Koui Takahashi

Relationship between structural properties of intramuscular connective tissue and toughness of various chicken skeletal muscles

The shear-force value, the total amount of collagen, heat-solubility of collagen and thickness of the perimysium were measured on six kinds of chicken skeletal muscle. Toughness of the meat was significantly correlated with both total amount of collagen (r(2) = 0.94) and thickness of the perimysium (r(2) = 0.95). Although differences in heat-solubility of collagen were observed among the six muscles, heat-solubility of collagen was not correlated to the shearforce value. These results suggest that the total amount of collagen and structures of the perimysium are the major factors determining toughness of chicken.

Structural weakening of intramuscular connective tissue during conditioning of beef

The structural changes in intramuscular connective tissues endomysium and perimysium during conditioning of beef were investigated using an improved technique of scanning electron microscopy. In beef conditioned for 28 days of 4°C, the endomysium resolved into individual collagen fibrils and the thick sheets of perimysium separated into collagen fibres of 4-8 μm in diameter. These results provide direct evidence for the structural weakening of endomysium and perimysium during conditioning. The structural changes in the intramuscular connective tissue were minimal until 10 days post mortem, but clearly observable after 14 days post mortem. Therefore, it is concluded that intramuscular connective tissue shows the effect on tenderisation of extended conditioning (2-4 weeks) of beef.

Calcium binding to an elastic portion of connectin/titin filaments.

Abstractα-Connectin/titin-1 exists as an elastic filament that links a thick filament with the Z-disk, keeping thick filaments centered within the sarcomere during force generation. We have shown that the connectin filament has an affinity for calcium ions and its binding site(s) is restricted to the β-connectin/titin-2 portion. We now report the localization and the characterization of calcium-binding sites on β-connectin. Purified β-connectin was digested by trypsin into 1700- and 400-kDa fragments, which were then subjected to fluorescence calcium-binding assays. The 400-kDa fragment possesses calcium-binding activity; the binding constant was 1.0 × 107 M−1 and the molar ratio of bound calcium ions to the 400-kDa fragment reached a maximum of 12 at a free calcium ion concentration of approximately 1.0 μM. Antibodies against the 400-kDa fragment formed a sharp dense stripe at the boundary of the A and the I bands, indicating that the calcium-binding domain constitutes the N-terminal region of β-connectin, that is, the elastic portion of connectin filaments. Furthermore, we estimated the N-terminal location of β-connectin of various origins (n = 26). Myofibrils were treated with a solution containing 0.1 mM CaCl2 and 70 μM leupeptin to split connectin filaments into β-connectin and a subfragment, and chain weights of these polypeptides were estimated according to their mobility in 2% polyacrylamide slab gels. The subfragment exhibited a similar chain weight of 1200 ± 33 kDa (mean ± SD), while α- and β-connectins were variable in size according to their origin. These results suggest that the apparent length of the 1200-kDa subfragment portion is almost constant in all instances, about 0.34 μm at the slack condition, therefore that the C-terminus of the 1200-kDa subfragment, that is, the N-terminus of the calcium-binding domain, is at the N2 line region of parent filaments in situ. Because the secondary structure of the 400-kDa fragment was changed by the binding of calcium ions, connectin filaments could be expected to alter their elasticity during the contraction–relaxation cycle of skeletal muscle.

Relationship between degradation of proteoglycans and weakening of the intramuscular connective tissue during post-mortem ageing of beef.

Changes in proteoglycans (PGs) during post-mortem ageing of bovine m. semitendinosus were studied. Electron microscopic observations made it clear that there were two types of PGs in bovine m. semitendinosus immediately post-mortem: PGs were arranged regularly in the basement membrane and PGs associated with collagen fibrils in the perimysium. After 28 days ageing at 4 °C, no PG was observed in the basement membrane, and the greater part of PGs in the perimysium had disappeared. The total amount of PGs decreased with time post-mortem. SDS-polyacrylamide gel electrophoresis indicated that PGs with a high molecular weight disappeared within 7 days post mortem. These results suggest that PGs are degraded during post-mortem ageing of beef. The degradation of PGs seems likely to be the main factor in the weakening of intramuscular connective tissues, i.e., separation of collagen fibrils and fibres from the endomysium and the perimysium, which results in the partial tenderization of beef during post-mortem ageing.

Non-enzymatic weakening of myofibrillar structures during conditioning of meat: calcium ions at 0.1 mM and their effect on meat tenderization.

The tenderness of meat is set by the properties of connective tissue and myofibrils. Skeletal muscle connective tissues become firm with chronological aging concomitantly with the increase in intermolecular non-reducing cross-links of collagen, and this process toughens meat, however, connective tissues hardly change during conditioning of meat. Therefore, the tenderization of meat during post mortem aging, or to put it more precisely, during post rigor aging, stems for the most part from changes in myofibril structures. My research derives its origin on findings of two kinds of post mortem changes in myofibril structures; i) fragmentation of myofibrils; and ii) restoration of rigor-shortened sarcomeres. These results were published in 1967 [1], and were, thereafter proved by many workers to be closely related to meat tenderization. I report in this paper the essential molecular mechanisms of these phenomena, and of structural changes in connectin or titin filaments. All of them are non-enzymatically induced by 0.1 mM calcium ion, which is the ultimate concentration of sarcoplasmic calcium ion in post mortem muscles.

Spontaneous aggregation of locust lipophorin during hemolymph collection

Abstract Density-gradient ultracentrifugation, gel permeation chromatography, polyacrylamide gel electrophoresis and electron microscopy were used to provide an experimental basis for the proposal that locust lipophorin aggregates spontaneously during the collection of hemolymph through a puncture in the neck or coxal region of the insect. The evidence presented in this paper supports the above proposal and clearly demonstrates that the “puncture” method promotes the aggregation of lipophorin molecules and results in the formation of large aggregates as an artificial product. Thus, the use of this method should be discouraged for the study of lipophorin in locusts.

Developmental expression of extracellular matrix components in intramuscular connective tissue of bovine semitendinosus muscle

Abstract We have investigated the expression patterns of extracellular matrix components in intramuscular connective tissue during the development of bovine semitendinosus muscle by means of indirect immunofluorescence techniques. Types I, III, V, and VI collagen and fibronectin were located in the endomysium and the perimysium. Type IV collagen, laminin, and heparan sulfate proteoglycans (PGs) were exclusively located in the endomysium, and dermatan sulfate PGs existed only in the perimysium. The localization of these components in the intramuscular connective tissue of semitendinosus muscle remained unchanged throughout prenatal and postnatal growth of cattle, suggesting that they are essential for forming and maintaining structures of the endomysium and perimysium in bovine semitendinosus muscle. On the other hand, decorin was undetectable in the endomysium of neonates, although other matrix components were already expressed. It was expressed slightly in the endomysium of 2-month-old calves, and clearly detectable in the endomysium of cattle more than 6 months old. Chondroitin sulfate PGs were barely detectable in the perimysium of fetuses and neonatal calves, and progressively appeared during postnatal development of the muscle. It seems likely that these PGs are closely related to the postnatal development of the endomysium and perimysium.

Purification and characterization of a biliverdin-binding cyanoprotein from the locust hemolymph

Abstract The purification of a cyanoprotien from locust hemolymph was achived using a simple preparative electrophoretic method. The preparation was homogeneous, as judged by polyacrylamide gel electrophoresis and electron microscopy. The cyanoprotein molecule appeared to have a flattened cylindrical shape with a diameter of about 120 A and height of about 100 A. The molecular weight, as determined by a sedimentation equilibrium method, was approximately 350000. The apoprotein consisted of identical subunits with molecular weights of 83000; thus, the native cyanoprotein molecule was assumed to be a tetramer, and this was also supported by electron microscopic observations. The cyanoprotein contained about 3.5% mannose associated covalently with the apoprotein, but there was no appreciable lipid component. The blue coloration of cyanoprotein was due to the presence of biliverdin pigments that associate non-covalently with the apoprotein. Each molecule of cyanoprotein contained eight molecules of biliverdin, suggesting that two molecules of biliverdin associate with each subunit.

Structural changes in endomysium and perimysium during post-mortem aging of chicken Semitendinosus muscle—Contribution of structural weakening of intramuscular connective tissue to meat tenderization

Post-mortem changes in endomysium and perimysium were investigated during aging of chicken semitendinosus muscle at 4°C. Although the shear-force value of raw meat decreased rapidly within 5 h post mortem and gradually thereafter, the solubility of collagen and the ratio of each chain of soluble collagen remained unchanged during 24 h post mortem. Light microscopic studies showed that structures of endomysium and perimysium disintegrated into several thin sheets within 12 h post mortem, and that many gaps opened in the cross-section of endomysium and perimysium. While endomysium and perimysium were not stained by periodic acid Schiff reagent in fresh muscle, they were markedly stained in muscle 12 h post mortem. These results provide direct evidence for the structural weakening of endomysium and perimysium during post-mortem aging of chicken. Therefore, we conclude that the structural weakening of the intramuscular connective tissue is closely related to tenderization of chicken.

Ultrastructure of the Intramuscular Connective Tissue in Bovine Skeletal Muscle

The three-dimensional arrangement of intramuscular connective tissues in bovine semitendinosus muscle was investigated using the cell-maceration method for scanning electron microscopy, by which cellular elements were eliminated and collagen fibrils and fibers were exposed. The 60-100 microns diameter honeycomb structures of the endomysium housing individual muscle fibers were clearly observed. The sheath of endomysium was membranous and consisted of tightly arranged collagen fibrils 30-70 nm in diameter. The perimysium was composed of several layers of 100-200 microns-thick sheets surrounding the sheaths of the endomysium. The sheets of the perimysium were wavy and consisted of collagen fibers made up of tightly bundled fibrils. The epimysium was composed of two distinct layers of thick sheets. The inner layer consisted of thousands of collagen fibers lying in an extremely regular wavy pattern parallel to the axis of the muscle fiber. The outer layer of epimysium was composed of several wavy sheets of collagen fibers which ran transversely to the axis of the muscle fiber. In a region of the epimysium, there was a thick wall of about 1 mm thickness, where fiber bundles of 200-300 microns in diameter ran longitudinally parallel to the axis of muscle fiber.