Characterization of fast skeletal myosin from white croaker in comparison with that from walleye pollack

ABSTRACT:   Enzymatic and structural properties of white croaker fast skeletal muscle myosin were determined and compared with those of walleye pollack counterpart. Ca²⁺-ATPase activity of white croaker myosin was decreased to approximately 70% of the original activity during 1 day of storage at 0°C and pH 7.0 in 0.5 M KCl and 0.1 mM dithiothreitol, whereas that of walleye pollack was decreased to approximately 20% under the same condition. The activation energy ( E _a) for inactivation of white croaker myosin calculated by the Arrhenius plot for inactivation rate constant (K_D) was 1.2-fold higher than that of walleye pollack. While Ca²⁺-ATPase showed a similar KCl-dependency for the two species, the maximal activity was observed at pH 6.2 and 6.3 for white croaker and walleye pollack, respectively. Actin-activated myosin Mg²⁺-ATPase activity of white croaker was approximately half that of walleye pollack at 0.05 M KCl and pH 7.0, although the two myosins showed a similar affinity to F-actin with K _m of 1.7 and 1.4, respectively. Limited proteolysis with α-chymotrypsin cleaved heat-denatured white croaker myosin mainly at heavy meromyosin/light meromyosin (HMM/LMM) junction, whereas walleye pollack myosin was cleaved at several sites in LMM as well as at the HMM/LMM junction.     

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.     

Isolation of ice-nucleating active bacterium from mackerel and its properties

ABSTRACT: An ice-nucleating bacterium, designated MACK-4, was isolated from ice-stored mackerel ( Scomber australasicus ) and identified as a Pseudomonas fluorescens . The optimal temperature and pH for its growth in nutrient broth with 2.5% glycerol (NB-G) were 15°C and 6.5, respectively. The maximal ice-nucleating activity (INA) was obtained after 54 h incubation at 15°C. However, the INA was almost completely lost after 48 h incubation at 25°C or higher. The growth and INA decreased with increase of NaCl added in NB-G within 0.0–4.0%. The INA of MACK-4 was very stable at 5–25°C, pH 4.0–9.5, while that of isolated ice-nucleating matter from MACK-4 was stable at 5–25°C, pH 5.5–9.0.     

Changes in enzymatic and structural properties of grass carp fast skeletal myosin induced by the laboratory-conditioned thermal acclimation and seasonal acclimatization

ABSTRACT:   Myosins were prepared from fast skeletal muscles of grass carp thermally acclimated to 10, 20 and 30°C in the laboratory as well as from those seasonally acclimatized and collected in January (winter) 2003 and May (spring), August (summer) and November (autumn) 2002. The maximal initial velocities ( V _max) of actin-activated Mg²⁺-ATPase activity for myosins from the 10°C-acclimated and winter grass carp were 1.7–1.8-fold as high as those from the 30°C-acclimated and summer fish. The inactivation rate constant ( K _D) of Ca²⁺-ATPase for myosin from the 10°C-acclimated grass carp was three to fourfold higher than those for myosins from the fish acclimated to 20°C and 30°C, whereas myosin from winter grass carp was about sevenfold as high as that for myosin from summer fish. Myosins from spring and autumn fish showed K _D values comparable to those of the fish acclimated to 30°C and 10°C, respectively. In differential scanning calorimetry analysis, the transition temperature ( T _m) was observed near 38°C and 45–46°C with most myosins. However, the lowest T _m at 32–33°C was given as one of the major endotherms in myosins from the 10°C-acclimated, autumn and winter fish. These responses of grass carp to changed environmental temperatures were almost similar to those for common carp reported previously.     

Degradation of myofibrils in cultured yellowtail <Emphasis Type="Italic">Seriola quinqueradiata</Emphasis> burnt meat: effects of a myofibril-bound EDTA-sensitive protease

“Burnt meat” is a term used to describe the white (pale, grainy, exudative) muscle of yellowtail or tuna. It (with lightness parameter, L* ≥ 55) was observed after 2 h storage in the suffocate in air (SA) 29°C group and after 4 h storage in the spinal cord destruction (SCD) 29°C group. In the SA 17°C group, burnt meat was also observed after 4 h storage. In contrast, the meat in the SCD 17°C group was normal until after 12 h storage. The myosin heavy chain (MHC) was more degraded than the other myofibrillar proteins, and some protein bands increased in the burnt meat. The protease that leads to the degradation of MHC was investigated using myofibrils from the meat. EDTA completely suppressed the degradation, indicating that a myofibril-bound EDTA-sensitive protease (MBESP) may exist in yellowtail muscle and this caused the degradation of MHC. The optimum pH and temperature of MBESP in yellowtail were 5.0 and 50–60°C, respectively.     

Inhibiting effect of polymerization and degradation of myosin heavy chain during preheating at 30°C and 50°C on the gel-forming ability of walleye pollack surimi

ABSTRACT: To confirm the contribution of polymerization and degradation of myosin heavy chain (MHC) during preheating to the gel-forming ability of fish meat paste, walleye pollack surimi paste was preheated at 30°C and 50°C prior to heating at 80°C in the presence of various inhibitors. At 30°C, ethyleneglycol bis(2-aminoethyl ether) -N,N,N ', N '-tetraacetic acid (EGTA) and ethylenediaminetetraacitic acid (EDTA) inhibited gel formation as well as the polymerization of MHC, whereas dithiothreitol (DTT) and leupeptin promoted gel formation, which was accompanied by the enhancement of MHC polymerization and decreased MHC degradation, respectively. At 50°C, leupeptin inhibited MHC degradation and improved gel strength, whereas EGTA, EDTA and DTT had no effect on MHC polymerization and degradation and did not affect gel formation. The results demonstrate that the gel strength of cooked gel (80°C) is not affected by preheating at 30°C and 50°C and does not inhibit polymerization and degradation. Results suggest that the gel strength of cooked gel is dependent on the polymerization and degradation of MHC during preheating.     

鲤肌肉肌原纤维结合型丝氨酸蛋白酶的分子克隆

肌原纤维结合型丝氨酸蛋白酶(myofibril-bound serine proteinase,MBSP)是最近发现的一种蛋白酶。该酶参与肌原纤维蛋白的降解及鱼糜制品弹性的下降。但是,对该酶一级结构的研究,迄今为止,未有报道。本文根据已测定的鲤MBSP N-末端氨基酸序列以及丝氨酸蛋白酶活性中心保守序列设计兼并引物,结合RT-PCR技术实现了MBSP基因片段的扩增。再根据克隆到的MBSP片段序列设计基因特异引物,用于MBSP基因的5′和3′末端快速扩增。综合以上结果,鲤MBSP的全长被确定。序列分析表明,MBSP cDNA含有一732 bp的开放阅读框,编码243个氨基酸残基,其中信号肽长度为21个氨基酸残基。组成丝氨酸蛋白酶活性中心的氨基酸残基(His61,Asp107和Ser197)在MBSP中保守存在。成熟MBSP含有222个氨基酸残基,分子量为24.5 ku,比其天然蛋白的分子量30 ku略小。成熟MBSP的等电点为10.43。鲤MBSP与鲫MBSP,猪胰蛋白酶,牛胰蛋白酶,美洲鲽胰蛋白酶的同源性分别为80.6%,55.8%,55.3%和53.9%。而与仓鼠肌肉中具有胰凝乳蛋白酶性质的蛋白酶的同源性为39.2%。MBSP有高含量的赖氨酸残基(11.93%),此特性可能与该酶的肌原纤维结合特性有关。     

Properties of extracellular ice-nucleating substances from <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> MACK-4 and its effect on the freezing of some food materials

ABSTRACT:   Pseudomonas fluorescens MACK-4, isolated from mackerel surface, has an ice-nucleating activity (INA). In addition to bacterial cells, this strain could also produce an extracellular ice-nucleating substance (INS) with a maximal INA at pH 6.0. The extracellular INS (EINS) was stable at pH 6–9 during 1-h incubation with 3–5% of saccharides including maltose, trehalose and sucrose at 15°C. However, glycerol dramatically lowered the INA in both bacterial cells and the EINS. The addition of either the EINS or bacterial cells significantly elevated the ice-nucleating temperatures of pure water, full-cream milk, and 10% starch solution, but not orange juice and mackerel mince. The EINS produced from this strain could serve as crystal nuclei and accelerate ice crystallization during freezing.     

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.     

Temperature and salinity tolerances of yellowfin sea bream, Acanthopagrus latus, at different salinity and temperature levels

Abstract Yellowfin sea bream Acanthopagrus latus (Houttuyn) (0.98 ± 0.27 g) were examined for temperature and salinity tolerances at nine different environmental regimes (0.3, 15 and 33 ppt combined with 10, 25 and 32 °C), in which the fish were subjected to gradual and sudden changes in temperature and salinity respectively. The 50% CTMax (critical thermal maximum) and the UILT (upper incipient lethal temperature) were in the range of 34.8–38.2 °C and 32.8–36.4 °C respectively. The 50% CTMin (critical thermal minimum) and the LILT (lower incipient lethal temperature) were in the ranges of 4.9–9.4 °C and 7.1–17.6 °C respectively. The 50% CSMax (critical salinity maximum) and the UILS (upper incipient lethal salinity) were in the ranges of 54–69‰ and 9–44‰, respectively. The fish at lower temperature (10 °C) and salinity of 33‰ tolerated temperature as low as 6 °C when the temperature was decreased gradually. The fish at 33 and 15‰ and lower temperature (10 °C) tolerated salinity as high as 66–67‰, whereas those at freshwater salinity level (0.3‰) and 32 °C tolerated salinity to 50‰ when salinity was increased gradually. The relationships among UILT, LILT, 50% CTMax, 50% CTMin, UILS, 50% CSMax, salinity and temperature are given.     

Growth of the fourspine sculpin <Emphasis Type="Italic">Cottus kazika</Emphasis> in the Gonokawa River,Japan, and effects of water temperature on growth

ABSTRACT:   The fourspine sculpin Cottus kazika is indigenous to Japan and found in Honshu except for the waters facing the Seto Inland Sea, and was also found in southern Shikoku and eastern Kyushu. This species has a catadromous lifestyle and migrates as juveniles from the sea to the middle reaches of rivers to grow. The growth pattern of this fish was investigated by a mark-and-recapture method from July 1994 to December 1996, in the Nigorikawa River, a tributary of the Gonokawa River system, Shimane Prefecture. 0-year-old fish of 50–70 mm total length ( TL ) occurred in the study area from June to July, grew to 90–140 mm  TL by the following April, and attained 160–210 mm  TL by December. This fish grew rapidly in September–November and April–July, almost ceasing to grow in July–September. It seems that this stagnant growth phase in summer is a characteristic of the seasonal growth pattern of C. kazika . A rearing experiment indicated that the growth rate of C. kazika was higher at 16–22°C than at 12–14 and 24–26°C. This result supports the field evidence of a stagnant growth phase in summer in the Nigorikawa River.     

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.     

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.     

Improved solubility and stability of carp myosin by conjugation with alginate oligosaccharide

ABSTRACT:   Carp myosin was conjugated with alginate oligosaccharide (AO) through the Maillard reaction under low relative humidity, and the functional properties of the myosin-AO conjugate were investigated to clarify the role of myosin in the functional improvement of fish myofibrillar proteins (Mf) by the glycosylation. The findings were as follows. First, myosin became highly solubilized at lower NaCl concentrations by conjugation with AO and NaCl-dependence of the solubility was lost when > 12% of the available lysine residues were reacted with AO and 50 µg/mg of AO was attached to myosin. Second, the thermal stability of myosin was effectively improved by conjugation with AO. Heat-treatment at 50°C for 6 h has no effect on the solubility of the myosin-AO conjugate regardless of the NaCl concentration. Third, the improved functionalities of myosin conjugated with AO remained even at a nearly isoelectric point. The improving effect of AO-conjugation on the characteristics of myosin was almost the same as Mf reacted with AO. Therefore, it is apparent that that improved functionalities of the glycosylated Mf reflect the functional changes of myosin.     

Purification and characterization of lysozyme from purple washington clam Saxidomus purpurata

ABSTRACT:   Lysozyme was purified from purple washington clam Saxidomus purpurata by sequential procedures using Chitopearl Basic BL-01 affinity and TSKgel ODS-120T column chromatographies. Molecular mass of the purified enzyme was estimated to be 12 kDa by SDS-PAGE. Optimum pH of the enzyme was 5.2 toward Micrococcus lysodeikticus cells. The optimum temperature was 50°C. The enzyme was stable in the range of pH 4.8–6.8 and 20–90°C. Further, the N-terminal amino acid sequence of the enzyme showed similarity to lysozymes from invertebrates. However, the specific activity of the enzyme toward M. lysodeikticus cells and p -nitrophenyl penta- N -acetyl- β -chitopentaoside was 143 times and 12 times higher than that of hen egg white lysozyme, respectively.     

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.     

Effects of fish size and water temperature on the acute toxicity of boron to Japanese flounder <Emphasis Type="Italic">Paralichthys olivaceus</Emphasis> and red sea bream <Emphasis Type="Italic">Pagrus major</Emphasis>

ABSTRACT:     The acute toxicities of boron were examined for Japanese flounder Paralichthys olivaceus and red sea bream Pagrus major in terms of fish size and water temperature. Japanese flounder of 0.1–70.0 g and red sea bream of 0.6–20.3 g were exposed to different concentrations of boron for 96 h at 20°C under semi-static conditions. In both fish species, the median lethal concentration (LC₅₀) for 96 h of boron increased linearly with increasing fish size, ranging from 108 to 252 mg B/L for the flounder, and from 97 to 172 mg B/L for the sea bream. The effect of water temperature on acute toxicity was examined for Japanese flounder of 0.6 and 1.5 g at 10, 15, 20 and 25°C, and for red sea bream of 0.6 and 2.4 g at 12, 15, 20 and 25°C. The toxicity of boron for the flounder increased linearly with increasing water temperature. The 96 h LC₅₀ values ranged from 299 to 108 mg B/L for the 0.6 g flounder and from 350 to 113 mg B/L for the 1.5 g flounder. A similar trend was shown for the 2.4 g red sea bream; however, the relationship for the 0.6 g red sea bream was not significant.     

A comparative study of the effects of high pressure on proteolytic degradation of sardine and blue whiting muscle

ABSTRACT:   High-pressure technology is used as an alternative to heat processing because of its inactivating effect on microorganisms and enzymes. However, it can also alter the structure of other muscle proteins. The present study compares the effects of high pressure (300 MPa, 7°C, 20 min) on the proteolytic degradation and alterations in the myofibrillar proteins of sardine and blue whiting muscle. Also, muscle homogenates and enzyme extracts were pressurized in order to evaluate the high-pressure effects on unprotected proteolytic enzymes outside the whole muscle structure. Peak proteolytic activity was found to occur at 55°C in both species. The peak activity pH was pH 3 for the sardine and pH 8 for the blue whiting; the main enzyme families being aspartic proteases in the former and alkaline serine proteases in the latter. Pressurization lowered activity levels at the peak activity pH and temperature in the fish muscle (by 30.8% in the sardine and by 9.5% in the blue whiting) and also slightly in the enzyme extracts (by 16.8% in the sardine and by 19.4% in the blue whiting). The electrophoretic profiles disclosed higher protein degradation in the pressurized muscle. Overall, the observed changes in proteolytic activity can be attributed not only to the effect of high pressure on the enzymes, but also and mainly, to the effect on other muscle proteins.     

Effect of Chinese Rice Wine on the Endogenous Protease Activity,Myofibrillar Degradation,and Quality Characteristics in Topmouth Culter (Culter alburnus)

ABSTRACT

The aim of this study was to investigate the influence of rice wine (Jinbiao, Yinbiao, Wunianchen, Nv’erhong) on the endogenous protease activity, myofibrillar degradation, and quality characteristics of topmouth culter (Culter alburnus). Rice wine had a pH of 4.3 and calcium ions ranging from 89.30 to 123.70 μg/mL. Nv’erhong rice wine had the highest total phenolic content of 583.38 μg gallic acid equivalent (GAE)/mL, while Wunianchen rice wine (WNC) showed the strongest total antioxidant capacity of 87.29 U/mL. WNC presented the optimum inhibitory effect on endogenous proteases of calpain and cathepsin B, D, and L, delaying the degradation of myosin heavy chain and α-actinin, which are important to muscle softening. The quality characteristic evaluation indicated that the addition of rice wine could maintain the hardness and chewiness, increase the springiness of the fish fillet, as well as inhibit the total volatile basic nitrogen production. These results suggest that rice wine had effects of inhibiting endogenous proteases, delaying myofibrillar degradation, and maintaining the sensory quality of topmouth culter.