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1.
ABSTRACT: Nucleotides and Ca2+ binding to α-actin prepared from ordinary skeletal muscle of carp Cyprinus carpio was studied. When bound Ca2+ was removed with ethylenediaminetetraacetic acid, carp α-actin denatured more rapidly than chicken α-actin. Kinetic studies of the denaturation process showed that in the absence of divalent cations, the binding constants of ATP to carp and chicken actin were 5.0 × 104 /M and 1.2 × 105 /M, respectively. Competitive binding of Ca2+ between actin and 8-amino-2-[(2-amino-5-methylphenoxy)methyl]-6-methoxyquinoline-N,N,N',N'-tetraacetic acid (Quin 2) showed that affinity of Ca2+ for carp actin was also lower than that for chicken actin by a factor of 1.6. These results indicated that carp actin could relatively easily denature due to the low affinities of these ligands. Enthalpy changes upon ATP binding to carp and chicken actin were −65 kJ/mol and −110 kJ/mol, respectively. Thermodynamic analyses of our results revealed that the entropy change associated with ATP binding to carp actin was significantly smaller than that to chicken actin, suggesting that structural stabilization upon ATP binding was less effective in carp actin. 相似文献
2.
Reduction in the IgE reactivity of Pacific mackerel parvalbumin by mutations at Ca2+-binding sites 总被引：1，自引：0，他引：1
ABSTRACT: Parvalbumin is a sarcoplasmic Ca2+ -binding protein of 12 kDa and represents the major fish allergen. Several peptide segments are identified as immunoglobulin E (IgE)-binding epitopes of cod parvalbumin. However, carp parvalbumin (Cyp c 1) shows a markedly reduced IgE-binding ability upon depletion of Ca2+ , suggesting the importance of conformational epitopes associated with Ca2+ -chelating. In this study, the IgE reactivity of Pacific mackerel Scomber japonicus parvalbumin (Sco j 1) was demonstrated to be markedly reduced (60–100% reduction) by Ca2+ -depletion, similar to Cyp c 1. Three Sco j 1 mutants (D51A, D90A, D51/90A), with modifications in either one or both of the two Ca2+ -binding sites, were then constructed by site-directed mutagenesis, followed by expression in Escherichia coli , and evaluated for their IgE reactivity. Interestingly, the double mutant (D51/90A), probably devoid of Ca2+ -binding capacity, exhibited a significantly reduced IgE reactivity (equivalent to 0.0–7.5% of the IgE reactivity of natural Sco j 1). The results suggest that the IgE-binding ability of Sco j 1 largely depends on the solid conformation mediated by Ca2+ -chelating, and that the hypoallergenic D51/90A will be a useful tool for the specific immunotherapy of fish allergy. 相似文献
3.
ABSTRACT: This study incorporated the 43 kDa Zn-binding membrane protein isolated from common carp into liposome. The specificity and strength of the binding of 65 Zn to the 43 kDa protein-liposomes, and the binding of the 65 Zn-labeled 43 kDa protein-liposomes to laminin were studied. It was found that 65 Zn was bound to the external side of the 43 kDa protein-liposomes. Specific binding of 65 Zn to the protein-liposomes was detected. The binding parameter of Zn to the protein was found to be: maximum binding site (Nmax ), 76.7 pmole/µg protein (approx. 3 mole of Zn2+ /mole); and equilibrium dissociation constant (Kd), 0.19 µM. Of the cations introduced (Ca2+ , Cd2+ ,Co2+ , Cr2+ , Cu2+ , Fe2+ , Hg2+ , Mg2+ , Mn2+ , Ni2+ , Pb2+ ), only Co2+ competed significantly with Zn. The protein-liposomes were also found to bind specifically to laminin with a Nmax of 1.1 pmole/µg laminin, and Kd of 4.79 µM. No significant protein-liposome binding occurred to other extracellular matrix proteins (fibronectin, fibrinogen or vitronectin). Furthermore, the binding was specifically inhibited by the Arg-Gly-Asp (RGD) peptide or GRGDSPG, while two other analogs (GRGESPG and GRADSPG) were without effect. 相似文献
4.
ABSTRACT: To examine the involvement of chloride cells in the uptake of Ca2+ in freshwater (FW) killifish, chloride cell morphology was compared in fish acclimated to different defined FW environments with Ca2+ concentrations of either 0.1 mM, 0.5 mM, or 2.5 mM. Numerous chloride cells were detected in whole-mount preparations of the gill filaments, which were stained with an antiserum specific for Na+ , K+ -ATPase. Chloride cells, located mostly on the afferent–vascular edge of the filaments, were larger at lower Ca2+ concentrations. Electron microscopic observations showed that in the 0.1 mM and 0.5 mM Ca2+ experimental groups, the apical membrane of chloride cells were flat or slightly projecting and equipped with numerous microvilli. In the 2.5 mM Ca2+ group, some chloride cells formed an apical pit, whereas other cells were similar to those observed in the 0.1 mM and 0.5 mM Ca2+ groups. Plasma osmolality decreased with decreasing ambient Ca2+ concentration, suggesting that environmental Ca2+ affects the permeability of the body surfaces. Gill Na+ , K+ -ATPase activity in the 0.1 mM and 0.5 mM Ca2+ groups were significantly higher than that in the 2.5 mM Ca2+ group, implying the involvement of the Na+ –Ca2+ exchanger in Ca2+ uptake in the gills. These findings suggest that chloride cells function as the site for Ca2+ uptake in killifish acclimated to low Ca2+ environments. 相似文献
5.
ABSTRACT: The present study used squid gill as a source of transglutaminase (TGase) because it has extremely high TGase activity compared with other tissues. The enzyme was purified using successive chromatographies of Sephacryl S-300 and hydroxyapatite columns. The yield and purification-fold of the enzymatic activity was 12.6% and 14.1-fold, respectively. The molecular mass of the purified enzyme was estimated to be 94 kDa by using sodium dodecylsulfate–polyacrylamide gel electrophoresis analysis. Enzyme activity was enhanced 15-fold with an increase in NaCl concentration. Although the activity was dependent on Ca2+ concentration, it was not sufficiently activated even by 50 mM CaCl2 in the absence of NaCl, but could be fully activated with 10 mM CaCl2 in 0.7 M NaCl. However, in the absence of substrates, the enzyme was rapidly inactivated. The pH and temperature optima of the enzyme were approximately pH 8.0 and 20°C, respectively. It was stable in the absence of Ca2+ at pH 7.5–9.0 and had a rate constant (K D ) of 1.6 × 10–5 s–1 for thermal inactivation at 50°C. These results in which squid gill TGase could be activated at higher concentrations of Ca2+ and NaCl than at a physiological concentration, suggest that contact with seawater or body fluid seems to activate the enzyme if the tissue is disrupted. 相似文献
6.
ABSTRACT: Incubation of squid mantle muscle homogenate caused a selective cleavage of myosin into heavy meromyosin (HMM) and light meromyosin (LMM). HMM was isolated from the incubated homogenate by using ammonium sulfate fractionation. The purified HMM retained two types of light chain components. Its Mg2+ -ATPase activity with or without F-actin showed a Ca-sensitivity. HMM was cleaved into subfragment-1 and subfragment-2 upon chymotryptic digestion with or without Ca2+ , possessing different light chain composition. Two types of light chain component were kept intact when digested in the presence of Ca2+ . Ca2+ stabilized HMM especially in a bound form to F-actin. 相似文献
7.
Thermal denaturation and autolysis profiles of myofibrillar proteins of mantle muscle of jumbo squid Docidicus gigas 总被引：1，自引：0，他引：1
KUNIHIKO KONNO CHO YOUNG-JE TAKEYA YOSHIOKA PARK SHINHO NOBUO SEKI 《Fisheries Science》2003,69(1):204-209
ABSTRACT: Jumbo squid was very similar to Japanese common squid in terms of myofibrillar Ca2+ -, Mg2+ - and K+ (EDTA)-ATPase activities. Myofibrils of jumbo squid were significantly stabilized upon addition of Ca2+ and destabilized by increasing KCl concentration for heating. Incubation of muscle homogenate of jumbo squid induced a selective cleavage of myosin into two major fragments and the cleavage was inhibited by EDTA. Autolysis was prominent at and above 0.3 M NaCl where myosin filaments dissolve. The enzyme involved in the autolysis was proved to be unstable showing maximal autolysis rate at 25°C. Washing the homogenate partially reduced the autolysis activity. 相似文献
8.
ABSTRACT: Amylase, with MW of 59 kDa, was purified from small abalone Haliotis sieboldii by ammonium sulfate fractionation, CM Sepharose Fast Flow and Sephacryl S-100 HR chromatographies. The optimal pH and temperature of purified amylase were 6.0 and 37°C, respectively. The purified enzyme was stable at pH 6.0–8.0 and low temperatures. It was activated by Ba2+ , Mg2+ , Ca2+ , Co2+ , Ni2+ , Mn2+ , K+ , Ag+ , Na+ and Li+ , but completely or partially inhibited by Al3+ , Cu2+ , Cd2+ , Hg2+ and Zn2+ . EDTA could completely inhibit, while iodoacetamide, N-ethylmaleimide and urea partially inhibit the purified amylase. According to the digestion mode of various polysaccharides, the purified enzyme was considered to be an α-amylase. 相似文献
9.
ABSTRACT: The low salt extract from scallop striated adductor muscle contained extra proteins in addition to sarcoplasmic proteins and was turned into a gel upon standing or immediately by the addition of Ca2+ . The amount of extra protein, which was composed of a large amount of actin and a small amount of myosin, decreased with a decrease in adenosine triphosphate (ATP) content in the muscle during storage at 5°C. Actin was easily extracted from scallop myofibrils in low salt solution with ATP at 1 mM or above. Furthermore, ATP was required to induce the gelation of the low salt extract. A visual observation of the gelation of low salt extract is therefore a simple and easy method to inspect prerigor state of scallop adductor muscle. 相似文献
10.
11.
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. 相似文献
12.
Abstract. Changes in the properties of urine and blood of carp with sekoke disease were studied. With the progress of this disease, the parameters of blood and particularly urine changed significantly. After 120 days, urine flow, osmolarity and concentrations of inorganic ions (K+ , Ca2+ , Mg2+ , Cl− ) and organic compounds (ammonium, creatine and creatinine, protein, glucose) in the experimental group showed higher values than those of the controls. Even after 60 days, when clinical signs of sekoke disease were not evident, abnormally high concentrations of ammonium, protein and glucose in the urine were found in the experimental group. 相似文献
13.
Satoru TANAKA Tomoko UTOH Yoshiaki YAMADA Noriyuki HORIE Akihiro OKAMURA Atsushi AKAZAWA Naomi MIKAWA Hideo P OKA Hisashi KUROKURA 《Fisheries Science》2004,70(5):780-787
ABSTRACT: In order to find out the role of sodium bicarbonate (NaHCO3 ) on the initiation of sperm motility in the Japanese eel Anguilla japonica , interactions were investigated between NaHCO3 and various reagents (K+ channel blocker 4-aminopyridine [4-AP], ammonium chloride [NH4 Cl], sodium acetate and calcium chloride [CaCl2 ]) that could regulate internal factors (intracellular K+ , intracellular pH [[pH]i ] and intracellular Ca2+ ) in sperm motility. Contradictory effects of NaHCO3 were observed (i.e. an inhibitory effect when 4-AP was absent and a promoting effect when 4-AP was present). Sodium bicarbonate inhibited the initiation of sperm motility in the Japanese eel. However, NaHCO3 restored the motility of immotile sperm that 4-AP inhibited. The inhibitory effect of NaHCO3 disappeared with the addition of NH4 Cl, which raised [pH]i , but the promoting effect was not affected by [pH]i . Although NaHCO3 recovered motility in the presence of 4-AP, this recovery was also observed with the addition of CaCl2 instead of NaHCO3 . In the initiation of sperm motility in the Japanese eel, two roles for NaHCO3 are suggested: an inhibitory role relating to the regulation of [pH]i and a promoting role relating to the uptake of another initiation factor, which could be Ca2+ . 相似文献
14.
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 (KD ) 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. 相似文献
15.
The influence of dietary cation–anion difference (CAD, Na+ + K+ – Cl– , mEq kg–1 ) on energy metabolism and nitrogen losses in juvenile African catfish Clarias gariepinus (Burchell) was examined in fish exposed to different dietary CAD levels (–146, 116, 497, 713 and 828 mEq kg–1 diet). The experiment was conducted in open circuit balance respiration chambers over a 3-week period. Five 24-h monitoring periods were carried out at 3-day intervals during the experimental period with O2 consumption, ammonia and nitrate + nitrite (NOx ) and CO2 production being measured at 5-min intervals for each chamber. The negative dietary CAD (–146 mEq kg–1 ) resulted in the highest energy expenditures (83 kJ kg–0.8 · d–1 ). With increasing dietary CAD levels, heat loss gradually decreased to minimum values of 56 kJ kg–0.8 day–1 at a dietary CAD level of 713 mEq kg–1 . Consequently, metabolizable energy utilization efficiency (MEU, percentage of retained energy over metabolizable energy) quadratically ( P < 0.05) increased and reached a maximum at a dietary CAD of 713 mEq kg–1 . 相似文献
16.
Comparison of characteristics of actomyosin from white, pink, and red muscle fiber types in cultured carp 总被引：1，自引：0，他引：1
Qin Wang Mutsuyosi Tsuchimoto Osamu Yada Kyung H EE Abdul Jabarsyah Paula Andrea Gomez Apablaza Tosio Misima and Katsuyasu Tachibana 《Fisheries Science》2001,67(4):682-689
ABSTRACT: The present study compared and examined the characteristics of actomyosin among white (W), pink (P), and red (R) muscle fiber types in carp (cultured). Both the superprecipitation reaction and the Mg2+ -ATPase activity of actomyosin became higher with increased Ca2+ concentration (pCa 7.0–pCa 5.0) and with decreased adenosine triphosphate (ATP) concentration (3.0–0.5 mM) in all three muscle fiber types. A comparison of the three fiber types shows that the superprecipitation reaction of actomysoin was lower in the order of W < P < R and, in contrast, was higher for Mg2+ -ATPase activity in the order of W > P > R. A significantly positive correlation between both values was found for each of the three muscle fiber types, but these correlations were clearly different among the three muscle fiber types, and the superprecipitation reaction of actomyosin was lower in the order of W < P < R when Mg2+ -ATPase activity was at the same level. In conclusion, the characteristics of actomyosin were remarkably different among white, pink, and red muscle fiber types. 相似文献
17.
In a 80-day feeding trial, a total of 1050 juvenile Jian carp ( Cyprinus carpio var. Jian) with an average initial weight of 10.71 ± 0.05 g were fed semi-purified diets containing seven graded levels of pyridoxine (0.20, 1.71, 3.23, 4.96, 6.32, 8.58 and 12.39 mg pyridoxine kg−1 diet). Results indicated that with increasing dietary pyridoxine levels up to 4.96 mg kg−1 diet, percent weight gain (PWG) and specific growth rate (SGR) were improved, and no differences were found with further increase of pyridoxine levels. Feed intake also followed the similar pattern to that observed with PWG and SGR when dietary pyridoxine levels were ≤6.32 mg kg−1 diet. But feed efficiency and protein efficiency ratio were not affected by pyridoxine levels. Crude protein of carcass, productive protein value and plasma ammonia concentration were improved with increasing dietary pyridoxine levels up to 4.96 mg kg−1 diet. Amylase activities in the intestine were improved with increasing dietary pyridoxine levels up to 4.96 mg kg−1 diet, but protease and lipase activities in the intestine were not affected by pyridoxine levels. Na+ , K+ -ATPase and Gamma-glutamyl transpeptidase activities in proximal intestine, mid intestine (MI) and distal intestine (DI) were lowest when fed the diet containing 1.71 mg pyridoxine kg−1 diet. The alkaline phosphatase activities in MI and DI followed the same pattern. The dietary pyridoxine requirement of juvenile Jian carp based on PWG estimated by broken line model was 6.07 mg pyridoxine kg−1 diet. 相似文献
18.
We investigated the thermal denaturation of carp F-actin by measuring the loss of polymerization ability. The thermal denaturation
rates of carp F-actin were at least 10-fold higher than those of chicken F-actin. Binding of tropomyosin thermally stabilized
carp F-actin with no appreciable change in activation energy, suggesting the instability was caused by a high frequency of
fragmentation of the actin filaments. A comparison of the critical concentration for polymerization suggested that the subunit–subunit
interactions of carp F-actin were indeed weaker than those of chicken F-actin. Furthermore, using fluorescence quenching we
showed that the nucleotide binding region of carp F-actin was in a more open conformation. Together, our results suggest that
the instability of carp F-actin is a function of both the rate of fragmentation and the dissociation of bound nucleotides. 相似文献
19.
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. 相似文献
20.
Four diets (T0 –T3 ) were formulated reducing the fishmeal (Indian) component by 100 g kg–1 from 300 to 0 g kg–1 and including proportionately increasing quantities of maize. Diets were fed for 120 days at 50 g kg–1 body weight to triplicate groups of common carp (av. wt. 2.11–2.18 g) stocked at 1 m–2 in mud bottomed cement tanks (18 m2 ), fertilized with poultry manure. Fish growth, SGR and FCR in the different treatments were statistically not significantly different ( P > 0.05). PER was lowest for the 300 g fishmeal kg–1 diet treatment (diet T0 ), increasing with decrease in dietary fishmeal content (diets T1 –T3 ). Fish survival ranged from 96.29 to 100%. Diets influenced carcass composition and digestive enzyme activity. A significant increase in lipid deposition was recorded with increasing dietary carbohydrate content. Amylase, protease and lipase activities were higher in fish fed with diets T2 and T3 . The protein sparing effect of dietary carbohydrate and the economic implication of eliminating fishmeal from the diet are discussed. 相似文献