Muscle protein expression of pikeperches (Stizostedion lucioperca and S. volgense)

The purpose of the present study was to investigate the muscle protein expression in two pikeperches (Stizostedion lucioperca and S. volgense) through intra‐ and intermyomeric composition of white muscles. Using denaturing 10% sodium dodecylsulfate‐polyacrylamide gel electrophoresis, muscle protein expression was studied in relation to within‐ and between‐species morphological development, sex, maturity and age of pikeperches. Myosin, actin and troponin have a distinct role in the contraction and length tension of muscle fibers of these species. No obvious intramyomeric differences were found in the myosin heavy chain of both species. Myosin light chains (15–38 kDa) have different expression in different age groups. The muscle protein of the fingerling and adult S. lucioperca had high molecular weight (50 kDa) myosin in contrast to the other Percid species. The molecular weight of actins increased comparatively in low‐age‐group fish. ATP is stored in myosin and released to cause contraction when myosin comes in contact with actin of the experimental fish. Troponin regulates increasing concentration of light‐chain myosin in mature fish. Because troponin T has been implicated in the regulation of skeletal muscle kinetics, muscle contraction kinetics was predicted in different age groups. The muscle proteins of both sexes of these species have polymorphism in various age groups but have no difference in similar aged fish. No muscle protein dimorphism was found in these Percid species. The white muscle protein composition and contractile properties affect power production during fast, unsteady movement and swimming.     

cDNA cloning and complete primary structures of myosin heavy chains from brushtooth lizardfish Saurida undosquamis and wanieso lizardfish S. wanieso fast skeletal muscles

ABSTRACT:   The complete cDNA sequences encoding predominant types of myosin heavy chain (MYH) in the fast skeletal muscle were determined for brushtooth lizardfish Saurida undosquamis and wanieso lizardfish S. wanieso , which are used as materials for preparing high-quality surimi-based products. The cDNA consisted of 5973 and 5987 bp, respectively, and both encompassed an open reading frame encoding a polypeptide of 1936 amino acid residues. Brushtooth and wanieso lizardfish MYH showed the amino acid sequence identity of 92–93% to white croaker MYH, which was higher than that of 90% to walleye pollack MYH. The putative binding sites for ATP, actin, and regulatory and essential light chains in the subfragment-1 region of brushtooth lizardfish MYH exhibited a high identity with white croaker counterparts as well as the sequences of subfragment-2 and light meromyosin. In contrast, phylogenetic tree, constructed by the neighbor-joining method based on mitochondrial 16S rRNA gene, revealed that the two lizardfish species formed a cluster with walleye pollack, which was paraphyletic with white croaker. Therefore, a good reputation for lizardfish and white croaker to have a high thermal-gel forming ability seemed to be reflected by MYH rather than biological similarity as revealed by the mitochondrial 16S rRNA gene.     

Determination of primary structure of amberjack myosin heavy chain and its relationship with structural stability of various fish myosin rods

The structural stability of fish myosin depends upon species and temperatures of water in which fish live. Primary, secondary, and quaternary structures of myosin heavy chain (MyHC) from three species of fish living at different temperature ranges have been compared with those of rabbit MyHC in order to investigate the differences in stability. Primary structure of MyHC, although being accessible for warm-water and cold-water fish (carp and walleye pollack), was not available in previous for tropical-water fish literature; so in this study primary structure of MyHC of the tropical-water fish amberjack has been determined by cloning and sequencing its cDNA. The MyHC has 1938 amino acid residues (AA), which are almost as much as as those of carp and walleye pollack. The amberjack MyHC is 91–95% homologous with other fish and rabbit MyHCs. There is a discernible difference between animal species with stable myosin rod (amberjack, carp, and rabbit) and walleye pollack with unstable rod. Stable rod species have a high probability of forming coiled-coil around the COOH-terminal end of the rod, while the pollack has a low coiled-coil formation probability. In addition, the average scores of the coiled-coil for myosin rod were rabbit (1.738) > amberjack (1.691) > carp (1.680) > walleye pollack (1.674) which correlated exactly with the observed stability. The results suggest that coiled-coil forming ability, particularly around the COOH-terminal end, directs structural stability of fish myosin rod.     

Mechanochemical properties of ordinary and dark muscle myosins from seawater fish

Myosin was isolated from two types of muscle, ordinary and dark muscles, of three species of fish living in sea water. The compositions of light chains were visualized by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the mechanochemical activity was examined by in vitro motility and ATPase assays. Ordinary muscle myosin of either species had three species of light chain, whereas dark muscle myosin had another two species of light chain judged by SDS-PAGE. Sliding velocity of ordinary muscle myosin was in the range of 4.92–6.89 μm/S, whereas that of dark muscle myosin was in the range of 3.07–4.25 μm/s. Therefore, ordinary muscle myosin showed 1.26–1.95 times higher sliding velocity than dark muscle myosin in either species. The ratios of V_max of actin-activated Mg²⁺-ATPase activity of ordinary to dark muscle myosins were correlated quite well to the ratios of sliding velocity. Activity of ordinary muscle myosin was comparable to that of mammalian fast muscle myosin, but that of dark muscle myosin was twice of that of mammalian slow muscle myosin. These results may reflect the essential role of fish dark muscle myosin always used in slow cruising.     

Comparative study on thermal denaturation modes of myosin in walleye pollack and carp myofibrils as affected by salt concentration

ABSTRACT:   The effect of salt concentration on the thermal denaturation profile of myosin in walleye pollack and carp myofibrils was compared by studying the subfragment-1 (S-1) and rod denaturation rates upon heating. Species-specific denaturation mode observed at 0.1 M KCl was no longer detected when samples were heated above 0.5 M KCl, where S-1 and rod denaturation rates were identical to each other. As the heating of the chymotryptic digest of myofibril formed practically no rod aggregates, S-1 denaturation in a form of myosin was the rate limiting step for rod aggregate formation. As the aggregate formation by rod was remarkably suppressed by lowering the temperature, the free movement of myosin tail upon heating was suggested to play an important role in the rod aggregate formation in a high salt medium.     

Spawning induces a shift in energy metabolism from glucose to lipid in rainbow trout white muscle

Enzymatic changes that occur in the white somatic muscle of rainbow trout (Oncorhynchus mykiss) in response to spawning were investigated, and the evenness of their distribution across the ventral-dorsal plane of this muscle was assessed. Four enzymes that are involved in energy metabolism were measured (phosphofructokinase: glycolytic capacity, 3-hydroxyacyl-CoA dehydrogenase: -oxidation, citrate synthase: citric acid cycle, cytochrome oxidase: oxidative capacity). The enzyme activities were followed in different parts of the white muscle of non-spawning female rainbow trout from May, four months after their first spawning, until December, at second spawning. Samples were taken from white epaxial muscle along the lateral line, on the dorsum, and in between. Samples were also taken from red muscle of non-spawning fish. The isoforms of myosin heavy chains (MyHC) were electrophoretically identified on 6% SDS-PAGE gel to study possible changes in contractile properties of the muscle.Transformation from the non-spawning to spawning phase was associated with dramatic changes in the activity of the enzymes studied in white muscle: glycolytic capacity decreased to less than half, whereas oxidative metabolism increased about two- to four-fold in all areas. Significant quantitative differences in enzyme activities were found between the three epaxial muscle areas: in the non-spawning fish lateral line samples differed from those taken in the other two areas, whereas in spawning fish the dorsal sample difered from the other two. No difference in the expression of MyHC-isoforms was found between spawning and non-spawning fish. Co-expression of both slow and fast isoforms was found in single fibres isolated from red muscle.The results show that the energy metabolism in white muscle of domestic rainbow trout is altered during spawning; i.e., the metabolism becomes increasingly aerobic, with an increased capacity for fatty acid utilization, concomitant with phenotypic changes associated with sexual maturation. These changes are especially pronounced in ventral, superficially located fibres.     

Comparative sequence analysis of four myosin heavy chain isoforms expressed in porcine skeletal muscles: Sequencing and characterization of the porcine myosin heavy chain slow isoform

A nucleotide sequence including the full coding region for the myosin heavy chain (MyHC) slow isoform was determined from the longissimus skeletal muscle. The deduced amino acid sequence was 1935 residues, which was the same length as the human and rat MyHC-slow isoforms. The porcine MyHC-slow isoform showed 97.6% and 97.4% amino acid identities to the human and rat isoforms on their entire regions, respectively. The functional regions were also highly conserved among mammalian MyHC-slow isoforms. Amino acid substitutions between the porcine MyHC-slow and MyHC-fast isoforms were concentrated on the functional regions. Loop 1, the controlling region of nucleotide binding and release, was conserved among the fast isoforms, but not between the slow and fast isoforms. Loop 2, a part of the actin binding region, was not conserved among any of the isoform types, and the most substitutions in this region were found in the slow isoform. The myosin essential light chain binding region was conserved among the fast isoforms, except for some substitutions in the 2b isoform, but was clearly different in the slow isoform. The myosin regulatory light chain binding region was conserved among the fast isoforms, but not between the slow and fast isoforms. These results indicate that the functional difference between the porcine MyHC-slow and -fast isoforms are controlled by the sequence diversity at the four functional regions compared.     

Segmental axonopathy of Merino sheep in New Zealand

AIM: To investigate an axonopathy of Merino sheep that caused progressive hindlimb ataxia and slight to moderate paresis, with the purpose of understanding its pathogenesis.

METHODS: Tissues were fixed in buffered paraformaldehyde or paraformaldehyde and glutaraldehyde, processed into wax and epoxy resin, respectively, and examined by light and electron microscopy. Fresh frozen spinal cord and trigeminal nerve roots were subjected to homogenisation, centrifugation and two-dimensional electrophoresis. Selected protein spots were identified using matrix-assisted laser desorption ionisation (MALDI) mass spectrometry.

RESULTS. By light microscopy, there were large pale foamy spheroidal axonal swellings affecting peripheral as well as central axons. By electron microscopy, these were shown to contain many membrane-bound vesicles. The main abnormalities in expressed proteins involved cytoskeletal elements and myosin heavy chain, the latter interpreted as associated with the molecular motor myosin Va.

CONCLUSIONS: The disorder is the same as that described in Merinos in Australia as segmental axonopathy, and believed to have an inherited aetiology. The lesions and protein changes indicate abnormalities of the cytoskeleton, its relationship with the myelin sheath, and myosin Va molecular motor. The consequence appears to be abnormal axonal transport and inability to maintain the integrity of axons and their myelin sheaths.