Purification and characterization of cathepsin S from hepatopancreas of carp Cyprinus carpio

SUMMARY: Cathepsin S was purified from carp hepatopancreas to homogeneity up to 300-fold. The amino acid sequence of its NH₂-terminus was determined to be V-P-D-A-M-D-W-Y-N-K-G-Y-V-T-D-V-K-N-Q. On the contrary, that of purified cathepsin L from carp hepatopancreas was to be V-P-N-S-L-D-W-R-E-K-G. Purified cathepsin S consisted of a single chain with 37 kDa estimated by sodium dodecylsulfate–polyacrylamide gel electrophoresis. The enzyme had strong hydrolytic activity toward Z-Phe-Arg-MCA with the pH optimum of 7.0, but this lacked the ability to hydrolyze most of the other MCA substrates. The optimum pH of cathepsin S for protein substrate (carp myosin heavy chain) was also to be pH 7.0. These properties of purified cathepsin S obviously differ from cathepsins B and L. The enzyme activity was totally inhibited by E-64, leupeptin, 5–5'-dithiobis (2-nitro-benzoic acid) and p -tosyl-lys chloromethylketone as well.     

白鲢骨骼肌丝氨酸蛋白酶抑制剂的提取、纯化及其特性

从白鲢（Hypophthalmichthys molitrix）骨骼肌中分离纯化到一种丝氨酸蛋白酶抑制剂，该抑制剂的分子晕大约50kD，用PAGE-明胶电泳证明，该抑制剂对胰蛋白酶具有抑制作用；当温度超过60℃时，抑制剂的活性下降70％左右，但在pH3～11的范围内均有活性。用MTT法分析白鲢骨骼肌丝氨酸蛋白酶抑制剂对急性T细胞白血病细胞Jarkat、人骨髓瘤细胞Sp20和人骨瘤细胞MG63生长的影响，结果发现该抑制剂对以上3种肿瘤细胞的增殖都有抑制作用，而且抑制作用呈剂量依赖性。本研究旨为进一步研究白鲢骨骼肌丝氨酸蛋白酶抑制剂的纯化及其抗肿瘤作用提供科学依据。     

Anionic Trypsin from the Pyloric Ceca of Pacific Saury (Cololabis saira): Purification and Biochemical Characteristics

Anionic trypsin from Pacific saury (Cololabis saira) pyloric ceca was purified to homogeneity by ammonium sulfate precipitation, ion-exchange chromatography, and gel filtration chromatography. It was purified to 53.7-fold with a yield of 6.1%. The apparent molecular weight of the enzyme was about 24 kDa, as determined by size exclusion chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). On native-PAGE, trypsin showed a single band. The purified anionic trypsin displayed optimal activity at pH 8.5 and 55°C. The enzyme was stable at neutral and alkaline pH and in the temperature range of 20–50°C. The stability was affected by the calcium ion. The activity of purified anionic trypsin was completely inhibited by soybean trypsin inhibitor and N-p-tosyl-L-lysine chloromethyl ketone (TLCK) and partially inhibited by ethylenediaminetetraacetic acid (EDTA). NaCl (0–30%) decreased the activity in a concentration-dependent manner. The kinetic trypsin constants K_m and K_cat were 0.19 mM and 210 s^?1, respectively, while the catalytic efficiency (K_cat/K_m) was 1105.26 s^?1 mM^?1. The N-terminal amino acid sequences of anionic trypsin, IVGGYECQAH, were found and were homologous to those of trypsin from other fish species.     

大豆胰蛋白酶抑制剂对鲢丝氨酸蛋白酶的作用

研究了大豆胰蛋白酶抑制剂(STI)对鲢肌肉中肌原纤维结合型丝氨酸蛋白酶(MBSP)引起的肌原纤维蛋白降解作用的影响。大豆胰蛋白酶抑制剂经高度纯化后加入肌原纤维中以判断它对MBSP的抑制效果。在不添加STI的情况下,55℃加热30min即可观察到肌球蛋白重链的分解,延长加热时间则可检测到肌动蛋白及原肌球蛋白的降解。相反,在终浓度为0.75μg·mL-1的STI存在下,包括肌球蛋白重链在内的肌原纤维蛋白降解均能得到有效抑制,表明STI是MBSP的特异性抑制剂。由于肌球蛋白重链及肌动蛋白的完整性是构成鱼糜制品凝胶强度的主要因素,因此,STI的添加有可能提高鲢鱼糜制品的凝胶强度。     

草鱼肝胰脏组织部分EST的鉴定及分类

本研究构建了草鱼（Ctenopharyngodon idellus）肝胰脏组织的cDNA文库，并对768个随机挑选的克隆进行部分测序，获得了567个ESTs。BLAST结果表明，457个Esrs与GenBank中已经鉴定的130个基因具有较高的同源性，其中有5个基因是已经报道的，125个是草鱼中首次鉴定的基因；110个和未鉴定的基因具有相对较低的同源性，称为假定蛋白。根据编码产物的功能可以将这些基因分为：代谢（41），基因表达及其蛋白质合成（56），细胞及机体防御（18），细胞信号传导与细胞通讯（11），细胞结构与运动（4）。     

Amino acid composition of edible parts of three-year-old experimental scaly crossbreds of common carp (Cyprinus carpio, Linnaeus 1758)

The aim of this study was to compare the amino acid (AA) composition of edible parts of three experimental groups of carp, i.e. a pure line of Přerov scaly carp (PS), a hybrid line of Přerov scaly carp and Northern mirror carp (PS × M72), and a hybrid line of Přerov scaly carp and Ropsha scaly carp (PS × ROP), with the quality of the edible parts of control hybrids of Hungarian and Northern mirror carp (M2 × M72) in harvest size (K₃). A comparison between the controls (M2 × M72) and experimental carp (PS, PS × M72, PS × ROP) showed that their muscle tissues contained the same amounts of 10 AA [essential amino acids (EAA): Thr, Val, Leu, Phe, Lys, His; non‐essential amino acids (NEAA): Asp, Gly, Ala, Tyr] of the 16 AA determined. Glu, Asp, Lys and Leu were the AA with the highest muscle concentrations. The total EAA_sum and NEAA_sum contents in the fastest‐growing PS × ROP hybrid, in spite of specific differences found (P<0.05: Arg, Met; P<0.01: Pro), were practically identical to those found in the control group of M2 × M72 mirror carp. PS × ROP hybrid female and male muscle tissues differed (P<0.05) only in Met and Ala levels. Hard roes of experimental female carp (PS, PS × M72, PS × ROP) contained the largest quantities of Glu and Val, and that of control female carp (M2 × M72) the largest quantities of Glu and Gly. Hard roes of PS × ROP hybrids contained the largest quantities (P<0.01) of EAA_sum (52.44±0.19%). Compared with hard roes, soft roes from all groups of carp contained more EAA_sum (PS × ROP: 55.03±0.26%). The two most abundant AA in soft roes were Lys and Arg. The most abundant AA in the hepatopancreas in all carp groups were Glu, Asp, Leu and Arg. Hepatopancreas EAA_sum levels in experimental carp (PS, PS × M72, PS × ROP) were significantly (P<0.05) lower than those in controls (M2 × M72).     

Purification of serine carboxypeptidase from the hepatopancreas of Japanese common squid Todarodes pacificus and its application for elimination of bitterness from bitter peptides

ABSTRACT:   An acidic serine carboxypeptidase (CPase Tpa) from the hepatopancreas of Japanese common squid Todarodes pacificus was purified. Purified CPase Tpa had a molecular mass of 36 kDa on sodium dodecylsulfate–polyacrylamide gel electrophoresis, and an isoelectric point of 6.0. The optimum pH of CPase Tpa was pH 4.0. In investigating the specificity of CPase Tpa for several peptide substances, it was found that peptides with hydrophobic or bulky amino acid residues at the P₁ position reacted well. The enzymatic activity was almost completely inhibited by p -chloromercuribenzoic acid, monoiodoacetic acid, diisopropylfluorophosphate and HgCl₂. This is the basis for its grouping in the serine carboxypeptidase family (EC 3. 4. 16. 5). The substrate specificity of CPase Tpa can be used to eliminate the bitterness of bitter peptides. In this study, the bitterness-reductive effect using bitter peptides prepared by hydrolyzing soy protein, casein and corn gluten with pepsin or trypsin was tested. The bitterness of soy peptide digested with pepsin was completely eliminated by treatment with CPase Tpa, whereas the bitterness of casein digested with trypsin and corn peptide digested with pepsin were somewhat less efficient. On the basis of these results, it is anticipated that CPase Tpa would be effective in eliminating the bitterness of some bitter peptides.     

Occurrence of two distinct molecular species of cathepsin B in carp Cyprinus carpio

ABSTRACT:   We purified cathepsins B₁ and B₂ from the ordinary muscle of carp Cyprinus carpio . The N-terminal amino acid sequences (12 residues) of 29 kDa bands of cathepsins B₁ and B₂ are the same and showed high homology of 75% and 83%, respectively, with the heavy chain of rat and human cathepsins B. Based on conserved sequences of other cathepsins B and the N-terminal amino acid sequences of 29 kDa bands, we cloned carp cathepsin B cDNA. The nucleotide sequence of carp cathepsin B cDNA consists of 1470 bp including a 993 bp open reading frame, encoding a deduced protein of 330 amino acids. The deduced amino acid sequence of carp cathepsin B has similarity of 80% to rainbow trout cathepsin B and of 76–78% to other vertebrate cathepsins B. The sequence of its isoform was also determined during molecular cloning, which has 94.8% similarity with first cloned cathepsin B. They are completely same in N-terminal amino acid sequence of heavy chain, active site and potential N-glycosylation site. This indicates there are at least two kinds of cathepsin B functioning in vivo in carp.     

酶解植物蛋白对建鲤生长、肝胰脏和肠道发育及消化酶活性的影响

试验考察了酶解植物蛋白对建鲤生长性能、肝胰脏和肠道发育及消化酶活性的影响.体质量为(9.18±0.21)g的建鲤900尾,随机分成6个处理组,每组3个重复,每个重复50尾,分别饲喂酶解植物蛋白替代相应完整植物蛋白0、20%、40%、60%、80%和100%的试验饵料,试验期为60 d.结果表明,酶解蛋白替代完整蛋白40%时,生长速度最快,饵料系数最低,摄饵量、蛋白和灰分沉积率最高;替代比例在60%以下时,肠重和肠皱襞高度、肠道脂肪酶、碱性磷酸酶、Na ,K -ATP酶的活性随着替代比例的上升而显著增加;替代比例超过60%以后,生长速度显著降低、饵料系数升高,肠道酶活、皱襞高度、蛋白沉积率下降,而肝胰脏谷草转氨酶活性、血氨浓度和脂肪沉积率升高(P<0.05).结果表明,适宜比例的酶解植物蛋白能促进建鲤生长、肝胰脏和肠道发育,提高饲料利用率.     

The purification and partial characterization of carp,Cyprinus carpio,vitellogenin

A procedure is described for the isolation of intact vitellogenin (c-VTG) from the carp, Cyprinus carpio. VTG was induced in juvenile females using oestradiol-17β and purified from the plasma using a combination of gel-filtration chromatography on Sepharose 6B and ion exchange chromatography on DEAE-cellulose. Purification procedures were conducted at low temperatures (below 9°C) in the presence of the proteolytic enzyme inhibitor aprotinin to prevent degradation. Intact c-VTG had an apparent molecular mass of 390,000 Daltons, but when extracted from plasma in the absence of aprotinin it underwent proteolysis into at least 2 protein fragments (apparent molecular masses of 230,000 and 96,000 Daltons), showing an instability of the native dimer. An amino acid analysis of c-VTG showed that its composition was almost identical to goldfish VTG, a species closely allied to the true carps and also similar to other oviparous vertebrate VTGs. Collectively, these data indicate that using these purification procedures VTG from carp, and probably other teleost species, can be isolated in an intact, highly purified form.     

Dietary phosphorus prevents oxidative damage and increases antioxidant enzyme activities in intestine and hepatopancreas of juvenile Jian carp

This study was conducted to investigate the effect of dietary phosphorus on the intestine and hepatopancreas antioxidant capacity of juvenile Jian carp (Cyprinus carpio var. Jian). Jian carp, with an average initial weight of 7.17 ± 0.01 g, were fed with diets containing graded concentrations of available phosphorus, namely 1.7 (control), 3.6, 5.5, 7.3, 9.2 and 11.0 g kg^?1 diet for 9 weeks. Results showed that, in intestine and hepatopancreas, content of malondialdehyde (MDA), protein carbonyl (PC) and glutathione (GSH), capacity of anti‐superoxide anion (ASA) and anti‐hydroxyl radical (AHR), and glutathione reductase (GR), catalase (CAT), glutathione S‐transferase (GST), superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities were significantly affected by dietary phosphorus levels (P < 0.05). Regression analysis showed that significant quadratic responses occurred in MDA content and ASA, GST, GPx and AHR activities in intestine, GSH content and CAT and SOD activities in hepatopancreas (P < 0.05). These results indicate that optimal level of dietary phosphorus prevented oxidative damage and increased antioxidant enzyme activities in the intestine and hepatopancreas of juvenile Jian carp. The phosphorus requirement estimated from MDA using quadratic regression analysis was 5.7 g kg^?1 diet.     

基于肝脂和1H-NMR技术的草鱼肝胰脏及血清代谢组学分析

为了探讨草鱼肝胰脏脂质过量蓄积的生化基础和促进草鱼的健康养殖,采用1H-NMR分析技术对肝脂含量分别为3 %~4 %和14 %~16 %的两组草鱼的肝胰脏和血清进行了代谢组学分析。结果表明,从草鱼肝胰脏和血清中分别检测到了58种和47种代谢物。葡萄糖、别嘌呤二醇、牛磺酸、乳酸、谷氨酸、肌酸、丙氨酸、sn-甘油-3-磷酸胆碱、甘氨酸、石碳酸、次黄嘌呤、乙酸、赖氨酸、亮氨酸、缬氨酸为两组草鱼肝胰脏主要差异代谢物;其中,肝脂含量为14 %~16 %的草鱼肝胰脏中葡萄糖和别嘌呤二醇的含量明显高于肝脂含量为3 %~4 %的草鱼组,但乳酸和多种氨基酸含量则低于肝脂含量为3 %~4 %的草鱼组。乳酸、葡萄糖、肌酸、柠檬酸、亮氨酸、丙酮酸、缬氨酸、脯氨酸、鸟嘌呤核苷、异亮氨酸、谷氨酸、sn-甘油-3-磷酸胆碱、丙氨酸、赖氨酸、甘氨酸为两组草鱼血清主要差异代谢物;其中肝脂含量为14 %~16 %的草鱼血清中葡萄糖、柠檬酸、脯氨酸、谷氨酸、sn-甘油-3-磷酸胆碱、精氨酸以及甘氨酸7种代谢物含量高于肝脂含量为3 %~4 %的草鱼组,血清乳酸等含量则明显低于肝脂含量为3 %~4 %的草鱼组。肝脂含量为14 %~16 %的草鱼组其体内糖的有氧氧化和糖酵解途径均受到了明显的抑制,同时存在氨基酸代谢异常,这应该是导致草鱼肝胰脏脂质过量蓄积的重要原因之一。     

鲢鱼骨骼肌过敏原小清蛋白的分离纯化及多克隆抗体制备与应用

小清蛋白是鱼类的主要过敏原,对该蛋白的研究不仅有利于过敏原检测方法的建立也可为低致敏性水产品的开发提供理论依据。通过组织捣碎、冷冻离心、热处理、Superdex75凝胶过滤等方法从鲢白色肉中纯化得到过敏原小清蛋白。Tricine-SDS-PAGE显示,在非还原条件下,该蛋白呈分子量分别为12ku、14ku、24ku的3个条带。而在还原条件下,仅有分子量为12ku的条带。Western-blotting分析表明,分子量为12ku、14ku和24ku的这3个条带都与小鼠抗蛙小清蛋白单克隆抗体(PARV-19)发生特异性反应,提示它们均为小清蛋白的不同形态。用纯化的小清蛋白制备多克隆抗体,经Protein A Sepharose亲和层析纯化得到高纯度的免疫球蛋白G(IgG)。Dot-blot检测发现,抗体稀释至1/51200时仍能与纯化的小清蛋白有显色反应。用制备的多克隆抗体进行Western-blotting分析,能特异地检测4种鱼(鲤、鲢、鲫、黄鳍鲷)中的小清蛋白。     

Partial characterization and activity distribution of proteases along the intestine of grass carp, Ctenopharyngodon idella (Val.)

A series of studies based on biochemical assays and electrophoretical observations has been conducted in order to investigate activity distributions and partially characterize various types of proteinases in the digestive tract of grass carp, Ctenopharyngodon idella (Val.). The casein digestion assays revealed that the presence of acidic proteinase had the highest activity at pH 2.5–3.0 and the alkaline proteinases at pH 10.0. The acidic proteinase activity distribution was found to decrease gradually from the oesophagus to the anus. Pepstatin A and EDTA inhibited the acidic proteinases activity. The SDS‐substrate‐PAGE showed that crude extraction of grass carp intestine contained an acidic proteinase active component with molecular mass of 28.5 ku. The substrate‐PAGE at neutral pH condition showed the presence of two acidic proteinase active components. The activity distribution of alkaline proteinase was found to slightly fluctuate along the intestine. And the whole intestine had very high activity. The inhibition assays and substrate specificity assays showed that trypsin was the main active component of the alkaline proteinases. The SDS‐substrate‐PAGE further showed that the crude extraction of grass carp intestine had four types of alkaline proteinase with molecular mass of 26.4, 30.8, 43.0 and 105.0 ku respectively. They were characterized to be trypsin (26.4, 30.8 and 43.0 ku) and un‐serine proteinase (105.0 ku) respectively. No chymotrypsin was detected.     

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.     

Purification and characterization of phospholipase A2 isoforms from the hepatopancreas of red sea bream, Pagrus major1

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.     

鲢肌球蛋白重链球状结构域的cDNA克隆及结构解析

根据已经获得的两种鲢肌球蛋白重链同工型基因(低温型sc-w和高温型sc-s)在3′端展现的明显差异,设计了2个特异性的反向引物,以鲤科鱼类肌球蛋白重链5′端的保守序列为正向引物,通过Long-PCR对编码鲢两种肌球蛋白重链同工型的球状结构域(Subfragment-1,S1)的全长基因进行了克隆和测序,并推断出它们一级结构的氨基酸序列。研究结果表明,sc-w与sc-s在S1的初级结构上显示80.5%的同源性、与已经报道的草鱼低温型(gc10)有97.2%的高同源性;sc-s则与草鱼中间型(gcI)和高温型(gc30)显示了分别为98.4%和97.1%的高同源性。低温型的sc-w和gc10在S1初级结构上展现的特有变异主要发生在43个氨基酸残基位点,其中15个属保守性残基。对S1区域中两个功能性的表面环loop1(与ATP结合位点有关)和loop2(与肌动蛋白结合位点有关)的结构解析发现,sc-w和gc10在两个表面环的长度、残基电荷分布和氨基酸组成等方面与其它同工型之间存在明显差异,揭示了这两个表面环的结构差异可能影响了栖息于不同环境温度下的淡水鱼的肌球蛋白分子马达功能。分子系统树的分析结果进一步证明,鱼类栖...     

天然滁州鲫氨基酸成分分析与营养价值评价

应用生化分析方法对滁州鲫鱼中氨基酸成分进行了定量分析,并对氨基酸含量进行营养分析评价。结果显示：滁州鲫鱼中氨基酸总量为86.35%（占干重）,其中8种必需氨基酸和2种半必需氨基酸总量为36.76%,4种呈味氨基酸含量为29.29%,必须氨基酸总量和呈味氨基酸含量均高于草鱼、鲢、鳙鱼等鱼类。表明滁州鲫较之其他鱼类是一种营养价值高,肉质鲜美的鱼类。     

Purification and characterization of a nucleoplasmin-like protein from carp (Cyprinus carpio) eggs

ABSTRACT: Nucleoplasmin, first isolated from Xenopus laevis eggs, promotes nucleosome assembly. Hereby, we have purified a nucleoplasmin-like protein from carp ( Cyprinus carpio ) eggs using ion exchange and subsequent gel filtration columns. The protein was recognized by a polyclonal antiserum against Xenopus laevis nucleoplasmin and had an amino acid composition similar to other member of the nucleoplasmin family proteins. Partial amino acid sequences from the cyanogen bromide (CNBr)-cleaved fragments showed high homology with Xenopus nucleoplasmin. The protein was also found to form an oligomeric complex and to be phosphorylated. Moreover, this protein promoted sperm nuclear decondensation as well as that of nucleoplasmin from Xenopus laevis eggs. These results suggest that the fish protein isolated here is a member of nucleoplasmin family.