White Myosin Research Articles

Myosins from red and white bovine muscles: Differences measured by turbidimetric, calorimetric and rheological techniques

AbstractThe aim of this study was to elucidate the functional performance of the most abundant protein component in meat, ie myosin, which is recognised as important for binding in meat products. As several genetic variants of skeletal myosin exist, myosins from two bovine muscles of different fibre type composition, M masseter and M cutaneus trunci were compared with respect to filament forming properties and denaturation characteristics. The principal methods used were turbidimetric measurements, which were used to monitor filament formation, calorimetry and rheology. The myosin systems were examined at two different salt levels (0.2 and 0.6 M NaCl) and at pH 5.5–7.0. The method of preparing myosin suspensions/solutions was also examined. Differences in the filament‐forming process for the two myosins were detected. Measurements of turbidity revealed that at conditions of low pH and low ionic strength white myosin had a higher ultimate turbidity compared with red myosin. Early in the transition from low to high turbidity, red myosin had a higher turbidity than white myosin corresponding to reduced solubility. The turbidity increased with time of storing the myosin suspensions/solutions. This change was attributed to formation of filaments and further association of filaments. White myosin had a smaller apparent enthalpy of denaturation than red myosin. The calorimetric measurements recorded in 0.2 M NaCl suggested that the head and the rod of white myosin were less stable than the corresponding parts of red myosin. However, exceptions to this rule were found at pH 6.0. In 0.6 M NaCl the identification of the transitions for red myosin was more difficult. The method of preparing myosin suspensions affected calorimetric and rheological measurements. In 0.6 M NaCl and pH 6.0 calorimetric thermograms of both myosins were affected by the preparation method. At pH 5.5 this change was interpreted as caused by denaturation promoted by the dilution/rapid titration technique compared with dialysing the systems to pH 5.5 from pH 7. Differences in the filaments formed might, however, also contribute to the variations seen in the calorimetric ther‐mograms. The gelling properties of white myosin were most sensitive to the preparation method used. Systems prepared by dialysis gave stronger heat‐induced gels than those prepared by ‘dilution’. White myosin always produced stronger gels than red myosin independent of the preparation technique. The rheological properties (at 80°C) of red myosin were less affected by the preparation method than were those of white myosin. At lower temperatures, however, there was more variation in the shapes of the rheological thermograms (Ǵ versus temperature) for red myosin than in the corresponding thermograms of white myosin.

Myofibrillar proteins in skeletal muscles of parr, smolt and adult atlantic salmon ( Salmo salarl.). Comparison with another salmonid, the arctic charr Salvelinus alpinus ( l.)

The heavy and light subunits of myosin from white and red muscles of Atlantic salmon parr, smolt and adult individuals were analyzed by SDS-PAGE and two-dimensional electrophoresis. Tropomyosin was identified by comigration with rat tropomyosins in two-dimensional gels in the presence and absence of urea. These myofibrillar proteins were compared to those of Arctic charr. 1. 1. The myosin heavy chain from Atlantic salmon red muscles was associated with two types of light chain, 1S and 2S, that comigrated with the light chains 1S and 2S of Arctic charr. 2. 2. As in the Arctic charr, four myosin light chain spots were detected in white muscles: two fast myosin light chains type 1, one of which comigrated with its analogous in the Arctic charr; one fast myosin light chain type 2, differing slightly in isoelectric point from that of Arctic charr; and one fast myosin light chain type 3 with higher electrophoretic mobility than that of Arctic charr. 3. 3. Three tropomyosin spots were detected. White muscles contained only one type of β-tropomyosin and red muscles two types of α-tropomyosin. These three tropomyosin spots comigrated with those of Arctic charr. 4. 4. Two myosin heavy chain bands were observed in red muscles of salmon parrs but only one in the rest of the red muscles analyzed. 5. 5. Only one myosin heavy chain band was detected in white muscles by SDS-glycerol-polyacrylamide gel electrophoresis. Alfa-chymotryptic peptide mapping of these white myosin heavy chain bands revealed differences attributed to the presence of a new type of myosin heavy chain first detected several months after smoltification.

Myosins from red and white bovine muscles: Part 1—Gel strength (elasticity) and water-holding capacity of heat-induced gels

Myosins were isolated from bovine M. masseter (red) and M. cutaneus trunci (white) and characterized in terms of solubility, electrophoretic pattern and susceptibility to tryptic digestion. Dynamic rheological measurements showed that white myosin is generally the superior gel former: gel strength, expressed as storage modulus, is consistently higher (a) at all temperatures above the gel-inducing minimum, (b) in both 0·2 m and 0·6 m NaCl, (c) at all concentrations investigated (≤ 20 mg/ml) and (d) at pH ≥ 5·8. In 0·6 m NaCl at pH < 5·8 red myosin appears to perform better. All gels formed in 0·2 m NaCl lost about 15% liquid on centrifugation; gels in 0·6 m NaCl displayed an inverse relationship between storage modulus and loss of liquid. The gel strength and water-holding capacity of gels made from mixtures of red and white myosin were a linear function of mixture proportions in the case of 0·2 m NaCl; 0·6 m NaCl gave gels with unpredictable properties.

Electrophoretic and immunological study of myosin light chains from freshwater teleost fishes

White muscle myosin light chains from nine freshwater teleosts show a qualitative and quantitative variability on PAGE without phylogenetic correlation. They look different from their higher vertebrate counterparts mainly with regard to electric charge and relative amounts of alkali light chains corresponding to various contents of isoenzymic forms of white muscle myosin. Antibodies against carp white myosin LC1 recognize almost entirely white muscle LC1 from the other fishes and to a lesser degree LC1 from other muscles and vertebrates. The primary structure of this light chain is thus relatively constant. LC2 from carp cardiac muscle and mammalian slow and cardiac muscle do not react at all.

The analysis of a chicken myosin heavy chain cDNA clone.

A cDNA library has been constructed in the plasmid pBR322 using a large size class of RNA derived from chicken embryonic leg muscle as the template material. A clone containing a 2350-base pair insert was selected and identified as coding for the myosin heavy chain sequence, based upon its ability to hybridize to genomic myosin heavy chain clones, and by direct nucleotide sequencing. Cross-hybridization experiments with myosin heavy chain genomic clones, and mRNAs derived from different muscle types were used to explore the heterogeneity of the various myosin heavy chain isoforms at the level of the coding sequences. Although extensive sequence homology with the other isoforms was observed, a fast white isoform-specific subclone was constructed, and used to demonstrate that different genes code for the adult and embryonic fast white myosin heavy chain proteins.

Comparison of adult, embryonic, and dystrophic myosin heavy chains from chicken muscle by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and peptide mapping.

Chicken myosin heavy chains from adult fast white muscle fibers (both normal and dystrophic), adult slow red fibers, and embryonic presumptive fast white fibers were compared by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and by peptide mapping. The heavy chain of slow red myosin migrated electrophoretically more slowly than the heavy chains of the other myosins and differed markedly from them in its peptide maps. The heavy chain of dystrophic fast white myosin was similar to its normal counterpart by peptide mapping but showed slight differences. The peptide map of the heavy chain of embryonic presumptive fast white myosin had the general features of that of the heavy chain of fast white, not slow red, fibers but contained definite differences from the former. The results are consistent with the existence of a separate gene for the heavy chain of embryonic presumptive fast white myosin.

Ultrastructural identification of muscle fiber types by immunocytochemistry.

In a fast-twitch muscle, three types of fibers (red, intermediate, and white) can be distinguished on the basis of mitochondrial content. Red fibers, identified by abundant mitochondria, can be further differentiated on the basis of a positive or negative response to antibodies specific for white ("fast") myosin. Because there is also a difference in Z-line width among fibers of the same muscle, the possibility existed that the two red fibers, which differ in type of myosin, might also differ in dimensions of the Z line. We therefore examined, with the electron microscope, fibers which had been exposed to antibody against white myosin. In those fibers which react with the antibody, an electron-opaque band is evident in the H-band region, thereby distinguishing reactive from unreactive fibers. The red fiber can now be subdivided on the basis of a positive or negative response to anti-white myosin visualized directly with the electron microscope. Both categories of red fibers ("fast" and "slow") have wide Z lines, and thus are distinguished from white and intermediate fibers, which react with the antibody but which have narrow Z lines. On the basis of combined immunocytochemical and ultrastructural characteristics, four types of fibers can be recognized in a single muscle. Moreover, it is demonstrated here that a wide Z line does not necessarily imply a slow speed of contraction.

Distribution of myosin isoenzymes among skeletal muscle fiber types.

Using an immunocytochemical approach, we have demonstrated a preferential distribution of myosin isoenzymes with respect to the pattern of fiber types in skeletal muscles of the rat. In an earlier study, we had shown that fluorescein-labeled antibody against "white" myosin from the chicken pectoralis stained all the white, intermediate and about half the red fibers of the rat diaphragm, a fast-twitch muscle (Gauthier and Lowey, 1977). We have now extended this study to include antibodies prepared against the "head" (S1) and "rod" portions of myosin, as well as the alkali- and 5,5'dithiobis (2-nitrobenzoic acid) (DTNB)-light chains. Antibodies capable of distinguishing between alkali 1 and alkali 2 type myosin were also used to localize these isoenzymes in the same fast muscle. We observed, by both direct and indirect immunofluorescence, that the same fibers which had reacted previously with antibodies against white myosin reacted with antibodies to the proteolytic subfragments and to the low molecular-weight subunits of myosin. These results confirm our earlier conclusion that the myosins of the reactive fibers in rat skeletal muscle are sufficiently similar to share antigenic determinants. The homology, furthermore, is not confined to a limited region of the myosin molecule, but includes the head and rod portions and all classes of light chains. Despite the similarities, some differences exist in the protein compositions of these fibers: antibodies to S1 did not stain the reactive (fast) red fiber as strongly as they did the white and intermediate fibers. Non-uniform staining was also observed with antibodies specific for A2 myosin; the fast red fiber again showed weaker fluorescence than did the other reactive fibers. These results could indicate a variable distribution of myosin isoenzymes according to their alkali-light chain composition among fiber types. Alternatively, there may exist yet another myosin isoenzyme which is localized in the fast red fiber. Those red fibers which did not react with any of the antibodies to pectoralis myosin, did react strongly with an antibody against myosin isolated from the anterior latissimus dorsi (ALD), a slow red muscle of the chicken. The myosin in these fibers (slow red fibers) is, therefore, distinct from the other myosin isoenzymes. In the rat soleus, a slow-twitch muscle, the majority of the fibers reacted only with antibody against ALD myosin. A minority, however, reacted with antiboddies to pectoralis as well as ALD myosin, which indicates that both fast and slow myosin can coexist within the same fiber of a normal adult muscle. These immunocytochemical studies have emphasized that a wide range of isoenzymes may contribute to the characteristic physiological properties of individual fiber types in a mixed muscle.

Polymorphism of myosin among skeletal muscle fiber types.

An immunocytochemical approach was used to localize myosin with respect to individual fibers in rat skeletal muscle. Transverse cryostat sections of rat diaphragm, a fast-twitch muscle, were exposed to fluorescein-labeled immunoglobulin against purified chicken pectoralis myosin. Fluorescence microscopy revealed a differential response among fiber types, identified on the basis of mitochondrial content. All white and intermediate fiber but only about half of the red fiber reacted with his antimyosin. In addition, an alkali-stable ATPase had the same pattern of distribution among fibers, which is consistent with the existence of two categories of red fibers. The positive response of certain red fibers indicates either that their myosin has antigenic determinants in common with "white" myosin, or that the immunogen contained a "red" myosin. Myosin, extracted from a small region of the pectorlis which consists entirely of white fibers, was used to prepare an immunoadsorbent column to isolate antibodies specific for white myosin. This purified anti-white myosin reacted with the same fibers of the rat diaphragm that had reacted with the white, intermediate, and some red fibers are sufficiently homologous to share antigenic determinants. In a slow-twitch muscle, the soleus, only a minority of the fiber reacted with antipectoralis myosin. The majority failed to respond; hence, they are not equivalent to intermediate fibers of the diaphragm; despite their intermediate mitochondrial content. Immunocytochemical analysis of two different musles of the rat has demonstrated that more than one isoenzyme of myosin can exist in a single muscle, and that individual fiber types can be recognized by immunological differences in their myosin. We conclude that, in the rat diaphragm, there are at least two immunochemically distinct types of myosin and four types of muscle fibers: white, intermediate, and two red. We suggest that these fibers correspond to the four types of motor units described by Burke et al. (Burke, R. E., D. N. Levine, P. Tsairis, and F. E. Zajac, III 1973. J. Physiol. (Lond) 234:723-748.)in the cat gastrocnemius.;