Carp White Muscle Research Articles

Purification and characterization of parvalbumins from silver carp (Hypophthalmichthy molitrix)

As the largest producer and consumer of freshwater fish in the world, many people suffer from allergy for consuming freshwater fish in China. However, the allergen profiles of freshwater fish are rarely known. Parvalbumins (PVs) from the white muscle of silver carp (Hypophthalmichthy molitrix) were purified by ammonium sulfate fractionation and column chromatography including DEAE-Sepharose and Superdex 75. Three PV isoforms-PV-I, PV-II, and PV-III-were obtained and their molecular masses as estimated by tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis were 12, 11, and 14 kDa, respectively. All the PVs could be detected by anti-frog PV monoclonal antibody. PV-I and PV-II were quite possibly glycoproteins, while PV-III was not glycosylated, as analyzed by periodic acid-Schiff (PAS) staining. Thermal stability revealed that PV-I and PV-II easily formed polymers, while these proteins were stable in a pH range of 4.0-10.0. A PV gene encoding 110 amino acid residues was cloned and it revealed high identity with PVs from other species of fish. Three isotypes of PV were purified to homogeneity and one distinct PV gene was cloned in silver carp white muscle.

Differential recovery from exercise and hypoxia exposure measured using 31P- and 1H-NMR in white muscle of the common carpCyprinus carpio

Phosphocreatine (PCr) was reduced to equivalent levels in carp white muscle by high-intensity exhaustive exercise and exposure to hypoxia at 15 degrees C and 25 degrees C in order to assess the influence of intracellular pH (pH(i)), temperature and lactate levels on PCr recovery in vivo. High-intensity exercise resulted in a significantly lower pH(i) compared with hypoxia exposure and the rate of PCr depletion and tissue acidification during hypoxia exposure was significantly higher in carp held at 25 degrees C compared with those at 15 degrees C. During recovery, PCr and pH(i) returned towards normoxia/resting levels at a faster rate following hypoxia exposure than after exercise. The lower pH(i) in exercised carp caused a greater perturbation to cellular energy status (assessed as the free energy of ATP hydrolysis; DeltafG') and resulted in a higher [ATP]/[ADP(free)] ratio, which may limit mitochondrial ATP production and contribute to the slower recovery from exercise compared with recovery from hypoxia exposure. Rates of recovery from exercise and hypoxia exposure were not affected by acclimation temperature (15 and 25 degrees C), suggesting that the processes involved in acclimation compensate for the Q(10) effects of temperature on metabolic processes. Finally, using a dual 31P-NMR and 1H-NMR analysis technique, we demonstrated that the greater tissue acidification observed after high-intensity exercise compared with hypoxia exposure occurred at similar white muscle lactate concentrations.

Structural Requirements of the Dihydropyridine Receptor α1S II-III Loop for Skeletal-type Excitation-Contraction Coupling

Residues Leu720-Leu764 within the II-III loop of the skeletal muscle dihydropyridine receptor (DHPR) alpha1S subunit represent a critical domain for the orthograde excitation-contraction coupling as well as for retrograde DHPR L-current-enhancing coupling with the ryanodine receptor (RyR1). To better understand the molecular mechanism underlying this bidirectional DHPR-RyR1 signaling interaction, we analyzed the critical domain to the single amino acid level. To this end, constructs based on the highly dissimilar housefly DHPR II-III loop in an otherwise skeletal DHPR as an interaction-inert sequence background were expressed in dysgenic (alpha1S-null) myotubes for simultaneous recordings of depolarization-induced intracellular Ca2+ transients (orthograde coupling) and whole-cell Ca2+ currents (retrograde coupling). In the minimal skeletal II-III loop sequence (Asp734-Asp748 required for full bidirectional coupling, eight amino acids heterologous between skeletal and cardiac DHPR were exchanged for the corresponding cardiac residues. Four of these skeletal-specific residues (Ala739, Phe741, Pro742, and Asp744) turned out to be essential for orthograde and two of them (Ala739 and Phe741) for retrograde coupling, indicating that orthograde coupling does not necessarily correlate with retrograde signaling. Secondary structure predictions of the critical domain show that an alpha-helical (cardiac sequence-type) conformation of a cluster of negatively charged residues (Asp744-Glu751 of alpha1S) corresponds with significantly reduced Ca2+ transients. Conversely, a predicted random coil structure (skeletal sequence-type) seems to be prerequisite for the restoration of skeletal-type excitation-contraction coupling. Thus, not only the primary but also the secondary structure of the critical domain is an essential determinant of the tissue-specific mode of EC coupling.

Effects of thermal acclimation on the relaxation system of crucian carp white myotomal muscle

Many cyprinid fish are able to compensate for a decrease in ambient temperature by process of physiological adaptation in the function of muscles. In the winter habitat of crucian carp (Carassius carassius L.), low temperature is associated with simultaneous oxygen shortage. Because of the oxygen deprivation, there is probably little space for compensatory adaptation because positive thermal compensation would increase energy demand and accelerate depletion of glycogen reserves. Thus, we assumed that the crucian carp, unlike many other cyprinid fish, would not show positive thermal compensation but either no compensation or inverse compensation in muscle function. To test this hypothesis in the relaxation system of skeletal muscles, we determined the parvalbumin content and the activity of sarcoplasmic reticular (SR) Ca-ATPase in white myotomal muscle of winter- and summer-acclimated crucian carp. In the laboratory, the winter fish were kept at 2 degrees C and the summer fish at 22 degrees C for a minimum of 3 weeks before the experiments. The specific activity of SR Ca-ATPase at low experimental temperature (2 degrees C) was similar in summer- and winter-acclimated fish (0.26 +/- 0.04 vs. 0.25 +/- 0.04 mM/mg/min; P > 0.05). Because of the bigger Q(10) of cold-acclimated carp, the enzyme activity at 30 degrees C was higher in cold-acclimated winter fish than in warm-acclimated summer fish (7.42 +/- 0.90 vs. 5.18 +/- 0.53 mM/mg/min; P < 0.05). In contrast, the yield of SR protein was 70% higher in summer than winter fish (0.315 +/- 0.045 vs. 0.187 +/- 0.017 mg/g; P < 0.001). Because of these opposing changes, total Ca-ATPase activity of SR (per gram muscle weight) remained relatively constant. Similarly, the parvalbumin content of the myotomal muscle was not different between summer (4.09 +/- 0.95 mg/g) and winter (3.70 +/- 0.60 mg/g) fish. Although there were no seasonal changes in the total relaxing system of the crucian carp white myotomal muscle, the same activity of SR Ca-ATPase in winter fish was obtained with less amount of SR pump protein, owing to the increased catalytic activity of the enzyme. The higher catalytic activity of winter fish SR Ca-ATPase might be caused by differences in fatty acid composition noted in membrane lipids; i.e., fewer saturated fatty acids and more n-6 polyunsaturated fatty acids (PUFAs), at the expense of n-3 PUFAs, were present in the SR of cold-acclimated winter fish. Temperature-induced changes in enzyme protein, however, cannot be excluded. Thus, the present results indicate the absence of positive thermal compensation in the relaxing system of crucian carp white muscle. It seems, however, that lipid composition of SR membranes and temperature dependence of SR Ca-ATPase are altered by seasonal acclimation.

Effects of thermal acclimation on the relaxation system of crucian carp white myotomal muscle

Effects of thermal acclimation on the relaxation system of crucian carp white myotomal muscle

Growth of carp (Cyprinus carpio) white axial muscle; hyperplasia and hypertrophy in relation to the myonucleus/sarcoplasm ratio and the occurrence of different subclasses of myogenic cells

In white axial muscle of carp addition of new fibres to the muscle mass (hyperplasia) decreased with increasing length of the fish. This was deducted from the decrease in the amount of small fibres. In carp larger than about 40 cm standard length (s.l.) hyperplasia no longer occurred (small fibres were absent) and muscle growth only occurred by means of hypertrophy (growth of existing fibres). The stage of growth in which many new fibres were added showed a relatively fast increase in trunk weight, as calculated from growth curves. During the stage of fast growth with a high occurrence of hyperplasia, the DNA/protein ratio decreased. The high percentage of postmitotic myosatellite cells isolated from carp of 5 cm s.l. suggests that in hyperplasia a subpopulation of already differentiated myosatellite cells formed in an earlier stage of development is incorporated in new muscle fibres. The increase of the relative importance of hypertrophy appears to be correlated to an increase in the percentage of proliferating myosatellite cells 17 h after isolation in vitro. This suggests that in hyperplasia and in hypertrophy different subpopulations of myosatellite cells are involved.

Growth of carp (Cyprinus carpio) white axial muscle; hyperplasia and hypertrophy in relation to the myonucleus/sarcoplasm ratio and the occurrence of different subclasses of myogenic cells

In white axial muscle of carp addition of new fibres to the muscle mass (hyperplasia) decreased with increasing length of the fish. This was deducted from the decrease in the amount of small fibres. In carp larger than about 40 cm standard length (s.l.) hyperplasia no longer occurred (small fibres were absent) and muscle growth only occurred by means of hypertrophy (growth of existing fibres). The stage of growth in which many new fibres were added showed a relatively fast increase in trunk weight, as calculated from growth curves. During the stage of fast growth with a high occurrence of hyperplasia, the DNA/protein ratio decreased. The high percentage of postmitotic myosatellite cells isolated from carp of 5 cm s.l. suggests that in hyperplasia a subpopulation of already differentiated myosatellite cells formed in an earlier stage of development is incorporated in new muscle fibres. The increase of the relative importance of hypertrophy appears to be correlated to an increase in the percentage of proliferating myosatellite cells 17 h after isolation in vitro. This suggests that in hyperplasia and in hypertrophy different subpopulations of myosatellite cells are involved.

Calcium channels from Cyprinus carpio skeletal muscle.

The complete amino acid sequence of the L-type calcium channel alpha 1 subunit from the carp (Cyprinus carpio) white skeletal muscle was deduced by cDNA cloning and sequence analysis. The open reading frame encodes 1852 amino acids (Mr 210,060). A 155-amino acid COOH-terminal sequence (after the fourth internal repeat) is evolutionarily preserved (90% homology) and may represent an important functional domain of L-type calcium channels. The photolabeled, membrane-bound, and purified carp alpha 1 subunits have masses of 211 and 190 kDa. The purified channel could not be phosphorylated by cAMP-dependent protein kinase. Two glycoproteins (alpha 2 subunits) are associated with the alpha 1 subunit and change their apparent masses from 235 and 220 kDa to 159 kDa upon reduction of disulfide bonds. Nucleic acid hybridization with alpha 2 cDNA revealed an 8.0-kilobase transcript in carp skeletal muscle. Evidence for a copurification of subunits similar in size to mammalian beta or gamma subunits was not obtained.

Effect of growth on the concentration of L-histidine and anserine in the white muscle of carp and rainbow trout.

Accumulation patterns of L-histidine and related dipeptides were examined in carp and rainbow trout white muscle as a function of body weight. Young carp (n=30) increased their body mass from 1.8 to over 200g during the growth period of a year. White muscle free L-histidine increased almost in a linear fashion from about 5 to 20μmol/g wet weight over the body weight range of 1.8 to 30-40g. Then the level of L-histidine decreased slightly to reach an almost constant level at 14.5-18.6μmol/g wet weight over the remaining growth period. On a double logarithmic plot, this histidine increase was linear related to body weight for small carp weighing below 32g (n=16, slope=0.52, r=0.88, p<0.001) and almost zero for larger cazp weighing from 15 to 247g (n=21, slope=-0.06, r=-0.28). In rainbow trout weighing from 1.8 to 321g (n=77), the Canadian trout weighing 10-150g (n=29) exhibited significantly higher amount of anserine in white muscle than their Japanese counterpart (n=48) over the same weight range. Both trouts, however, showed almost the same hyperbolic pattern of anserine increase over the concentration range of 3 to 18μmol/g wet weight. This increase was linear in double logarithmic scale for both Canadian and Japanese trout (slope=0.32, r=0.91, p<0.001 for Japanese trout; slope=0.15, r=0.82, p<0.001 for Canadian trout).

Thermoacclimatory response of nucleic acid and protein content of carp muscle tissue: influence of growth rate and relationship to glycine uptake by scales

RNA concentration in carp (Cyprinus carpio) white muscle tissue increased with higher growth rate for all acclimation temperatures, while DNA, protein content, and tissue hydration were unaffected. For a given growth rate, RNA concentration per unit tissue weight and per unit DNA (RNA per cell) was higher in colder acclimated fish as a probable compensation for decreased RNA activity. Maximum growth resulted in an approximate doubling of the RNA/DNA ratio while, over the range of 12–30 °C, cold acclimation caused an increase of approximately 50%. Changes in DNA concentration and protein/DNA ratios at different acclimation temperatures indicated an increase in cell mass with warmer acclimation temperatures. Muscle RNA/DNA ratios and the rate of glycine uptake by scales were highly correlated. Regression equations describing this relationship for each acclimation temperature were not significantly different when scales were incubated at each fish's acclimation temperature, but were significantly different when glycine uptake was expressed for incubation at the temperature of maximum uptake. These results suggest a whole-body integration between thermoacclimatory changes in the RNA component of muscle protein synthesis and in the rate of amino acid transport by scales.

Electrophoretic analysis of connectin from the muscles of aquatic animals.

Myofibrils or muscle homogenates from aquatic animals, including various fishes, whale, lobster, cuttlefish, and scallop, were analyzed for connectin by SDS-polyacrylamied gel electrophoresis. Vertebrate striated myofibrils were shown to contain considerable amounts of connectin much largerin molecular weight than myosin heavy chain. On the other hand, no protein bands characteristic of connectin were foud in the striated and smooth myofibrils of scallop adductor or in the striated muscles of lobster tail and cuttlefish mantle. However, a trace of small amount of connectin-like components was present in some invertebrate muscles. Post-mortem changes in connectin of myofibrils were studied using carp white muscle. No significant changes were detected by SDS-gel electrophoresis, whereas a rapid decrease in connectin contents was observed without any detectable cleavage of other myofibrillar proteins by proteases. This decrease was considered to be due to the increased solubility of connectin during post-mortem ageing.

筋肉アルカリ性プロテアーゼ ＶＩＩ かまぼこの火もどりに及ぼすシログチおよびマアジの筋肉アルカリ性プロテアーゼとたんぱく質画分の影響

The purified alkaline protease from the white muscle of horse mackerel, Trachurus japonicus, induced the “Himodori” (the collapse of elasticity) phenomenon during the Kamaboko prepara-tion using the white muscle of horse mackerel and of carp, Cyprinus carpio. However, the purified alkaline protease from the white muscle of white croaker, Argyrosomus argentatus, did not induce the “Himodori” phenomenon during the Kamaboko preparation using the white muscle of white croaker and of carp. A fraction obtained from the white croaker muscle which corresponds to the “Himodori-inducing fraction” of the carp white muscle did not induce the “Himodori” phenomenon in Kamaboko preparation using the white muscle of white croaker. However, a elasticity-reducing material was recognized in another fraction from the white croaker muscle. The “Himodori-inducing proteins” from the white muscle of horse mackerel were also obtained in the corresponding fraction from the carp white muscle.

A comparative study of glycolysis in red and white muscles of the trout (Salmo gairdneri) and mirror carp (Cyprinus carpio)

The activities of some glycolytic and associated enzymes have been determined in the muscles of trout and carp to investigate the possibility that the discrepancies previously reported between lactate accumulation and anoxic tolerance in these two fish result from underlying differences in glycolytic potential. Steady state concentrations of certain glycolytic intermediates were also determined in freeze‐clamped muscles from tankrested fish. The activities of hexokinase, phosphorylase and phosphofructokinase were approximately 2–3 times lower in carp than trout white muscles. Pyruvate kinase and lactate dehydrogenase activities were 5 times lower in carp white muscle. The lower, broader pH optima of lactate dehydrogenase and pyruvate kinase from carp compared to trout muscles is thought to be correlated with the greater anoxic tolerance of the carp. Glycolytic enzyme profiles were markedly different between the red and white muscles of the rainbow trout but broadly similar, with the exception of hexokinase activity, for the corresponding muscles of the carp. The results are discussed in relation to what is known about anaerobiosis in these two species and the comparative physiology of red and white muscles in fish.

Isolation and characterization of the three light chains from carp white muscle myosin.

Myosin from carp white muscle contains two mol of "DTNB" light chain (mol. wt 17 500 daltons) and two mol of "alkali" light chains (mol. wt 25 000 and 16 400 daltons). The three light chains have been isolated in pure state and characterized by electrofocusing, ultraviolet absorption, amino acid analysis and tryptic peptide mapping. Our results show a great homology between the two carp alkali light chains whereas LC2 seems chemically more different. But the homology of LC1 and LC3 is not so extensive as in the case of the higher vertebrate myosin.

Some enzymatic properties of cathepsin a from carp white muscle.

Some enzymatic properties of the highly purifted cathepsin A from white muscle of carp were examined. The optimal pH of the cathepsin A was 4.6 in 0.2M acetate buffer solution. Approximate Km values of the cathepsin A for carbobenzoxy (CBZ)-Glu-Phe, CBZ-Glu-Tyr and CBZ-Gly-Phe were 0.42, 0.75 and 14 mM, respectively, in 0.2M acetate buffer solution, pH 5.0. Acetyl-Phe-Tyr (I2) was hardly hydrolyzed by the enzyme. The thermostability of the enzyme was increased in the presence of NaCl and sucrose. Hg2+, pCMB and diisopropyl fluorophosphates were inhibitory on the enzymatic activity. The inhibition of the enzyme by antipan was competitive foe CBZ-Glu-Tyr, with Ki value of 17 μM. The enzymatic activity was inhibited by all the thiol compounds tested at high concentrations. Some discussions were presented as to whether carp cathepsin A and catheptic carboxypeptidase among cathepsins are the same enzyme or not. In the enzyme assay, the fluorometric method using fluorescamine showed some advantages to the conventional ninnhydrin method in simplicity, rapidity, and sensitivity.

Cellular distribution of sarcoplasmic calcium-binding proteins by immunofluorescence

Specific antibodies against carp paravalbumin, crayfish calcium binding protein and crayfish arginine kinase were used for indirect immunofluorescence localization of the respective proteins. Simultaneous staining of the same muscle sections with human serum containing anti-actin autoantibodies served as a probe to identify the isotropic band. Parvalbumin appears to be evenly distributed in carp white muscle. The crayfish calcium binding protein however shows a distinct localization, in the isotropic band, coincident with the actin staining. Arginine kinase, which has the same molecular weight and is extractible in the same way as the calcium binding protein does not show this distinct localization, but is evenly present in crayfish tail muscle, similarly to parvalbumin. The possible meaning of the different distribution of the two calcium binding proteins is discussed.

Control of energy metabolism in fish white muscle.

Concentrations of key metabolites were determined in carp white muscle before exercise and after maximal activity. It was found that the concentration of ATP decreases by about 65%, ADP decreases slightly, and AMP remains unchanged. Consequently, the level of the free adenylate pool decreases. Simultaneously there is an increase in the concentration of IMP and NH4+. The increase in IMP level and the decrease in adenylate pool are essentially in 1:1 stoichiometry, a result showing that the adenylate pool is decreased by the reaction catalyzed by 5'-AMP deaminase (EC 3.5.4.6.). During exercise there is an increase in levels of glucose-6-phosphate, fructose 6-phosphate, and fructose 1,6-diphosphate that, along with the decrease in ATP levels, can account for the increase in glycolytic flux by activation of phosphofructokinase and pyruvate kinase.

The unanswered question of high anaerobic capabilities of carp white muscle.

Carp white muscle is considered to have a very high anaerobic capability; however, the accumulation of lactate is often not proportional to the amount of energy which must be generated. On the basis of whole-animal studies it is known that some fish produce NH4+ and CO2 anaerobically. This is interesting in light of the concept that many invertebrate facultative anaerobes simultaneously utilize carbohydrates and amino acids with the production of a variety of end products. We tested the hypothesis that in carp white muscle (a) lactate was not the sole end product of anaerobic metabolism and (b) amino acids were utilized as an anaerobic energy source. After an anaerobic work period induced by hypoxia we could detect none of the end products known to build up in invertebrate tissues and no mobilization of the free amino acid pool. Thus, carp white muscle appears to depend solely on glycogenolysis. The low level of lactate in carp white muscle even after severe hypoxic stress may be due to the transfer of this compound out of the tissue despite poor vascularization.

筋肉アルカリ性プロテアーゼ Ｖ かまぼこの“もどり”におよぼすコイの筋肉内アルカリ性プロテアーゼの影響

These investigations were performed to clarify the effects of the alkaline protease purified from white muscle of carp, Cyprinus carpio and its potent inhibitors upon the “Modori” phenomenon in Kamaboko prepared from carp white muscle. The enzymatic activity was not inhibited by chicken egg white, soybean trypsin inhibitor, carp plasma, TLCK or t-AMCHA, but was considerably inhibited by leupeptin, ovomucoid, TPCK, antipain, K3, NEM and NBS. Therefore, the enzymatic properties of this protease seem to be similar to those of α-chymotrypsin. The degree of “Modori” of Kamaboko prepared from the washed muscle was considerably less than that from the unwashed muscle, though about 70% of the enzymatic activity still remained in the washed muscle. This result could be explained by the inhibitory effect of added sodium chloride on the enzymatic activity. However, the “Modori” phenomenon in Kamaboko prepared from the unwashed muscle was not prevented by the addition of enzyme inhibitors such as K3, NEM, or NBS, and only partially by leupeptin. And also the elasticity of Kamaboko prepared from the muscle which was treatd to remove most of the enzyme and sarcoplasmic proteins was not reduced by the addition of the enzyme. From these results it may be concluded that the enzyme in muscle does not take part in the “Modori” phenomenon in Kamaboko production.

Isolation and molecular properties of creatine kinase from carp white muscle

1. 1. Three creatine kinase isoenzymes analogous to the MM, MB and BB forms of enzyme in higher vertebrates, are present in adult carp skeletal muscles, heart and brain. 2. 2. A procedure is described to isolate the MM isoenzyme from carp white muscle. Purity of the enzyme preparations is established from starch gel electrophoresis and ultracentrifugation analysis. 3. 3. A 6-fold purification yields pure enzyme with low catalytic activity compared to that of rabbit muscle enzyme. The validity and implications of this finding are discussed. 4. 4. The molecular weight, diffusion and sedimentation coefficients as well as amino acid composition of carp MM creatine kinase have been determined. 5. 5. Peptide maps of the rabbit and carp MM enzymes are compared. As a first approximation, a maximum of 75% similarity in peptides may be expected. 6. 6. Eight cysteine residues are found on amino acid analysis, two of which are essential to catalytic activity.