Isolation and characterization of 1,200 kDa peptide of alpha-connectin.

When rabbit skeletal muscle myofibrils were kept for 12 h at 4 degrees C, alpha-connectin was partially degraded and 1,200 kDa peptide was newly formed [Takahashi, K. & Takai, H. (1988) Abst. 80th Jpn. Soc. Zootech. Sci. p-102]. The latter was isolated together with remaining alpha-connectin. Ultracentrifugation of the mixture at low ionic strength resulted in sedimentation of alpha-connectin, leaving the 1,200 kDa peptide in the supernatant. Physicochemical properties of the isolated 1,200 kDa peptide were investigated: UV absorption spectra, circular dichroism spectra, amino acid composition, and molecular size and shape. Polyclonal antibodies against the 1,200 kDa peptide [PcAb(1200)] bound the Z line and I-band. The position of the stripe in the I band near the N1 line due to the binding of PcAb(1200) moved both away from the Z lines and from the A band as sarcomeres were elongated. Therefore, it is considered that the 1,200 kDa portion of alpha-connectin is elastic.     

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.     

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.     

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.     

Identification of high molecular weight proteins in squid muscle by Western blotting analysis and postmortem rheological changes

The high molecular weight protein connectin (also called titin) in Japanese common squid (Todarodes pacificus) mantle muscle was identified by western blotting analysis with 3B9, the mouse anti-chicken skeletal muscle connectin monoclonal antibody. Similarly to vertebrate samples, there exists connectin in invertebrate squid mantle muscle, and the amino acid sequences are assumed to resemble those present in the A band of vertebrate connectin, judging by the specificity of 3B9. Moreover, the connectin in squid muscle migrated in this study as a closely spaced doublet of alpha and beta (titins 1 and 2). Between 5 and 7 h post-mortem, the SDS PAGE patterns of the squid sample indicated a change of the doublet bands into a single beta-connectin band. Simultaneously, the rheological properties of the squid muscle changed substantially. This degradation of alpha-connectin into beta-connectin in the muscle can explain the critical change that occurs during the post-mortem tenderization of squid muscle.     

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.     

Molecular size and shape of beta-connectin, an elastic protein of striated muscle

Connectin is an elastic protein of vertebrate striated muscle, and consists of doublet components, alpha and beta (also called titins 1 and 2). In the present study, beta-connectin isolated in the native state was investigated in order to characterize its molecular size and shape. The molecular weight was approximately 2.1 X 10(6) (SDS gel electrophoresis) or 2.7 X 10(6) (sedimentation equilibrium). The sedimentation coefficient (SO20, w) was 17S in 0.1 M phosphate buffer, pH 7.0. The intrinsic viscosity measured in an Ostwald-type viscometer was 1.8 dl/g. However, the viscosity was greatly dependent on the velocity gradient, and at a very low velocity gradient of 0.0007 s-1, a solution of connectin (0.3 mg/ml) showed a viscosity value of 17,000 cp. Flow birefringence measurements suggested a length distribution ranging from 300 to 450 nm. Electron microscopic observations revealed that connectin is a long flexible filament and the peaks of frequency of length distribution were at 150, 300, 450, and 600 nm. It was tentatively assumed that the connectin molecule is 300-400 nm long and 34-38 nm wide. It is likely that beta-connectin is derived from alpha-connectin, which has an apparent molecular weight of 2.8 X 10(6).     

Highly homologous filamin polypeptides have different distributions in avian slow and fast muscle fibers

The high molecular weight actin-binding protein filamin is located at the periphery of the Z disk in the fast adult chicken pectoral muscle (Gomer, R. H., and E. Lazarides, 1981, Cell, 23: 524-532). In contrast, we have found that in the slow anterior latissimus dorsi (ALD) muscle, filamin was additionally located throughout the l band as judged by immunofluorescence with affinity-purified antibodies on myofibrils and cryosections. The Z line proteins desmin and alpha-actinin, however, had the same distribution in ALD as they do in pectoral muscle. Quantitation of filamin and actin from the two muscle types showed that there was approximately 10 times as much filamin per actin in ALD myofibrils as in pectoral myofibrils. Filamin immunoprecipitated from ALD had an electrophoretic mobility in SDS polyacrylamide gels identical to that of pectoral myofibril filamin and slightly greater than that of chicken gizzard filamin. Two-dimensional peptide maps of filamin immunoprecipitated and labeled with 125I showed that ALD myofibril filamin was virtually identical to pectoral myofibril filamin and was distinct from chicken gizzard filamin.     

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.     

Localization of connectin-like proteins in leg and flight muscles of insects

In leg muscle sarcomeres of a beetle, approximately 6 mum sarcomere length at rest, projectin ( approximately 1200 kDa) was located on the myosin filament up to 2 mum from the both ends of the filament, using immunofluorescence and immunoelectron microscopy. On the other hand, projectin linked the Z line to the myosin filament and bound on the myosin filament in beetle flight muscle, approximately 3-4 mum sarcomere length at rest. Connectin-like protein ( approximately 3000 kDa) was detected by immunoblot tests in beetle, bumblebee and waterbug leg muscles. Immunofluorescence and immunoelectron microscopic observations revealed that the connectin-like protein linked the myosin filament to the Z line in beetle leg muscle.     

Immunolocalization of muscle and nonmuscle isoforms of actin in myogenic cells and adult skeletal muscle

In vertebrate skeletal muscle, the proliferating myoblasts synthesize nonmuscle isoforms of actin, and the cells begin to express muscle-specific actin isoforms during their myogenic differentiation. To study the distributions of the actin isoforms in myogenic cells and fully differentiated skeletal muscle, we prepared a peptide antibody specific for the skeletal alpha isoform of actin and used this antibody along with an antibody specifically reactive with nonmuscle gamma actin to stain cultured myotubes and adult skeletal myofibrils by double-indirect immunofluorescence. At this level of resolution, no differences in isoform localization were seen: Both muscle and nonmuscle actins were detected in the myotubes and in the striations of mature myofibrils. Myotubes were also double-stained using immunogold electron microscopy, and the isoform distributions were determined quantitatively by counting the two sizes of gold particles that corresponded to labeling with each antibody. A quantitative analysis of immunoreactivity revealed that, although both forms were present in all actin-containing structures, nonmuscle actin was relatively more prevalent along the edges (cortical microfilaments) of the myotubes, whereas the muscle isoform predominated in the interior regions (containing forming myofibrils). Thus, we have found evidence of a heterogeneous distribution of muscle and nonmuscle actin isoforms in differentiating myogenic cells, and we have demonstrated that a nonmuscle actin isoform is a component of the muscle contractile apparatus.     

Isolation, characterization, and immunochemical properties of a giant protein from sea urchin egg cytomatrix

A giant protein of apparent molecular weight (Mr) 2000 kDa, as determined by SDS-PAGE, was isolated and partially purified, under denaturing conditions, from the detergent-resistant cytomatrix of unfertilized sea urchin egg. Immunoblot analysis and indirect immunofluorescence microscopy observations indicated that this high-molecular-weight protein cross-reacted with the immunospecific serum raised against chicken breast muscle beta-connectin. However, rotary-shadowing electron microscopy images of the protein revealed short threadlike structures which appear morphologically different from beta-connectin structure. Indirect immunofluorescence localization of the protein with anti-beta-connectin serum showed a distribution throughout the whole unfertilized egg cytomatrix. This immunofluorescence pattern seems to change upon egg fertilization, since at metaphase the fluorescence stain appears to be excluded from the mitotic apparatus region as revealed by the double immunolabeling with anti-beta-connectin serum and monoclonal anti-alpha-tubulin antibody. Moreover, when egg cortical fragments were double-labeled with anti-beta-connectin serum and rhodamin-conjugated phalloidin, it was observed that the microfilaments assembled after fertilization seem to be in close association with the protein at the cleavage furrow and other locations. The possible significance of this sea urchin egg connectin(titin)-like protein is discussed.     

FITC-labeled I-protein specifically binds to A-bands and/or Z-lines of glycerinated myofibrils of chicken breast muscle

Ion-exchange column-purified I-protein was labeled by fluorescein isothiocyanate (FITC) at an equimolar ratio. When FITC-labeled I-protein was reacted with glycerinated myofibrils of chicken breast muscle in a phosphate-buffered saline, fluorescence was observed at the A-band and/or the Z-line of the sarcomere. However, FITC-labeled I-protein did not stain freshly prepared myofibrils. When FITC-I-protein was reacted with a nitrocellulose paper sheet on which muscle proteins were blotted after SDS-polyacrylamide gel electrophoresis, some peptide bands, including connectin and nebulin, were fluorescent. These facts can explain why anti-I-protein antibodies stain the A-I junctional region of fresh myofibrils and A-bands and/or Z-lines of glycerinated myofibrils. It is very likely that I-protein is transferred from the A-I junctions of myofibrils and translocates to A-bands and Z-lines, where some components that can bind to I-protein are localized, as myofibrils are degraded during the glycerination.     

The expression of multiple forms of troponin T in chicken-fast-skeletal muscle may result from differential splicing of a single gene

Troponin T isolated from chicken fast skeletal muscle has been shown to be present in three different molecular forms, one in breast and two in leg muscle. The three forms differ in both size and charge. Troponin T from breast muscle has a molecular mass of 33.5 kDa and a pI of about 7. Of the two leg muscle forms the larger has a molecular mass of 30.5 kDa and a pI of about 8.5 and the smaller a molecular mass of 29.8 kDa and a pI of about 10. Considerably more heterogeneity has been found in the leg than in the breast muscle proteins although this is not reflected in their N-terminal sequences. The reason for this is not clear. Troponin T from breast or leg muscle can be phosphorylated with troponin T kinase at the single serine residue at the N-terminus. No difference in the rate or extent of phosphorylation could be found between proteins from breast or leg muscle. The three proteins have been shown to differ only in the amino acid sequence of their N-terminal tryptic peptides. These peptides are of different length, that from breast troponin T being 58 residues and those from leg troponin T being 36 and 42 residues, these differences account for the difference in molecular mass of the parent proteins. Despite this difference the sequence of the first 12 and last 14 residues is identical in all three N-terminal peptides. The remainder of the sequence of the smallest peptide is also repeated in the other two but they each contain an extra piece of unique sequence. On the basis of these sequences it is proposed that chicken troponin T is coded for by a single gene containing, at the 5' end, a number of small exons and that three different mRNA molecules may be produced by alternative pathways of RNA splicing. The possible significance of these N-terminal sequence variations is discussed.     

Bovine type XII collagen: amino acid sequence of a 10 kDa pepsin fragment from periodontal ligament reveals a high degree of homology with the chicken alpha 1(XII) sequence

A 10 kDa collagenous peptide, derived from a 30 kDa disulfide bonded fragment, was purified from bovine periodontal ligament. Amino acid sequence analysis of tryptic peptides demonstrated a 92.8% homology with the chicken alpha 1(XII) cDNA derived sequence, demonstrating for the first time the presence of type XII collagen in a mammalian species and in an adult tissue.     

Immunohistochemical studies on regulation of alternative splicing of fast skeletal muscle troponin T: non-uniform distribution of the exon x3 epitope in a single muscle fiber

Troponin T (TnT) isoforms of chicken fast skeletal muscle are classified into two types, breast-muscle-type (B-type) and leg-muscle-type (L-type) isoforms. These isoforms are produced from a single gene by differential alternative splicing of pre-mRNA. We investigated immunohistochemically the distribution of B-type TnT isoforms in chicken leg muscle (musculus biceps femoris), using anti-exon x3 that was raised against a synthetic peptide corresponding to exon x3 and recognized B-type, but not the L-type, TnT isoforms. Mosaic patterns of immunostaining showing locally different expression of B-type TnT isoforms in a single fiber were observed among fibers, and the non-uniform distribution of the isoforms was also detected in sectioned fibers and myofibrils from the muscle. The results indicated that regulation of pre-mRNA splicing of fast skeletal muscle TnT was different not only among the muscle fibers but also within a single fiber, suggesting that heterogeneous myonuclei in regulation of alternative splicings occur in a single muscle fiber.     

Identification of a high molecular weight actin-binding protein in skeletal muscle.

A large polypeptide having a molecular weight of 240,000 as determined by electrophoresis in the presence of sodium dodecyl sulfate has been identified in whole cell homogenates from chick skeletal muscle myoblasts and the rat myoblast L6 cell line. A similar polypeptide was identified in both thigh and breast chicken skeletal muscle, but the latter contained less of this protein per g of tissue. Antibodies made to gizzard filamin (an actin-binding protein having a molecular weight of 240,000) cross-reacted with the partially purified Mr = 240,000 protein from chicken skeletal muscle. With use of the indirect immunofluorescence technique, the filamin antibody localized in the Z-line region of chicken skeletal muscle myofibrils. These results indicate that skeletal muscle contains a filamin-like protein that may form an integral part of the myofibril structure.     

M-protein in chicken cardiac muscle.

Chicken cardiac muscle myofibrils lack a visible M-line. Antibodies against chicken breast muscle M-protein, an M-line component with M_r = 165 000, were used to demonstrate the presence of a similar protein in chicken heart muscle. The immunoreplica technique showed the heart protein to have about the same molecular weight as the breast muscle M-protein on polyacrylamide slab gels in the presence of sodium dodecyl sulfate (SDS). Positive staining within the H-zone was observed when the indirect immunofluorescence technique was used to localize the M-protein in isolated heart myofibrils. This result was confirmed by electron microscopic investigations on longitudinal sections of antibody-incubated heart muscle fiber bundles showing the antibody against M-protein to be bound within a region corresponding to the M-line region of breast muscle myofibrils.     

A mouse monoclonal antibody to human alpha 2-macroglobulin (alpha 2M) crossreacts with alpha 2M from mouse: epitope mapping and characterization of subunit structure of murine alpha 2M

Mice were immunized with the isolated C-terminal heat-induced fragment of human alpha 2-macroglobulin (alpha 2M) and the spleens were used to prepare hybridomas. A monoclonal antibody (Mab) designated F17D5 was selected for further characterization. The epitope defined by Mab F17D5 was not expressed on alpha 2M, on alpha 2M-methylamine, or on alpha 2M-proteinase complexes. On the other hand, the antibody reacted avidly with denatured human alpha 2M and with some types of alpha 2M from other species, including mouse, on nitrocellulose-immunoblotting. The epitope of Mab F17D5 was mapped to less than 250 residues C-terminal of the internal thiolester of human alpha 2M. This was based on CNBr fragmentation of the 60 kDa C-terminal heat-fragment and on peptide mapping of alpha 2M, derivatized at the internal thiolester GLX-residue with 125I-labeled histamine. Murine alpha 2M was confirmed to contain two types of subunit: a 185 kDa subunit and a combination of 165 kDa and 35 kDa polypeptides. By partial disulfide bond reduction, heat-fragmentation and immunoblotting with Mab F17D5, the structure of murine alpha 2M was compared to that of human alpha 2M. The F17D5-epitope was mapped to a 30 kDa heat-induced fragment, which was released by denaturation without reduction. This fragment contained an intrachain disulfide bridge. By analogy with human alpha 2M, the 35 kDa subunit would be located at the C-terminal end of murine alpha 2M, disulfide-bonded to the major 165 kDa subunit.     

The primary structure of skeletal muscle myosin heavy chain: III. Sequence of the 22 kDa fragment and the alignment of the 23 kDa, 50 kDa, and 22 kDa fragments

The amino acid sequence of the 197-residue 22 kDa fragment from chicken pectoralis muscle was determined to be as follows: K-K-G-S-S-F-Q-T-V-S-A-L-F-R-E-N-L-N-K-L- M-A-N-L-R-S-T-H-P-H-F-V-R-C-I-I-P-N-E-T-K-T-P-G-A-M-E-H-E-L-V-L-H-Q-L-R- C-N-G-V- L-E-G-I-R-I-C-R-K-G-F-P-S-R-V-L-Y-A-D-F-K-Q-R-Y-R-V-L-N-A-S-A-I-P-E-G-Q- F-M-D-S- K-K-A-S-E-K-L-L-G-S-I-D-V-D-h-T-Q-Y-R-F-G-H-T-K-V-F-F-K-A-G-L-L-G-L-L-E- E-M-R-D- D-K-L-A-E-I-I-T-R-T-Q-A-R-C-R-G-F-L-M-R-V-E-Y-R-R-M-V-E-R-R-E-S-I-F-C-I- Q-Y-N-V-R-S-F-M-N-V-K-H-W-P-W-M-K-L-F-F-K, where h stands for 3-N-methylhistidine. The amino acid sequences of the 22 kDa fragment and its equivalent fragment from chicken ventricle and gizzard muscle myosins were also determined by our group. Predicted secondary structures of these 22 kDa fragment regions and of the reported chicken embryo myosin revealed some possible structural differences.(ABSTRACT TRUNCATED AT 250 WORDS)