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.     

Stabilization of myosin by ionic compounds as affected by F-actin

ABSTRACT:   Suppressive effects of non-ionic (sorbitol, maltose, and trehalose) and ionic (Na-glutamate, Na-acetate, Na-sulfate, and ammonium sulfate) compounds on the thermal inactivation of myosin subframgent-1 (S-1) and myofibril Ca²⁺-ATPase were compared. All compounds suppressed S-1 denaturation. When myofibrils were used (at 0.1 M KCl), sugars and sugar alcohol (non-ionic compounds) suppressed denaturation similar to S-1, while Na-glutamate, Na-acetate, and Na-sulfate weakly suppressed them. Ammonium sulfate accelerated denaturation, but suppressed denaturation when heated in 2 M KCl, at which myosin lost protection by F-actin. It was thus concluded that ionic compounds affected the denaturation of myofibrils in two ways; suppression as established with S-1, and acceleration as a result of loss of protection by F-actin caused by increase in ionic strength.     

Myosin denaturation in heated myofibrils of scallop adductor muscle

The thermal inactivation of Ca²⁺ ATPase of scallop myofibrils (0.1 M KCl, pH 7.5) was found to be unaffected by the presence of Ca²⁺. Monomeric myosin content and salt solubility decreased much faster than Ca²⁺ ATPase inactivation in both Ca and EDTA media, which was well explained by faster denaturation of the rod portion than subfragment-1 of myosin. In contrast, when the myofibrils were heated at 0.5 M KCl, a slow decrease in salt solubility was observed, which was also explained by slow denaturation of the rod portion of myosin. Myofibrils from scallop smooth muscle showed the same denaturation pattern as those from adductor muscle. These results show that mollusk myosin is not always stabilized by Ca²⁺.     

Thermal denaturation profiles of catfish and tilapia myofibrils as affected by pH for heating

Thermal denaturation profiles of catfish myosin when heated as myofibrils (Mf) were compared with those of tilapia. The Ca²⁺-ATPase inactivation rate of catfish myofibrils was the same as that of tilapia myofibrils. The conclusion was the same with isolated myosin. Catfish Mf was clearly distinguished from tilapia Mf in terms of subfragment-1 (S-1) and rod denaturation. Quick denaturation of rod relative to S-1 was characteristic of catfish Mf, while slower denaturation of rod relative to S-1 was the pattern for tilapia Mf. These patterns were greatly affected by the pH for heating. Rod denaturation was accelerated with increasing pH for heating and oppositely suppressed by lowering the pH, for both Mf. Tilapia Mf showed a S-1 and rod denaturation pattern similar to that for catfish Mf, but at 1 pH unit higher; for example, the pattern of catfish Mf at pH 7.5 was similar to that for tilapia Mf at pH 8.5. Less rigid filament structure of catfish Mf than tilapia Mf was demonstrated by studying chymotryptic digestion at various pH values. Accordingly, the difference in the S-1/rod denaturation patterns between the two fish species can be explained by the different rigidity of their myosin filaments.     

Species-specific thermal denaturation pattern of fish myosin when heated as myofibrils as studied by myosin subfragment-1 and rod denaturation rates

Thermal denaturation of myofibrils from various species of fish was investigated by measuring ATPase inactivation, myosin aggregation, myosin subfragment-1 (S-1) and rod denaturation rates as studied by chymotryptic digestion. Decrease in monomeric myosin (myosin aggregation) was always faster than the ATPase inactivation for all myofibrils tested. The relative denaturation rate of rod to that of S-1 differed from species to species. Preceded denaturation of rod was observed with some species, and the opposite was true with other species. The denaturation pattern was explained by the different magnitude of S-1 stabilization by F-actin in myofibrils at low salt medium. Myofibrils which receive a great stabilization by F-actin as studied by ATPase inactivation showed the preceded rod denaturation pattern, and vice versa. S-1 portion, not F-actin, determined the different stabilization of S-1 by F-actin in myofibrils.     

Suppressing effect of neocarrabiose 4-O-sulfate on thermal and freeze denaturation of myosin subfragment-1 and myofibrils

Suppressive effects of neocarrabiose 4-O-sulfate on the thermal and freeze denaturation of myosin subfragment-1 (S-1) and on myofibrils were investigated. The compound strongly suppressed the thermal denaturation of S-1. Its suppressive effect was greater than that of sorbitol and similar to that of maltose. However, it tended to accelerate the denaturation of myofibrils, suggesting a loss of protection by F-actin upon its addition. The compound suppressed the freeze denaturation of myofibrils and S-1. The effect was similar to that of sorbitol or maltose, and completely different from that of Na-sulfate. The compound solubilized myofibrils at concentrations similar to KCl. Therefore, it was concluded that neocarrabiose 4-O-sulfate behaved as an ionic salt in the thermal treatment process, whereas it behaved as a sugar in the frozen storage process.     

TMAOase, trimethylamine-N-oxide demethylase, isa thermostable and active enzyme at 80°C

ABSTRACT:   Purified trimethylamine- N -oxide demethylase (TMAOase)from walleye pollack muscle is a thermostable protein that was notinactivated after heating at 80°C for 30 min.The heated enzyme was electrophoresed in the same manner as fornative enzyme. Circular dichroism (CD) spectra for purified enzymechanged reversibly in the temperature range of 10–80°C.As the enzyme was still active at 80°C, the CD spectralchange did not directly relate to enzyme activity. TMAOase activity inthe myofibrillar fraction decreased sharply above 30°C,but was extracted and recovered from the heated myofibrillar fraction,suggesting that the activity seemed to be interrupted and apparently inactivateddue to the thermal alteration of myofibrillar proteins or some unknownfactors. The complicated profile found in dimethylamine (DMA) formationfrom trimethylamine- N -oxide (TMAO) in walleye pollack muscleduring heating consisted of both enzymic and non-enzymic processes.Most DMA was produced enzymatically below 40°C and interruptedabove 40°C. Therefore, DMA and trimethylamine was formednon-enzymatically at high temperatures regardless of the presenceof native enzyme. A new, simple and easy purification method wasproposed based on the thermostable nature of the enzyme.     

Effect of calcium ion on the thermal denaturation of subfragment-1 and rod regions of squid myosin upon the heating of myofibrils

Thermal inactivation of Ca²⁺ ATPase of squid myofibrils was significantly suppressed in the presence of Ca²⁺. Monomeric myosin content decreased much faster than Ca²⁺ ATPase inactivation in Ca medium, which was well explained by fast rod denaturation. In contrast, rod denaturation was slower than S-1 in EDTA medium. The decrease in monomeric myosin content was explained by faster S-1 denaturation. Comparing the S-1 and rod denaturation rates at a fixed temperature, it was concluded that S-1 denaturation was suppressed by Ca²⁺ whereas the rod denaturation was not. An unfolding experiment with isolated myosin rod confirmed that there was no stabilizing effect of Ca²⁺ on myosin rod. It was concluded that significant stabilization of the S-1 portion by Ca²⁺ generated the apparently different myosin denaturation patterns in the two media.     

Total activity of transglutaminase at various temperatures in several fish meats

ABSTRACT: Transglutaminase seems to be related to the setting phenomenon of fish meat paste that occurs at temperatures below 40°C. In many reports on the relationship between transglutaminase and setting phenomenon, the enzyme activity has been measured at 25°C. However, it is known that the setting phenomenon is complicated and the effect of calcium and the 3-D structure of myofibrils, which are sensitive to temperature, play important roles in the reaction. In the present study, total activities of transglutaminase of threadfin bream, white croaker, red sea bream and carp meats were measured at various temperatures. Total transglutaminase activities at 25°C of tested fish meats are arranged in order as follows; white croaker, red sea bream, carp, threadfin bream. In contrast, optimal temperature of carp meat is 30°C, red sea bream 40°C, threadfin bream 50°C and white croaker 50–55°C. In carp meat, the enzyme activity at optimal temperature (30°C) became 8.5-fold higher than at 25°C. The data of the total activity at various temperatures are useful in order to comprehend the details of the reaction.     

Different effects of ionic and non-ionic compounds on the freeze denaturation of myofibrils and myosin subfragment-1

The effects of non-ionic (sorbitol, maltose, trehalose) and ionic compounds (Na-glutamate, Na-acetate, Na-sulfate, ammonium sulfate) on freeze denaturation of myosin subfragment-1 (S-1) and of myofibrils were compared. Sugars, Na-glutamate and Na-acetate well suppressed the freeze denaturation of myofibrils as well as S-1 in a concentration dependent manner. Although sulfate suppressed freeze denaturation of S-1 irregularly, it accelerated myofibril denaturation. It was concluded that sulfate salts were useless as cryoprotectant for myofibrils. Stabilization extent by F-actin in frozen storage was much less than that in heating.     

Characteristics of burnt meat in cultured yellowtail <Emphasis Type="Italic">Seriola quinqueradiata</Emphasis>

ABSTRACT:   In order to understand the characteristics of burnt meat in cultured yellowtail Seriola quinqueradiata , fish were kept at two different temperatures (13 and 30°C) and slaughtered by either spinal cord destruction (SCD) or suffocation in air (SA). Early postmortem changes during storage at 32°C were analyzed by rheological, biochemical, and histological methods. The burnt meat (with lightness parameter, L* ≥ 55) was observed at 1-h storage in the SA 30°C group, at 2 h in SCD 30°C, and at 4 h in SA 13°C; meat was normal for the SCD 13°C group until 6 h of storage. Breaking strength scores were higher for the normal meat (200 g/cm²) than burnt meat (70 g/cm²) at 4 h of storage. Expressible water content was higher for the burnt meat than for the normal meat. Adenosine triphosphate concentrations for the SCD groups were higher than for the SA counterparts. Moreover, pH decrease was much faster in the 30°C groups, showing pH 5.6 at 2 h of storage. A negative correlation between the pH and lactic acid contents in muscle ( P  < 0.001) was found. Histological analysis evidenced a larger pericellular area (40%) in the burnt samples than in the normal samples (16%). It was confirmed that a higher fish-keeping water temperature and a stressful slaughter method (faster glycolytic process) were determinative factors that influence the occurrence of burnt muscle in yellowtail, and that the effect of the former is larger than the latter.     

Heat treatment affects protein quality and protease activity in decapsulated cysts of Artemia when used as starter food for larvae of African catfish Clarias gariepinus (Burchell)

Decapsulated cysts of Artemia subjected to different heat treatments (40, 60, 80 and 96 °C) were fed to African catfish Clarias gariepinus larvae. Heated cysts, untreated cysts and live Artemia nauplii as control constituted the experimental diets. Protein denaturation and solubility, total alkaline protease and specific trypsin activities in the cyst diets were evaluated. The growth of catfish larvae and the proteolytic activity of larval samples during development were also determined. Heat treatment of cysts increased protein denaturation and decreased protein solubility. The protease activity in the cyst diets decreased with higher heating temperatures. The growth of catfish larvae differed according to the diet; higher fish growth was achieved with nauplii and cysts heated at 40 °C. The digestive enzyme activity in larval samples remained similar in all dietary treatments during larval development. The quality of food protein and the way this protein is processed might be more important for successful larval growth than exogenous enzyme supply.     

Effects of constant and cyclical thermal regimes on growth and feeding of juvenile cutthroat trout of variable sizes

Abstract –  The effects of constant (12, 18, and 24 °C) and cyclical (daily variation of 15–21 and 12–24 °C) thermal regimes on the growth and feeding of Lahontan cutthroat trout ( Oncorhynchus clarki henshawi ) of variable sizes were examined. Higher constant temperatures (i.e., 24 °C) and more variable daily temperatures (i.e., 12–24 °C daily cycle) negatively affected growth rates. As fish mass increased (from 0.24 to 15.52 g) the effects of different thermal regimes on mass growth became more pronounced. Following 14 days exposure to the thermal regimes, feeding rates of individual fish were assessed during acute exposure (40 min) to test temperatures of 12, 18, and 24 °C. Feeding rate was depressed during acute exposure to 24 °C, but was not significantly affected by the preceding thermal regime. Our results indicate that even brief daily exposure to higher temperatures (e.g., 24 °C) can have considerable sublethal effects on cutthroat trout, and that fish size should be considered when examining the effects of temperature.     

Myosin and actin denaturation in frozen stored kuruma prawn Marsupenaeus japonicus myofibrils

Myosin and actin denaturation in kuruma prawn myofibrils stored frozen (0.1 M NaCl, pH 7.5) at ?20 °C was investigated. The inactivation profile of Ca²⁺-ATPase in the myofibrils was identical to that for myosin, indicating that myosin in myofibrils was not protected by actin. The presence of myosin detached from actin in the soluble fraction was proven by ammonium sulfate fractionation in the absence and presence of Mg-ATP. Actin denaturation in myofibrils was further confirmed by its increased susceptibility to chymotryptic degradation. In the frozen myofibrils, actin denatured more rapidly quicker than myosin: actin had completely denatured by storage day 1, followed by a gradual denaturation of myosin. Both myosin and actin in the frozen stored myofibrils retained their high salt-solubility, which decreased slowly during the frozen storage period. The presence of aggregated inactivated myosin in the salt-soluble fraction was proven by precipitation at 40 % saturation of ammonium sulfate in the presence of Mg-ATP, leaving active monomeric myosin in the soluble fraction. Almost no actin denaturation was observed with heated myofibrils.     

Stabilizing effect of Ca<Superscript>2+</Superscript> on myosin and myofibrils of squid mantle muscle as affected by heating conditions

The suppressive effects of Ca²⁺ on the thermal denaturation of myosin and myofibrils of squid mantle muscle were compared. The stabilization effect on myosin was smaller than that on myofibrils and was not affected by KCl concentration. The stabilizing effect of Ca²⁺ on myosin decreased as the heating temperature dropped, showing no stabilization at 20°C, while the effect on myofibrils was the same at all temperatures tested. The stabilizing effect of Ca²⁺ on myosin disappeared even at 30°C in the presence of sorbitol, where a small inactivation rate was found, while the effect of Ca²⁺ on myofibrils was equally detected irrespective of the reduction in inactivation rate in the presence of sorbitol. Stabilization of myosin by Ca²⁺ again appeared even in the presence of sorbitol when the heating temperature was raised to 38°C. It was suggested that Ca²⁺ confers stabilization on myosin only when myosin is under unstable conditions. The stabilization effect of Ca²⁺ on myosin was enhanced upon F-actin binding: Ca²⁺-bound myosin was more significantly stabilized by F-actin binding, and the effect was no longer affected by the conditions for heating.     

Suppression of fish meat softening by strict control of storage temperature

ABSTRACT:   Changes in meat firmness and structure of cultured yellowtail at several storage temperatures, from −1.5°C to 10°C, were compared. Firmness decreased less at −1.5°C and 10°C than at the other temperatures. During storage at temperatures from −0.5 to 4.0°C, expansion of intercellular space, which indicates weakening of the connecting force between muscle cells, began to increase after 8 h storage, and the ratio of space to the whole area increased by 3.4–4.9 times after 24 h of storage. However, at −1.5°C and 10°C, the increase of intercellular space was suppressed, and the ratio was 2.4–2.6 times, even after 24 h storage. Collagen fibrils connecting muscle cells disintegrated after 24 h storage at all storage temperatures. Conversely, in 10°C storage, the fine structure of muscle, except for collagen fibrils, was maintained better than at other storage temperatures. These findings indicate that −1.5 and 10°C storage could maintain fish-meat quality for longer periods than storage at −0.5, 1.0, or 4.0°C, the normal refrigeration temperature.     

Effects of dietary protein and lipid level, and water temperature, on the post-feeding oxygen consumption of Atlantic cod and haddock

Tank respirometry was used to study the interactive effects of protein:lipid level (55%:11% vs. 42%:16%; both diets isoenergetic) and temperature (11, 6 and 2 °C) on the magnitude and duration of specific dynamic action (SDA) in juvenile Atlantic cod and haddock. The protein:lipid level did not affect any measured variable. However, numerous temperature and species effects were observed. For example, although the maximum post-feeding oxygen consumption (30–50% above routine metabolic rate; RMR) and SDA duration (∼55–85 h; SDA_DUR) were not affected by temperature, SDA_DUR g⁻¹ of food increased from 11 to 2 °C (from ∼3 to 12 h g food⁻¹). While absolute SDA (mg O₂) decreased by ∼60–65% in cod and ∼75% in haddock from 11 to 2 °C, due to a concomitant decrease in food consumption from ∼2.0% to 0.6% body mass, SDA comprised between 3.3% and 5.2% of the dietary energy content at all temperatures. Finally, RMR at 11 and 2 °C and SDA_DUR at 2 °C were 25–35% and 25% greater in cod, respectively, as compared with haddock. These results suggest that feeding reduced protein diets at low water temperatures is unlikely to improve the growth of these species.     

Effects of hormone treatments and temperature on sex-reversal of Nigorobuna Carassius carassius grandoculis

ABSTRACT: Functional sex reversal of all-female nigorobuna Carassius carassius grandoculis to phenotypic males was examined by immersion exposure of fry to 17-methyltestosterone (17-MT) and controlled water temperature during early development. Fry were reared in water containing different concentrations of 17-MT at 24 and 30°C for 80 days starting 20 days after hatching. Although the fish exposed to 0.1 and 1.0 μg/L 17-MT at 24°C were all male, treatment with 10.0 μg/L 17-MT resulted in 43% females. Twenty-two percent males appeared in the control treatment at 30°C but the control at 24°C was entirely female. The proportion of males in treatments exposed to 0.01– 1.0 μg/L 17-MT at 30°C was slightly lower than in the respective treatments at 24°C. These results indicate that the phenotypic expression of sex in nigorobuna is thermolabile and that sex determination is under the control of genetic factors and temperature. Also, control of temperature during early development has been shown to be important for the production of all-female offspring for use as breeding stock for pond culture of fish suitable for preparation as 'funazushi'.     

Growth and mortality of common octopus Octopus vulgaris reared at different stocking densities in Mediterranean offshore cages

Four ongrowing cycles of common octopus ( Octopus vulgaris ), each with two stocking densities, were carried out in an offshore area in order to evaluate the effect of the following variables on growth ( G , % body weight day⁻¹) and mortality ( M , % day⁻¹): weight ( W , 0.5–3.0 kg), temperature ( T , 14–26 °C), stocking density (SD, 6–46 kg m⁻³), size dispersion, expressed as coefficient of variation (CV, 13–42%) and significant wave height (SWH: 0.4–1.2 m). The assays were performed in an 8 m³ stainless-steel floating cage divided into two compartments of 4 m³ each. For the range of temperatures considered (14–26 °C), G depended significantly on T and SWH, with maximum G values being obtained at 18.5 °C and with an important negative effect of SWH. M depended significantly on T, W and CV, mortality being minimal at 18 °C. The two other variables had an antagonistic effect, mortality increasing with greater size dispersion – suggesting that animals should be graded throughout the process – although the effect diminished as the sizes increased. The results point towards two alternatives for the commercial ongrowing of octopus under the particular conditions of the present study: (A) two ongrowing cycles of 3.5 months to reach a final weight of 2.5 kg or (B) one 5-month cycle to reach a final weight of 3.5 kg.     

Effect of temperature and cell density on ethanol fermentation by a thermotolerant aquatic yeast strain isolated from a hot spring environment

ABSTRACT: A thermotolerant, fermentative yeast strain named RND13 from a hot spring drainage was evaluated for its ethanol-producing ability at elevated temperatures at a high substrate concentration [15% (w/v) glucose] close to the level reflecting industrial practice. The RND13 was capable of utilizing glucose almost completely at 40°C with increasing inoculum size, producing ethanol up to 6.6% (w/v), which is comparable to levels (7.0–7.2%) at 30°C. The maximum rate of ethanol production by the RND13 was found to be 9.0 g/L per h at 40°C in an inoculum sized 5% (w/v). At 43°C, however, the RND13 could not utilize glucose to completion and showed a slight drop in the extent of produced ethanol [6.0% (w/v)]. Thus, the culture at 40°C with a 5% cell inoculum was considered to be the optimal condition for ethanol production at higher temperatures in terms of batch fermentation. In the phylogenetic analysis based on the small-subunit rDNA sequence, the strain was grouped together with both Candida glabrata and Kluyveromyces delphensis , which are relatively close to Saccharomyces cerevisiae .