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1.
郭川 《水产学报》2007,31(4):423-430
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。  相似文献   

2.
Gelatinolytic enzymes were partially purified from the skeletal muscle of red sea bream Pagrus major and characterized to obtain information on post mortem tenderization of fish muscle. Four gelatinolytic activities, G1 (90 kDa), G2 (65 kDa), G3 (60 kDa), and G4 (100 kDa), were detected in the Q Sepharose column. G1, the major gelatinolytic enzyme, and G4 were identified as serine proteinases from results of inhibitor spectrum and substrate specificity. By contrast, G2 and G3 were found to be metalloproteinases since these were inhibited by ethylenediamine tetraacetic acid and o-phenanthroline, and activated by 4-aminophenylmercuric acetate. The optimum pH and temperature of these enzymes were in the ranges of 7–9 and 20–40°C, respectively.  相似文献   

3.
A lipase was purified from the extract of the delipidated powder of red sea bream hepatopancreas to nar homogeneity by fractional precipitation with ammonium sulfate and sequential chromatography on first anion-exchange-, hydrophobic- and second anion-exchange columns followed by gel filtration and anion-exchange HPLC. The final enzyme preparation showed a single band with an apparent molecular mass of approx. 64 kDa by sodium dodecyl sulfate-polyacrylamid e gel electrophoresis. The purified enzyme had a pH optimum in the range of pH 7.0–9.0. Using -nitrophenyl myristate or triolein as a substrate, the enzyme required the presence of sodium taurocholate or sodium cholate for its activity. No activity was observed in the presence of sodium deoxycholate. The enzyme preferentially hydrolyzed ethyl esters of polyunsaturated fatty acid, such as arachidonic acid and eicosapentaenoic acid which were resistant to porcine pancreatic lipase. These results strongly suggest that the enzyme purified from the hepatopancreas of red sea bream is homologous to mammalian bile salt-activated lipase.  相似文献   

4.
Four semi-purified diets, containing crystalline amino acids (CAAs), were fed to juvenile red sea bream, Pagrus major in order to ascertain the ideal dietary amino acid pattern for this species. A control diet containing 50% casein–gelatin as protein sources, but no CAAs were fed to the fish. The other diets contained 30% casein–gelatin and 20% CAAs. CAAs were added to diets to simulate with amino acid pattern of the red sea bream eggs protein (REP), red sea bream larvae whole body protein (RLP), red sea bream juvenile whole body protein (RJP), and brown fishmeal protein (BFP). The juveniles (average initial body weight, 1.58 ± 0.01 g) were maintained in triplicate tanks and fed twice daily for 30 days. The highest weight gain was observed in juveniles fed the RJP diet. No significant difference was observed in juveniles fed the RLP and BFP diet. Feed efficiency ratio, protein efficiency ratio and amino acid retention in the whole body were significantly (p < 0.05) affected by the simulated dietary amino acid patterns. The essential amino acid profile and A/E ratios of the whole body after the growth trial showed little difference among the dietary treatments. The results suggest that red sea bream juveniles are able to utilize high amounts of CAA in coated form. The amino acid pattern of RJP could be used as an appropriate of reference dietary amino acid for this species.  相似文献   

5.
Cathepsin L, one of the cysteine proteases found in fish muscle, is considered to be the main cause of post-mortem autolysis of fish muscle. We have determined the presence of cathepsin L in the membranes of red blood cells of carp, amberjack, and red sea bream and measured its activity. Immunoblotting of an extract of the red blood cell membranes from these three fish species using human anti-cathepsin L antibody revealed the presence of cathepsin L of different molecular masses. The molecular masses of cathepsin L was estimated to be 120 and 85 kDa in the amberjack and 75 and 70 kDa in the carp. These proteins have higher molecular masses than the mature form of cathepsin L, suggesting that they are precursor forms. In contrast, the protein in the red sea bream was estimated to have a molecular mass of 30 kDa, suggesting that this cathepsin L is a mature form. The specific activity of cathepsin L was highest in the red blood cell membranes of the amberjack, followed by the carp and the red sea bream in descending order.  相似文献   

6.
KEN  TOUHATA  YUKI  TOKUDA  MORIHIKO  SAKAGUCHI  HARUHIKO  TOYOHARA 《Fisheries Science》2002,68(5):1118-1123
We previously cloned cDNA of type V/XI collagen α1 chain (ColVa1) gene from cultured cells derived from red sea bream embryo. We raised an antibody against the deduced C-telopeptide of ColVa1 in order to detect the translation products of this cDNA and their degradation products in red sea bream muscle. To improve its specificity, the antibody was purified from rabbit antiserum by use of an affinity column cross-linked with recombinant C-terminal peptide of ColVa1 produced by Epicurian coli. The purified antibody recognized a band corresponding to the α chain of type V/XI collagen in western blot analysis of the extract of cultured cells. The antibody also recognized two bands in acid-soluble and pepsin-solubilized collagens, indicating that the translation products of the ColVa1 gene are present in muscle and that bands correspond to α and β chains of type V/XI collagen. A band corresponding to a molecular weight of approximately 65 k was detected in the NaOH extracts of muscle, suggesting that type V/XI collagen α1 chain is restrictedly digested in red sea bream muscle.  相似文献   

7.
Two experiments were conducted to investigate the effect of dietary taurine and cystine on growth and body composition of juvenile red sea bream Pagrus major. In Experiment I, a casein-based semi-purified diet included a small amount of fish meal were supplemented with taurine at the levels of 0 (control) and 1.0%. The experimental diets in Experiment II were without fishmeal and supplemented with taurine at 0 (control), 0.5, 1.0 and 2.0% or cystine at 1.0 and 2.0%. These diets were fed three times a day for 6 weeks to fish (average body weight: 2.3 g in Experiment I and 2.5 g in Experiment II). In Experiment I, fish fed the taurine-supplemented diet showed significantly (P < 0.05) improved growth, feed efficiency and feed consumption relative to fish fed the unsupplemental diet. The whole body taurine content increased, whereas the non-essential amino acid contents decreased, in fish fed the taurine-supplemental diet compared to fish fed the unsupplemented diet. In Experiment II, the growth, feed efficiency and feed consumption of fish fed the taurine-supplemented diets, irrespective of the dietary taurine levels, were significantly higher than those of fish fed the control diet and the cystine-supplemented diets. Taurine content in the whole body increased with the dietary taurine level, while the taurine contents did not increase by the supplemental cystine. Other free amino acid contents in the taurine-supplemented diet groups followed similar trends to those in Experiment I. These results indicate that supplemental taurine to a casein-based semi-purified diet at more than 0.5% improved the growth and feed performance of juvenile red sea bream. It is also suggested that juvenile red sea bream cannot metabolize cystine into taurine.  相似文献   

8.
黑鲷幼鱼饲料蛋白源氨基酸平衡的研究   总被引：13，自引：1，他引：12  
以黑鲷幼鱼肌肉的氨基酸组成作为幼鱼饲料中必需氨基酸需要量的标准模式,通过必须氨基酸指数（ＥＡＡＩ）和生物饲养试验结果相结合,对黑钱财幼鱼的９种不同蛋白源酸比的饲料进行了比较研究,对黑钱财幼鱼配合饲料中氨基酸平衡和蛋白质营养价值进行了评估,筛选出个高效饲料蛋白源配方;表明ＥＡＡＩ是鱼用饲料蛋白质营养价值评估和氨基酸平衡研究的一种简便有效的评价指标。  相似文献   

9.
Olive leaf is known to have the high polyphenol content of 6–9% in dry weight. We investigated the effects of olive leaf powder (OLP) supplemented to fish feed on muscle protein of red sea bream (Pagrus major). Fish reared with feed containing 8% OLP for 40 days had 1.4 times higher myofibril content and 2.2 times higher acid-soluble collagen content than fish reared with control feed for the same period. On the other hand, sarcoplasmic protein content and collagenase activity of the muscle were almost the same between the control fish and OLP-diet fish. Microstructure observation of fish muscle showed that OLP-diet fish has more rigid endomysium structure than that of the control-diet fish. Since collagen fiber in endomysium is responsible for the texture of the muscle, feeding OLP to aquaculture fish will lead to a harder muscle texture. The present study suggests that OLP is a useful feed additive to enhance the texture of aquaculture red sea bream muscle through strengthening of the collagen structure in the muscle.  相似文献   

10.
Histidine decarboxylase (HDC) from Staphylococcus epidermidis TYH1, a halotolerant histamine-producing bacterium isolated from Japanese fermented fish-miso, was purified to homogeneity for the first time. The enzyme was purified 182-fold from cell-free extracts by ammonium sulfate precipitation, anion exchange chromatography and gel filtration chromatography. The N-terminal amino acid sequences of two polypeptide chains of 27–30 and 7–9 kDa were highly homologous with those of α- and β-chains of other staphylococcal HDCs. The optimum pH and temperature for the enzyme were 6.0 and 60 °C, respectively. This enzyme did not decarboxylate lysine, arginine, tyrosine, tryptophan or ornithine. The enzyme activity decreased with the addition of NaCl. At pH 4.8, the V max and K m values were 45.5 μmol histamine min?1 mg?1 and 1.10 mmol/L, respectively. Moreover, this enzyme was resistant to heat treatment (80 °C for 15 min) and was stable upon freezing at ?30 °C for 7 days. The very similar physiological properties of this enzyme and the almost identical N-terminal amino acid sequence to that of the HDC from S. capitis indicated that this enzyme may be evolutionally highly conserved in the genus Staphylococcus. The biophysical properties of staphylococcal HDC were elucidated using native purified enzyme.  相似文献   

11.
To establish the practical use of microbound diets (MBD) for larval fish in mass seedling production, rearing experiments of larval red sea bream, Pagrus major , and Japanese flounder, Paralichthys olivaeeus , were conducted. A mixture of various protein sources was used, and dietary amino acid patterns were approximated to those of larval whole body protein. Two thousand red sea bream larvae and 1,000 Japanese flounder larvae, all 10 days old, were placed in 100 liter tanks with running sea water under ambient water temperature, which ranged from 18 to 20C. The particle size of MBD was 125 μm at the beginning of the experiment and adjusted as fish size increased thereafter. Employing MBD together with a small amount of live food could sustain the growth and survival of larval red sea bream and Japanese flounder. Thus, data from the present study indicates that substitution of artificial feeds for live foods is possible for larval fish production, although improvements in MBD diets may be necessary before they are adequate for large scale seedling production.  相似文献   

12.
Expressed sequence tag (EST) analysis is an efficient tool for gene discovery and for profiling gene expression. In order to isolate functional genes involved in immunity in fish, a cDNA library was constructed from red sea bream (Chrysophrys major) spleen by unidirectional cloning. A total of 2010 ESTs from the library was sequenced and compared with sequences in the GenBank database. Of the 2010 ESTs, 320 ESTs (15.9%) were identified as orthologs of known gene from other organisms by BLAST searches, whereas 1690 ESTs (84.1%) appeared to be unknown and are likely to represent newly described genes. These identified clones were derived from at least 81 genes, which were categorized into eight categories: 9 in cell structure/motility (11.1%), 14 in metabolism (17.3%), 8 in cell defense/immunity (10%), 5 in cell division (6.2%), 7 in cell signal transduction/communication (8.6%), 30 in gene/protein expression (37%), 5 hemoglobin (6.2%) and 3 genes lacking enough information to be classified (3.7%). Several important cDNAs involved in immune functions, such as immunoglobulin light chain (IgL), MHC class II, MHC class IIβ and RAP2c, were identified in red sea bream and compared for their structure with those from other organisms. Alignment showed that the red sea bream IgL precursor was closer to that of spotted wolfish than to that of yellowtail, Europe sea bass, orange spotted grouper, Atlantic salmon, channel catfish, fugu and sterlet. Phylogenetic analysis indicated that the red sea bream MHC II and MHC IIβ were more related to those from striped sea bass than to those from cichlid, flounder, salmonids, zebrafish and carp. High identity (over 92%) in deduced amino acid sequence of RAP2c between red sea bream and mammals implied that RAP2c gene was highly conserved during evolution.  相似文献   

13.
Glucose-6-phosphate dehydrogenase (G6PD) and glutathione reductase (GR) are metabolically quite important enzymes. Within this study, these two enzymes were purified for the first time from the gills of Lake Van fish. In the purifying process, ammonium sulfate precipitation and 2′,5′-ADP Sepharose 4B affinity column chromatography techniques for glucose-6-phosphate dehydrogenase, temperature degradation and 2′,5′-ADP Sepharose 4B affinity column chromatography for glutathione reductase enzyme were used. The control of the enzyme purity and determination of molecular weight were done with sodium dodecyl sulfate polyacrylamide gel electrophoresis. K M and V max values were determined with Lineweaver–Burk plot. Besides, the effects of some chalcone derivatives on the purified enzymes were analyzed. For the ones showing inhibition effect, % activity–[I] figures were drawn and IC50 values were determined. K i value was calculated by using Cheng–Prusoff equation.  相似文献   

14.
The effects of dietary amino acid profile (based on muscle (M) or whole body composition (WB) and the balance between indispensable (IAA) and dispensable amino acids (DAA) in the diet, on plasma levels of insulin and glucagon, were analysed in rainbow trout and gilthead sea bream. Plasma insulin values (baseline and 6 h postfeeding) were higher in trout than in sea bream, but the relative postfeeding increase was more pronounced in sea bream. Within the same dietary amino acid profile, diets with lower IAA/DAA, had a lower effect on the postfeeding secretion of insulin in both species. Circulating levels of glucagon (baseline and postfeeding relative increases) were higher in sea bream. In trout, diets with WB amino acid profile had a greater secretory effect on postfeeding glucagon than did diets with M profile, while gilthead sea bream showed an inverse response to circulating glucagon with respect to diet. Muscle insulin and insulin growth factor-I binding parameters were not affected by the dietary regimen. The postfeeding glucagon response depends on both the dietary AA profile and the fish species, while that of insulin seems to be more uniform, and is affected in a similar way regardless of the species.  相似文献   

15.
Juvenile gilthead sea bream were fed to visual satiety with isonitrogenous diets based on fish meal and different plant ingredients (33–35% replacement) supplemented with free amino acids to meet the desired indispensable amino acid (IAA) profile and dispensable amino acid (DAA) content. In diets M and WB, IAA profile and DAA content resemble that of the muscle or whole body, respectively. In diets MGlu and WBGlu, DAA content was increased by adding -glutamic acid (Glu) and thus the IAA/DAA ratio varied from 1.13 (diet M) to 0.80 (diet WBGlu). Growth rates were not significantly different among experimental groups, but feed conversion ratio and nitrogen retention were impaired by the decrease of dietary IAA/DAA ratio. Postprandial ammonia excretion increased with the increase of dietary DAA content irrespective of IAA profile. Conversely, hepatic activity of glutamate dehydrogenase (GDH) was lower in fish fed diet WBGlu than in fish fed diet M. Hepatic growth hormone (GH) binding was not significantly affected by the dietary treatment, but circulating levels of insulin-like growth factor-I (IGF-I) and GH were, respectively, down- and up-regulated in fish fed diet WBGlu, which suggests some defect in the transmission of GH receptor signal. Fat retention and hepatic activities of lipogenic enzymes (glucose-6-phosphate dehydrogenase, G6PD; malic enzyme, ME) were decreased in fish fed diet MGlu. Key metabolic enzymes of hepatic glycolysis (glucokinase, GK) and gluconeogenesis (phosphoenolpyruvate carboxykinase, PEPCK) were also altered in this group of fish. Since soybean meal concentration was highest in diet MGlu, results on lipid and carbohydrate metabolism can be primarily attributed to this component of the diet. In contrast, data on growth performance, ammonia excretion and GH axis mainly reflect changes in the dietary amino acid profile, which reveals that a muscle IAA profile and a high IAA/DAA ratio are important in feeds for gilthead sea bream.  相似文献   

16.
Two phospholipase A2 (PLA2) isoforms, tentatively denoted as DE-1 and DE-2 PLA2s, were purified from the hepatopancreas of red sea bream (Pagrus major) to near homogeneity by sequential column chromatography on S-Sepharose fast flow, DEAE-Sepharose fast flow and butyl-Cellulofine, and by ion-exchange, gel-filtration and reversed-phase HPLC. The purified DE-1 and DE-2 PLA2s both showed a single band with the apparent molecular mass of approx. 13.5 kDa by SDS-polyacrylamide gel electrophoresis, and were found to be both related to group I PLA2 based on the N-terminal amino acid sequences. DE-1 PLA2 had a pH optimum in the alkaline region at around pH 10 and required approximately 10 mM of Ca2+ and 4-10 mM of sodium deoxycholate for its maximal activity, using 2 mM of phosphatidylcholine and phosphatidylethanolamine as substrates. DE-2 PLA2 also had a pH optimum in the alkaline region at around pH 8-9 and required >10 mM of Ca2+ and approximately 6 mM of sodium deoxycholate for its maximum activity with 2 mM of phosphatidylcholine as a substrate; its enzymatic activity towards phosphatidylethanolamine was greatly inhibited by the addition of sodium deoxycholate. The results demonstrate that red sea bream hepatopancreas contains two enzymatically distinct group I PLA2 isoforms.  相似文献   

17.
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.  相似文献   

18.
The objective of this study was to evaluate the effect of harvest practices and slaughter method on stress, quality and shelf life of whole fish (gilthead sea bream, European sea bass and red sea bream) towards the development of a humane slaughter practice. The use of hook and line resulted in significantly lower plasma cortisol and glucose in European sea bass and gilthead sea bream. Water temperature at harvest affected significantly the concentrations of stress indicators (plasma cortisol and glucose), mainly in European sea bass and gilthead sea bream. No significant differences were observed between the harvesting methods, regarding fish appearance, microbial spoilage and sensory scoring and subsequently shelf life. However, significantly lower hardness was recorded in fish harvested using the electric stunner, compared to the fish killed with slurry ice of the same harvesting period. Shelf life ranged between 13–18 days for gilthead seabream, 10–16 days for European sea bass and 10–14 days for red sea bream, depending on harvesting period.  相似文献   

19.
SUMMARY: Two trypsins, designated as trypsin A and trypsin B, have been purified from the hepatopancreas of carp. The purification procedures consisted of ammonium sulfate fractionation, and chromatographies on DEAE-Sephacel, Ultrogel AcA54 and Q-Sepharose. Trypsin A was purified to homogeneity with the molecular mass of approximately 28 kDa, while trypsin B gave two close bands of 28.5 kDa and 28 kDa on sodium dodecylsulfate polyacrylamide gel electrophoresis both under reducing and non-reducing conditions. On native-PAGE, both trypsin A and trypsin B showed a single band. Trypsin A and trypsin B revealed optimum temperature of 40°C and 45°C, respectively, and shared the same optimum pH 9.0 using Boc-Phe-Ser-Arg-MCA as substrate. Both enzymes were effectively inhibited by trypsin inhibitors and their susceptibilities were similar. The NH2-terminal amino acid sequences of trypsin A and trypsin B were determined to 37th and 40th amino acid residue, respectively. Their sequences were very homologous, but not identical to that of a trypsin-type serine proteinase from carp muscle and these of other trypsins. Immunoblotting test using the antibody raised against trypsin A cross-reacted with trypsin B positively.  相似文献   

20.
The acute toxicities of copper were examined for Japanese flounder, Paralichthys olivaceus, and red sea bream, Pagrus major, in terms of fish size and water temperature. Artificial seawater of low pH of 5.4–6.7 was used as testing water to keep dissolved copper concentration at 0.04–41 mg Cu/L. Japanese flounder of 0.3–17 g and red sea bream of 0.5–13 g were exposed to different concentrations of copper for 96 h at 20 C under semistatic condition. Median‐lethal concentration for 96 h of Japanese flounder and red sea bream were 8.7–12.2 and 2.0–5.2 mg Cu/L, respectively. No significant relationships were observed between median‐lethal concentrations for 96 h and fish size for Japanese flounder, while the value decreased significantly with increasing fish size for red sea bream. Effect of water temperature on the acute toxicity was examined for Japanese flounder of 0.3 and 0.4 g at 10, 15, 20, and 25 C and red sea bream of 0.5 and 1.0 g at 12, 15, 20, and 25 C. Ninety‐six‐hour median‐lethal concentrations for Japanese flounder and red sea bream were 5.1–11.2 and 1.0–5.3 mg Cu/L, respectively. No significant relationships were observed between median‐lethal concentrations for 96 h and water temperature for both fish species.  相似文献   

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