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1.
Ca^++离子能极大地激活五步蛇蛇毒PLA2的酶活力,Na^+离子只能在一定程度上激活,作者推测在测定PLA2酶活力的底物溶液中可以不加Na^+离子。 相似文献
2.
以嗜热脂肪芽孢杆菌为材料,通过PolyminP沉淀、硫酸铵分级及DEAE纤维素、磷酸纤维素、Blue-Sepharose、FPLCMonoQ、FPLC Superose12等柱层析,得到了部分纯化DNA解链蛋白BstH2。BstH2具有受DNA促进的ATP酶活力,没类型的核酸对BstH2的ATP酶活力的促进作用没。BstH2在55℃有最高ATP酶活力。这种活力受大肠杆菌单链DNA结合蛋白的抑制及随 相似文献
3.
嗜热脂肪芽孢杆菌DNA解链蛋白1的纯化及性质 总被引:1,自引:1,他引:0
以嗜热脂肪芽孢杆菌为材料,通过PolyminP沉淀,硫酸铵分级及Phenyl-Sepharose,DEAE纤维素,磷酸纤维素,FPLC MonoQ,FPLC Superose12等柱层析,得到部分纯化的DNA解链蛋白1。BstH1具有依赖DNA和Mg^2+的ATP酶活力,不同类型的核酸对BstH1的ATP酶活力的促进作用不同。 相似文献
4.
本文应用离子交换柱层析和凝胶过滤技术从福建产圆斑蝰蛇泰国亚种蛇毒中纯化出一个新的PLA,(PLA2-3)。经聚丙烯酰胺凝胶电泳,SDS-聚丙烯酰胺凝胶电泳和Ouchterlony双向免疫扩散证明为均一蛋白质。其理化及酶学性质如下:由约120个氨基酸残基组成(不包括半胱氨酸和色氨酸);分子量为14800;等电点为7.8;PLA2酶比活力用自动电位滴定法测定为255μmol/min·mg,用间接溶血法测定半数溶血量(HU50)在家兔和大白鼠分别为0.025μg/ml和0.05μg/ml;小鼠静脉注射的近似LD50为3154mg/kg。与从该蛇毒中分离出的毒性和碱性PLA,相比,具有PLA。酶比活力高,毒性小的特点。此酶具有明显降压作用。抗血小板聚集效应和可逆性负性心肌收缩力作用,但未观察到心肌挛缩现象. 相似文献
5.
产碱菌麦芽四糖淀粉酶的化学修饰 总被引:7,自引:0,他引:7
不同蛋白质侧链修饰剂对麦芽四糖淀粉酶进行修饰。在一定条件下,分别用IAA、NEM、EDC和NAI处理后,酶活力不受影响,仍为100%,说明巯基、羧基和酪氨酸残基与酶活力无关。用DEP、NBS和HNBB修饰后,酶活力大幅度下降,说明组氨酸和色氨酸基为酶活力所必需。 相似文献
6.
利用荧光光谱学对8PLA_2和Ca ̄(2+)相互作用的研究表明,Ca ̄(2+)在BPLA_2中十分重要。在Ca ̄(2+)存在时,底物与BPLA_2的结合使酶中色氨酸残基周围的环境变得较为疏水,荧光发射谱蓝移达9nm,而无Ca ̄(2+)存在时却无此现象发生;实验证明BPLA_2分子中有一较强的疏水区,Ca(2+)可明显增强这一区域的疏水性;另外,我们还发现Ga ̄(2+)与酶的结合与酶中唯一的His残基有关。 相似文献
7.
江浙蝮蛇毒酸性与碱性磷脂酶A_2溶液构象变化的差示扫描量热法研究 总被引:1,自引:0,他引:1
运用差示扫描量热法,在不同pH值的缓冲溶液内和各种浓度的碱土族氯化物溶液内,研究了来自江浙蝮蛇(AgkistrodonhalysPallas)毒的酸性与碱性磷脂酶外A2(PLA2)的热变性过程。得到表征这两种酶溶液构象变化的热力学参数。依据这些参数研究了两者的溶液构象及其变化。在pH4.5以下,分子净荷正电的这两种酶在溶液中不形成可热致伸展的有序构象;pH高于4.5时,Asp和Glu的侧链羧基以负离子形式存在有利于有序构象的稳定。His是决定PLA2活力和热稳定性的重要残基。磷酸根离子和这两种酶有结合作用而降低有序构象的热稳定性。碱土族阳离子除和这两种酶结合外,还以依赖于离子强度的方式复杂地影响酶的溶液构象,但其作用不完全是静电性的,是或多或少地随离子的不同而不同的。计算给出酸性PLA2的△Hcd. 相似文献
8.
9.
PEG修饰的辣根过氧化物酶及其在非水介质中的性质 总被引:3,自引:0,他引:3
酶的化学修饰可以明显提高酶在有机相中的活力。通过氧化过氧化物酶(HRP)的糖链后引入氨基再连接甲氧基聚乙醇(PEG)5000和在酶的肽链上连接PEG5000,发现HRP多肽链上修饰后的酶在水相中的活力几乎没有变化,但通过氧化糖链连接PEG的酶在水相中的活力下降近2倍。在甲苯及二氧六环含量较高的体系中,修和均呈上升趋势。特别在甲苯体系中两种修饰酶活力都比未经修饰的酶提高了近2倍。稳定性研究表明,不论 相似文献
10.
磷脂酶A2对中性粒细胞趋化和粘附的影响 总被引:1,自引:0,他引:1
胰源性14×10 ̄3u磷脂酶A_2(PLA_2)在体外同大鼠中性粒细胞(PMN)培养60min后,细胞对TNF趋化增强,培养10min后上清液中血栓素(TXB_2)含量比正常增高(P<0.01)。前列环素(PGI_2)含量不变。PLA_2激动剂A23187也能显著加强中性粒细胞对TNF的趋化,并伴有TXB_2释放增多(P<0.01)。此外,PLA_2和A23187还显著增强PMN对玻璃珠的粘附活性。使用PLA_2抑制剂二溴苯乙酮(PBPB)和PLA_2多克隆抗体可抑制外源性PLA_2对PMN趋化和粘附的增强作用,但对A23187的调节作用无效。以上结果表明PLA_2激活及其代谢产物可能介导PMN趋化和粘附作用。 相似文献
11.
Pei-Hsiu Kao Ku-Chung Chen Shinne-Ren Lin Long-Sen Chang 《Journal of peptide science》2008,14(3):342-348
Modification of His-47 and removal of the N-terminal octapeptide caused a different effect on the structure of Naja naja atra (Taiwan cobra) phospholipase A2 (PLA2). Unlike native enzyme, Ca2+ induced an alteration in the structural flexibility of His-modified PLA2. Moreover, the spatial positions of Trp residues in His-modified PLA2 were not properly rearranged toward lipid-water interface in the presence of Ca2+. CD spectra and fluorescence measurement showed that the dynamic properties of Trp residues and the gross conformation of N-terminally truncated PLA2 were totally different from native enzyme. Although a precipitous drop in the enzymatic activity was observed with modified PLA2, His-modified PLA2 and N-terminally truncated PLA2 retained cytotoxicity on inducing necrotic death of human neuroblastoma SK-N-SH cells. Our data suggest that structural perturbations elicited by the chemical modification cause a dissociation of enzymatic activity and cytotoxicity of PLA2. 相似文献
12.
Three monoclonal antibodies (mAbs) against Naja naja atra phospholipase A2 (PLA2) were prepared by hybridoma technique. Of which two mAbs, 1E531 and 5F92 inhibited the enzymatic activity of PLA2, but 5F6F10 did not. 1E531 and 5F92 nearly exhibited the same binding patterns which was different from that observed with 5F6F10 toward the chemically modified derivatives and homologous variants of PLA2 as revealed by enzyme immunoassay. This suggests that, based on the results of comparative analysis on their relative reactivity with modified derivatives and PLA2 homologues, the mAbs with different immunochemical properties could be differentiated. 相似文献
13.
Bianco ID Daniele JJ Delgado C Fisher D Francis GE Fidelio GD 《Bioscience, biotechnology, and biochemistry》2002,66(4):722-729
Secretory phospholipases A2 (PLA2) from Naja naja naja (cobra snake) venom, from Bothrops neuwiedii (crotalid snake) venom (two isoforms) and from bee venom were modified with tresylated monomethoxy poly(ethylene glycol) (TMPEG). The kinetic and inflammatory properties of the adducts (PEG-PLA2) were measured. As found by gel permeation chromatography, 95-100% of P-1 PLA2 from B. neuwiedii and PLA2 from N. naja naja venom change their chromatographic mobility after TMPEG treatment. By contrast, only 50-60% of both P-3-PLA2 from B. neuwiedii and PLA2 from bee venom modify their elution profile from Superdex 75. All the modified proteins preserved the enzymatic activity toward phospholipid monolayers, but with a reduced specific activity and greater lag times than the unmodified controls. These results suggest that the PEG-PLA2 complexes would have an altered interaction with lipid membranes. The PEG-linked proteins preserve their edema-inducing activity evaluated by the rat hind-paw edema test except for N. naja naja PEG-PLA2 in which inflammatory activity was significatively decreased. Altogether, the results show a partial dissociation of catalytic and inflammatory activities of Group II and III secretory PLA2s after their modification with PEG. 相似文献
14.
We have isolated, from canine pancreatic juice, two 14-kDa proteins with secretin-releasing activity that had N-terminal sequence homology with canine pancreatic phospholipase A2 (PLA2). In this study we have obtained evidence that secretin-releasing activity is an intrinsic property of pancreatic PLA2. Porcine pancreatic PLA2 from Sigma or Boehringer Mannheim was fractionated into several peaks by reverse phase high performance liquid chromatography. They were tested for stimulation of secretin release from murine neuroendocrine intestinal tumor cell line STC-1 and secretin cells enriched mucosal cell preparations isolated from rat upper small intestine. Each enzyme preparation was found to contain several components of secretin-releasing activity. Each bioactive fraction was purified to homogeneity by rechromatography and then subjected to mass spectral analysis and assays of PLA2 and secretin-releasing activities. It was found that the fraction with highest enzymatic activity also had the highest secretin-releasing activity and the same Mr as porcine pancreatic PLA2. Moreover, it also had the same N-terminal amino acid sequence (up to 30 residues determined) as that of porcine pancreatic PLA2, suggesting that it was identical to the enzyme. Purified porcine pancreatic PLA2 also stimulated secretin release concentration-dependently from both STC-1 cells and a mucosal cell preparation enriched in secretin-containing endocrine cells isolated from rat duodenum. Abolishment of the enzymatic activity by pretreatment with bromophenacyl bromide did not affect its secretin-releasing activity. The stimulatory effect of purified pancreatic PLA2 on secretin secretion from STC-1 cells was inhibited by an L-type Ca2+ channel blocker, by down-regulation of protein kinase C or by pretreatment of the cell with pertussis toxin. It is concluded that porcine pancreatic PLA2 possesses an intrinsic secretin-releasing activity that was independent of its enzymatic activity. This action is pertussis toxin-sensitive and is in part dependent on Ca2+ influx through the L-type channel and activation of protein kinase C. 相似文献
15.
The edema inducing activity of phospholipase A2 (PLA2) enzymes from snake venoms and porcine pancreas was investigated using mouse paw as experimental model. All ten PLA2 enzymes exhibited potent edema inducing activity. PLA2, however, is generally not the major edema inducing component of snake venom. Chemical modification studies indicated that enzymatic activity of PLA2 was required for its edema inducing activity. All PLA2 enzymes examined displayed a rapid onset edema which was suppressed by pretreatment of the mice with antihistamine. Dexamethasone pretreatment also inhibited edemas elicited by some PLA2 enzymes. 相似文献
16.
The goal of the present study is to elucidate the mechanism of quercetin on modulating Naja naja atra phospholipase A2 (PLA2) activities. Sphingomyelin inhibited PLA2 enzymatic activity and membrane-damaging activity against egg yolk phosphatidylcholine (EYPC), while cholesterol and quercetin abrogated the sphingomeyelin inhibitory effect. Quercetin incorporation led to a reduction in PLA2 enzymatic activity and membrane-damaging activity toward EYPC/sphingomyelin/cholesterol vesicles. Both cholesterol and quercetin increased detergent resistance and reduced membrane fluidity of EYPC/sphingomyelin vesicles. Quercetin reduced detergent insolubility but increased ordered lipid packing of EYPC/sphingomyelin/cholesterol vesicles. Acrylamide quenching studies and trinitrophenylation of Lys residues revealed that quercetin altered the membrane-bound mode of PLA2 differently upon absorption onto the membrane bilayers of different lipid compositions. However, 8-anilinonaphthalene sulphonate-binding assay revealed that quercetin marginally affected the interaction between active site of PLA2 with phospholipid vesicles. Collectively, our data indicate that membrane-inserted quercetin modulates PLA2 interfacial activity and membrane-damaging activity via its effects on membrane structure and membrane-bound mode of PLA2. 相似文献
17.
《Biochimica et Biophysica Acta (BBA)/Molecular and Cell Biology of Lipids》2017,1862(7):692-705
Lipid droplet (LD) accumulation in hepatocytes is a typical character of steatosis. Hepatitis C virus (HCV) infection, one of the risk factors related to steatosis, induced LD accumulation in cultured cells. However, the mechanisms of which HCV induce LD formation are not fully revealed. Previously we identified cytosolic phospholipase A2 gamma (PLA2G4C) as a host factor upregulated by HCV infection and involved in HCV replication. Here we further revealed that PLA2G4C plays an important role in LD biogenesis and refined the functional analysis of PLA2G4C in LD biogenesis and HCV assembly. LD formation upon fatty acid and HCV stimulation in PLA2G4C knockdown cells was impaired and could not be restored by complementation with PLA2G4A. PLA2G4C was tightly associated in the membrane with the domain around the amino acid residues 260–292, normally in ER but relocated into LDs upon oleate stimulation. Mutant PLA2G4C without enzymatic activity was not able to restore LD formation in PLA2G4C knockdown cells. Thus, both the membrane attachment and the enzymatic activity of PLA2G4C were required for its function in LD formation. The participation of PLA2G4C in LD formation is correlated with its involvement in HCV assembly. Finally, PLA2G4C overexpression itself led to LD formation in hepatic cells and enhanced LD accumulation in the liver of high-fat diet (HFD)-fed mice, suggesting its potential role in fatty liver disease. 相似文献
18.
Noetzel C Chandra V Perbandt M Rajashankar K Singh T Aleksiev B Kalkura N Genov N Betzel C 《Zeitschrift für Naturforschung. C, Journal of biosciences》2002,57(11-12):1078-1083
Vipoxin from the venom of Vipera ammodytes meridionalis is an unique neurotoxic complex between a toxic phospholipase A2 and a highly homologous non-toxic protein inhibitor. It is an example of evolution of a catalytic and toxic function into inhibitory and non-toxic one. The activity of the V. ammodytes meridionalis toxin is 1.7 times higher than that of the closely related (92% sequence identity) neurotoxic complex RV4/RV7 from the venom of Vipera russelli formosensis The enhanced enzymatic activity of vipoxin is attributed to limited structural changes, in particular to the substitutions G54R and Q78K in the PLA2 subunit of the complex and to the T54R substitution in the inhibitor. Oleyloxyethylphosphocholine, aristolochic acid and vitamin E suppressed the enzymatic activity of vipoxin and its isolated PLA2 subunit. These compounds influence inflammatory processes in which PLA2 is implicated. The peptide Lys-Ala-Ile-Tyr-Ser, which is an integral part of the PLA2 components of the two neurotoxic complexes from V. ammodytes meridionalis and V. russelli formosensis (sequence 70-74) activated vipoxin increasing its PLA2 activity by 23%. This is in contrast to the inhibitory effect of the respective pentapeptides with 70-74 sequences on other group II PLA2s. Surprisingly, the same peptide inhibited 46% of the V. russelli formosensis PLA2 activity. The limited changes in the structure of the two highly homologous neurotoxins lead to considerable differences in their interaction with native peptides. 相似文献
19.
Arachidonic acid (AA) mobilization by phospholipase A2 (PLA2) and subsequent prostaglandin synthesis is considered to be a pivotal event in inflammation. The purpose of this study was to assess the efficacy of a Type II PLA2 specific inhibitor, SB 203347, in reducing prostaglandin production in Type II PLA2-transfected Chinese hamster ovary (CHO) cells and in human placenta. In both experimental models utilised, Type II PLA2 represents the principal isozyme contributing to total PLA2 enzymatic activity. PLA2 enzymatic activity released into cell culture media and placental explant media was quantified by radiolabelled substrate assay [14C-phosphatidylethanolamine (PE)]. Immunoreactive prostaglandin F2alpha (PGF2alpha) concentrations were determined by radioimmunoassay. SB 203347 (at 0.1-10 microM final concentration) inhibited PLA2 enzymatic activity released by Zn++ -activated CHO cells by up to 60% (P<0.0001). The concentration of PGF2alpha present in culture media was concomitantly reduced by up to 90% (P<0.0001). Similar results were observed for human placental explants. Treatment of human placental explants with SB 203347 (1 microM final concentration) significantly reduced PLA2 enzymatic activity recovered in media after 24 h incubation (P<0.0001; n = 10). Incubation media PGF2alpha concentrations were also reduced by 60% (P<0.00001). The addition of endogenous arachidonic acid (30 microM final concentration) significantly attenuated SB 203347-inhibition of PGF2alpha release (P<0.01). The data obtained in this study are consistent with the hypothesis that Type II PLA2 contributes to the liberation of arachidonic acid for prostanoid formation in human placenta and in cells that abundantly express this isozyme. 相似文献
20.
采用微孔比色法及荧光分析法 ,研究抗男性生育化合物棉酚与猪胰腺磷脂酶A2 (phospho lipaseA2 ,PLA2 ,EC3 1 1 4 )温育并透析前后对酶活力及荧光的影响 .结果表明 ,棉酚与PLA2 不可逆地结合明显地降低了PLA2 活力及荧光强度 .棉酚对酶活力抑制作用的IC50 为 35μmol L ;当其浓度达到 80 μmol L时 ,能够完全抑制PLA2 ( 4 11μmol L)对合成底物 2 硫代十六酰乙基磷酸胆碱(HEPC ,0 .2 5mmol L)的水解作用 .PLA2 的最大激发波长与发射波长分别为 2 75nm ,34 3nm ,荧光强度与酶浓度呈良好的线性关系 .棉酚对PLA2 的荧光具有较强的淬灭作用 .由于PLA2 与男性生育密切相关 ,棉酚对PLA2 活力的影响可能是其避孕作用及伴随的副作用的一种新的重要机制 相似文献