase the length of myofibrils. Adhesion plaque proteins, e.g., vinculin, do not appear to play any role in assembling actin monomers at these sites on the inner surface of the sarcolemma. Immunofluorescence and immunoelectron microscopy of cardiomyocytes double-stained with antibodies against two distant domains of connectin (titin) filaments and other sarcomeric proteins showed that these domains of connectin filaments and myosin were synthesized almost simultaneously on large polyribosomes and/or associated immediately after the synthesis of these molecules. Connectin and myosin bands were formed after alpha-actinin striations (Z bands) were seen on preformed I-Z-I-like structures.(ABSTRACT TRUNCATED AT 250 WORDS)

Actinin↗

PubMed1999

Relation of nebulin and connectin (titin) to dynamics of actin in nascent myofibrils of cultured skeletal muscle cells.

Cultured embryonic chicken skeletal muscle cells microinjected with rhodamine (rh)-labeled actin were stained with antibodies against nebulin and connectin (titin). In premyofibril areas, nebulin was observed as dotted structures, many of which were arranged in a linear fashion. These structures were associated with injected rh-actin. Among these linearly arranged dots of nebulin and rh-actin, numerous small nebulin dots without rh-actin incorporation were scattered. It is probable that the dots of nebulin and/or its associated protein(s) represent a preformed scaffold upon which actin monomers accumulate; exogenously introduced actin associates initially with small nebulin dots, which in turn coalesce to form rh-actin dots and are arranged linearly. In developing myofibrils, two patterns of nebulin distribution were found: "singlets" and "doublets." Recovery of rh-actin's fluorescence after photobleaching was slowest in the nonstriated dotted portions, followed by the striated myofibrillar portions with nebulin singlets and those with doublets, in that order. Thus, the distribution patterns of nebulin seem to be related to the accessibility/exchangeability of actin into nascent myofibrils. It is possible that early nebulin filaments exhibiting singlets are not tightly associated with actin filaments and that this loose association allows myofibrils to exchange nonadult isoforms of actin and other proteins into adult types. Connectin formed a striated pattern before the formation of rh-actin/nebulin striations. It appears that connectin does not have any significant role in the accessibility of actin into nascent myofibrils.

Actins↗

PubMed1989

Behaviour of connectin (titin) and nebulin in skinned muscle fibres released after extreme stretch as revealed by immunoelectron microscopy.

Stretching of skinned fibres of frog skeletal muscle beyond the overlap of the thin and thick filaments followed by release to resting length results in disorganization of the thin filaments at the A-I junction of a sarcomere (Higuchi et al. (1988) J. Muscl. Res. Cell. Motility 9, 491-8). Immunoelectron microscopic observations showed that the binding sites of antibodies against connectin (titin) returned to the original position after extreme stretch and release but those of anti-nebulin antibodies were largely disorganized. The binding sites of anti-connectin antibodies moved within an I band with the change in sarcomere length, but those of anti-nebulin antibodies did not. Nebulin remained in the I band at extreme stretch. Thus connectin filaments appear to be responsible for maintaining mechanical continuity of a sarcomere and appear to behave independently of thin filaments. It is suggested that nebulin is localized in the I band but not in the A band and is associated with thin filaments but not with the elastic structure of myofibrils.

Animals↗

PubMed1992

Autophosphorylation of beta-connectin (titin 2) in vitro.

Phosphorylation of beta-connectin (titin 2), an elastic protein of chicken breast muscle, occurred in the presence of [gamma-32P] ATP, 0.2 mM CaCl2 and 25 mM phosphate buffer, pH 7.0. Addition of 3 mM MgCl2 did not affect the phosphorylation. However, Ca2+ ions were required for the phosphorylation and EGTA inhibited it even if MgCl2 were present. Myosin light chain kinase (gizzard MLCK), cAMP dependent protein kinase (A kinase), and protein kinase C (C kinase) did not phosphorylate beta-connectin in vitro under optimal conditions. Thus it appears that beta-connectin, possibly containing a domain homologous with MLCK, has an autophosphorylating action.

Adenosine Triphosphate↗

PubMed1991

Structural properties of connectin studied by ultraviolet resonance Raman spectroscopy and infrared dichroism.

Ultraviolet resonance Raman spectra of solubilized connectin indicated the presence of beta-sheets and hydrogen-bonded irregular structures. Some Trp and Tyr sidechains are located in hydrophobic environments and some NHs of mainchain amides and Trp indoles are not easily reached by solvent water, suggesting the presence of folded structures constructed of the beta- and irregular parts. Infrared spectra showed an abundance of beta-sheets in a connectin fiber, some of which were aligned with their mainchain axes parallel to the fiber axis. Thus, the beta-spiral structure proposed for elastin is improbable in connectin. This conclusion is also supported by their different amide III frequencies in the visible Raman spectra. A possible filamentous structure of repeated domains, consisting of beta-sheets and irregular parts, is discussed.

Animals↗

PubMed1999

Engrailed negatively regulates the expression of cell adhesion molecules connectin and neuroglian in embryonic Drosophila nervous system.

Engrailed is expressed in subsets of interneurons that do not express Connectin or appreciable Neuroglian, whereas other neurons that are Engrailed negative strongly express these adhesion molecules. Connectin and Neuroglian expression are virtually eliminated in interneurons when engrailed expression is driven ubiquitously in neurons, and greatly increased when engrailed genes are lacking in mutant embryos. The data suggest that Engrailed is normally a negative regulator of Connectin and neuroglian. These are the first two "effector" genes identified in the nervous system of Drosophila as regulatory targets for Engrailed. We argue that differential Engrailed expression is crucial in determining the pattern of expression of cell adhesion molecules and thus constitutes an important determinant of neuronal shape and perhaps connectivity.

Animals↗

PubMed2000

Inhibition of nebulin and connectin (titin) for assembly of actin filaments during myofibrillogenesis.

In order to examine the role of cytoskeletal scaffolding proteins, nebulin and connectin (titin), in actin dynamics during myofibrillogenesis, rhodamine (rh)-labeled actin was microinjected into cultured skeletal muscle cells in which the function of these proteins had been inhibited with their respective antibodies. In the nebulin function-inhibited cells, exogenously introduced actin formed irregularly distributed amorphous patches or bright foci inside the cells, but it was not incorporated into myofibrillar structures at any stage. Thus, the blockage of actin binding sites of nebulin seems to inhibit the association of actin monomers to the preexisting nebulin scaffold. In the cells inhibited with anti-connectin antibody, incorporation of rh-actin was similar to that in antibody-uninjected cells. These results support the idea that nebulin is related to the accessibility/exchangeability of actin into nascent myofibrils, but connectin does not have such a role in actin assembly. Since all antibodies recognizing different domains of nebulin filaments blocked actin incorporation along the entire length of actin filaments, inhibition of any domains of nebulin filaments seems to affect actin dynamics.

Actin Cytoskeleton↗

PubMed1995

Muscle-specific calpain, p94, responsible for limb girdle muscular dystrophy type 2A, associates with connectin through IS2, a p94-specific sequence.

p94, a muscle-specific member of calpain family, is unique in that it undergoes rapid and exhaustive autolysis with a half-life of less than 1 h resulting in its disappearance from muscle. Recently, p94 was shown to be responsible for limb girdle muscular dystrophy type 2A. To elucidate the muscular proteolytic system mediated by p94 and to solve the mystery of its unusually rapid autolysis, we searched for p94-binding proteins by the two-hybrid system. Although calpain small subunit plays a crucial role for regulation of ubiquitous calpains, it did not associate with p94. After a screening of skeletal muscle library, connectin (or titin), a gigantic filamentous protein spanning the M- to Z-lines of muscle sarcomere, was found to bind to p94 through a p94-specific region, IS2. The connectin-insoluble fraction of washed myofibrils contained full-length intact p94, suggesting that connectin regulates p94 activity.

Amino Acid Sequence↗

PubMed1987

Elastic properties and beta-sheet structure of connectin threads.

Connectin is a very long and flexible protein of striated muscle, linking myosin filaments to z discs in a sarcomere. Isolated native connectin in solution frequently forms elastic threads upon concentration of the solution, by side-by-side association of molecules. An X-ray diffraction study was performed to examine the presence of beta-sheet structure in artificially prepared threads. The elastic properties of such threads were measured at various temperatures. Negative temperature dependence of the elastic coefficient suggests that the elasticity of connectin threads is due to deformation of the three-dimensional structure and not to rubber-like behavior.

Animals↗

PubMed1988

Extensible and less-extensible domains of connectin filaments in stretched vertebrate skeletal muscle sarcomeres as detected by immunofluorescence and immunoelectron microscopy using monoclonal antibodies.

Two kinds of monoclonal antibodies (3B9 and SM1) against connectin, muscle elastic protein, reacted with both alpha- and beta-connectins. Immunofluorescence studies revealed that 3B9 stained both edges of the A band of chicken breast muscle myofibrils and remained as such upon stretching to a sarcomere length of 3.5 microns. On the other hand, SM1 stained the I band very close to the edges of the A band and the SM1-stained stripes moved considerably upon stretching to a sarcomere length of 3.5 microns. Immunoelectron microscopic observations with frog semitendinosus muscle revealed that three distinct stripes bound with 3B9 in the edges of the A band did not move on stretching up to 3.5 microns. On the other hand, the two stripes stained with SM1 in the I band clearly moved to the same extent as the stretching. However, when a sarcomere was stretched to 4.0 microns, all the stripes with 3B9 or SM1 disappeared and diffused deposits of the antibodies were observed. Thus it is concluded that connectin filaments in the I band region are more extensible than those at both edges of the A band.

Animals↗

PubMed1990

Isoelectrical focusing of connectin by agarose gel electrophoresis.

Two-dimensional gel electrophoresis using 0.5% agarose gel in the 1st dimension and 2-12% gradient polyacrylamide gel in the 2nd dimension succeeded in the isoelectrical focusing of connectin, a giant myofibrillar protein of approximately 3,000 kDa. Immunoblotting with an anti-connectin monoclonal antibody, SM1-36-2, following the two-dimensional gel electrophoresis, demonstrated that connectin was an acidic protein with an estimated pI of approximately 5.7.

Animals↗

PubMed1992

Changes in contractile properties with selective digestion of connectin (titin) in skinned fibers of frog skeletal muscle.

Changes in contractile properties of mechanically skinned fibers were examined when connectin in the fibers was selectively digested by a low concentration (0.25 microgram/ml) of trypsin. Resting tension and isometric active tension were reduced as the digestion of the connectin progressed; the rate of reduction of active tension was larger than that of resting tension. Maximum shortening speed and calcium ion sensitivity of active tension were not changed by the digestion. Electron micrographs showed that A-bands in the fibers treated with trypsin are dislocated from I-bands. These results suggest that the digestion of connectin does not directly influence the reaction of actin-myosin-regulatory proteins, and thus the resultant reduction in the active tension is mainly due to disordering of the regular structure in a sarcomere.

Animals↗

PubMed1995

Localization of three fragments of connectin in chicken breast muscle sarcomeres.

Connectin (titin) links the Z line to the myosin filament in sarcomeres of vertebrate skeletal muscle. An 800 kDa fragment of alpha-connectin runs from the Z line up to the N2 line region in the I band, and the following beta-connectin portion runs up to the edge of the M line on the myosin filament in chicken breast muscle sarcomeres. Immunoelectron microscopy showed that a 400 kDa fragment following the 800 kDa fragment reaches the edge of the myosin filament and, thereafter a 1,700 kDa fragment runs to the M line on the myosin filament in chicken breast muscle sarcomeres. When stretched, the epitopes to anti-400 kDa fragment antibodies outside the myosin filament moved toward the inside of the I band.

Animals↗