羊红细胞铜锌超氧化物歧化酶的纯化及部分性质研究

目的：从羊红细胞中分离纯化铜锌超氧化物歧化酶(Cu，Zn—SOD)．并对其部分理化性质进行研究．方法：采用有机溶剂去除血红蛋白，热变性，超滤浓缩，丙酮沉淀、DF—FF柱色谱的方法．对羊红细胞Cu．Zn-SOD进行分离纯化。结果：羊红细胞520g得Cu，Zn—SOD总活力为467000IU．比活为8126μ／mg.pro，纯化倍数为26．2，活性回收率为61％，理化性质分析表明，该酶的最大紫外吸收波长为285nm，亚基相对分子量为16．1ku，每个亚基含1个铜原子和1个锌原子，pH在6-11范围内该酶稳定性很好，在35℃-75℃范围内，保温20min，酶活基本没有损失。2mmol／L的SDS对Cu，Zn—SOD没有明显的抑制作用，而2mmol／L的H2O2对该酶表现出较强的抑制作用，结论：采用此方法分离纯化的Cu，Zn—SOD达到电泳纯，其理化性质与其它动物血液来源的Cu，Zn—SOD一致。     

一株海洋细菌Pseudomonas sp.7—11所产蛋白酶的纯化及部分性质研究

海洋假单胞菌（Psedomonas sp.7-11)菌株所产蛋白酶经盐析,透析,离子交换层析后,得到了聚丙烯酰胺凝胶电泳纯的一酶组分,该酶的比活力从19．46U.mg^-1提高到13953115U.mg^-1,提高了717．02倍,回收率为1．24％,酶水解酪蛋白的最适作用温度为45度,最适PH为8．0;酶在pH6-7,50度以下时比较稳定,EDTA和IAA强烈抑制酶的活性SDS也具有一定的抑制作用,而PMSF对该酶的抑制作用不明显,,酶被EDTA失活后,Mn2 ,Mg2 能部分恢复其活力,尤其是Mn2,Hg2 ,Fe2 ,Zn2 ,Cu2 和Pb2 对酶有抑制作用,而Ca2 ,Mg2 和（NH4）SO4则有一定激活作用,该酶对乙和尿素有较强的抗性,对吐温20也具有一定的抗性,纯酶的相对分子质量大约30000Da。     

大豆超氧化物歧化酶的分离和鉴定

采用磷酸盐缓冲液使样品匀浆,两次硫酸铵分级沉淀和DEAE-Cellulose离子交换层析从大豆中分离出超氧化物歧化酶。分离酶的聚丙烯酰胺凝胶电泳呈一条蛋白谱带。过氧化氢和乙醇-氯仿试验表明,该酶属铜锌超氧化物歧化酶。酶的比活力为4710u/mg Pr,活力回收率23％,紫外吸收峰在265.4nm,分子量为32.4kD。     

重组人铜锌SOD的部分理化性质研究

对rhCu，Zn-SOD进行SDS-PAGE测定该SOD的亚基分子质量为19ku，该酶对热稳定，在pH5左右的活力最高，对乙腈、EDTA等抑制剂敏感，但是对SDS、尿素等变性剂表现出很好的稳定性。对该酶进行光谱分析表明，在265nm和673nm具有吸收峰，原子吸收光谱分析表明每分子SOD含有2个铜原子和2个锌原子，ESR表明铜原子价态为2价。可认为该重组酶与天然Cu，Zn-SOD在理化性质和结构上基本相同。     

羊红细胞铜锌超氧化物歧化酶的纯化及部分性质研究

目的从羊红细胞中分离纯化铜锌超氧化物歧化酶 (Cu ,Zn SOD) ,并对其部分理化性质进行研究。方法采用有机溶剂去除血红蛋白、热变性、超滤浓缩、丙酮沉淀、DE FF柱色谱的方法 ,对羊红细胞Cu ,Zn SOD进行分离纯化。结果羊红细胞 5 2 0g得Cu ,Zn SOD总活力为 4 6 70 0 0u ,比活为 812 6u/mg·pro,纯化倍数为 2 6 .2 ,活性回收率为 6 1%。理化性质分析表明 :该酶的最大紫外吸收波长为 2 5 8nm ,亚基相对分子量为 16 .1kD ,每个亚基含 1个铜原子和 1个锌原子。pH在 6～ 11范围内该酶稳定性很好 ,在 35℃～ 75℃范围内 ,保温 2 0min ,酶活基本没有损失。 2mmol/L的SDS对Cu ,Zn SOD没有明显的抑制作用 ,而 2mmol/L的H2 O2 对该酶表现出较强的抑制作用。结论采用此方法分离纯化的Cu ,Zn SOD达到电泳纯 ,其理化性质与其它动物血液来源的Cu ,Zn SOD一致。     

血清铜蓝蛋白的遗传学研究

铜蓝蛋白(Ceruplasmin)是一种含铜的血清α_2球蛋白和二氧化酶,故又称之为铜氧化酶,其活性通常与血清含铜量成正比.Wilson氏病患者的铜蓝蛋白会出现病理性的减低,而通常认为这种遗传性酶的缺陷是导致该病的主要原因.近来,有作者报告在精神疾病患者中血清铜蓝蛋白的异常,但机制不清.本文报道采用双生子法对血清铜蓝蛋白进行遗传学研究的结果.     

影响铜锌超氧化物歧化酶活性的几个因素

影响铜锌超氧化物歧化酶(简称Cu·Zn-SOD)活性的因素很多。溶液介电常数的增加减弱了酶分子活性中心附近ε-NH3_ 对O_2_-的静电吸引力,从而导致酶活的降低。CI_-浓度对Cu·Zn-SOD有明显的抑制作用是由于CI_-引起活性部位构象的变化。H_2o_2作用于Cu·Zn-SOD造成铜锌的脱落,从而影响酶结构的稳定性,造成酶结构损伤。H_2O_2是活性氧的一种,它的存在会引起酶二级结构的改变和导致SOD肽链的“随机”断裂,继而影响SOD的活性。     

肝豆状核变性基因与肿瘤耐药的研究进展

肝豆状核变性基因(ATP7B)定位于13q14．3区，编码一种铜转运P型ATP酶。ATP7B的突变使其蛋白缺乏或丧失转运肝铜的功能，导致肝、肾和脑等组织铜累积过多，表现为慢性肝病和(或)神经损害，ATP7B蛋白对铜的转运机制尚未明了。ATP7B在多种肿瘤细胞中表达对顺铂耐药，研究表明ATP7B除了是铜的转运蛋白外，还可能转运顺铂，但转运位点不清楚。ATP7B可能是一个新的肿瘤化疗耐药指标。     

IN型吉妮宫内节育器和带铜V200宫内节育器的临床效果比较

目的探讨两种活性宫内节育器的避孕效果和副作用的比较,IN型吉妮宫内节育器和带铜V200宫内节育器,临床各取200例随访观察一年。结果使用一年时间IN型吉妮IUD和带铜IUD的有效避孕率分别为98．6％和96．3％,差异无统计学意义;其白带增多、脱落率、出血率均P〈0．01,副作用P〈0．05,有明显差别。结论吉妮IUD比V200IUD避孕率高,具有安全性大、副作用少的特点。     

一种产蛋白酶菌的菌种选育及发酵条件研究

一株具有溶解血纤维蛋白活性的菌株A1,经亚硝基胍和紫外线复合诱变获得一株纤溶活性较高的突变株C22,其半成品酶比活力可达到2947．60u.mg^-1蛋白,产酶活力为未诱变菌株的4．23倍,本文对其产酶的是适条件进行了优化,结果表明产酶最适培养条件为：培养基组成2．5％豆饼粉,0．1％酵母提取物,0．2％NaCl,0.05%MgSO4.7H2O,0.001?SO4.7H2O,用0．05mol.L^-1,pH7.4的磷酸缓冲液配制;培养基起始PH7．0－7．5,25度旋转摇床培养40h。     

Purification and characterization of chick corneal beta-D-glucuronyltransferase involved in chondroitin sulfate biosynthesis

Beta-D-Glucuronyltransferase, which transfers D-glucuronic acid (GlcA) from UDP-GlcA to N-acetyl-D-galactosamine (GalNAc) at the nonreducing end of chondro-pentasaccharide-PA (pyridylamino-), GalNAcbeta1-(4GlcAbeta1-3GalNAcbeta1)2-PA, was purified 339-fold with an 11.0% yield from 2-d-old chick corneas by chromatography on DEAE-Sepharose, WGA-agarose, heparin-Sepharose, and 1st and 2nd UDP-GlcA-agarose (in the presence of Gal) columns. The activity was detected by fluorescence of PA residues of the product. The purified enzyme has an optimum pH of 7.0 (Mes buffer), and much higher activity toward chondro-heptasaccharide-PA than toward the chondro-pentasaccharide-PA, but no activity toward p-nitrophenyl-beta-GalNAc. The enzyme activity was almost completely inhibited by GalNAc (20 mm). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the purified enzyme fraction showed one band of 38 kDa with many other bands. The amino acid sequence was determined for the tryptic digests of the 38 kDa band protein. The sequences determined showed no homology to those of several beta-glucuronyltransferases reported previously. It seems that the enzyme is involved in the elongation of chondroitin sulfate chains in vivo.     

Biochemical characterization of the soluble alkaline phosphatase isolated from the venomous snake W. aegyptia

A soluble form of alkaline phosphatase (ALP) has been identified and purified from Walterinnesia aegyptia venom using an HPLC system Gold 126/1667 equipped with Protein PAK 125 and Protein PAK 60 columns. The enzyme was purified 3.4 fold over crude venom with a yield of 37.3%. On SDS-PAGE under non-reduced conditions the purified enzyme showed three bands of 212 kD, 80 kD, and 55 kD. However, under reducing conditions, the enzyme showed two bands of 80 kD and 55 kD. The specific activity of ALP was 24 U/mg with p-nitrophenylephosphate as the substrate. During isoelectric focusing experiments the ALP exhibited two bands focused at pH 6.2 and 6.8, which suggests that either the enzyme exists as two different isoforms or the two bands in IEF may be two subunits of 80 kD and 55 kD. The kinetic parameters (Km and Vmax) and IC50 of ALP inhibition by L-phenylalanine, L-leucine, imidazole, caffeine, orthophosphate and permanganate were also investigated in the present study. Zinc and cyanide ions at a concentration of 15 mM and 10 mM, respectively, completely inhibited the activity of W. aegyptia ALP.     

Oxidases involved in biosynthesis of macrolide antibiotic patulolides from Penicillium urticae S11R59

Patulolides are a group of 12-membered macrolide antibiotics produced by Penicillium urticae S11R59. An enzyme involved in the conversion of patulolide C to patulolide A was purified from P. urticae S11R59 and characterized. The enzyme showed a single band on SDS-PAGE and molecular sieve HPLC both of which indicated a Mr of 86,000, indicating that the enzyme is monomeric. However, the enzyme was separated into two bands of very similar pI's (pI 4.2 and 4.3) by isoelectric focusing. Both bands catalyzed the conversion of patulolide C to patulolide A, as demonstrated by activity staining. The two isoenzymes were proved to be oxidases by the simultaneous production of H2O2 during the conversion of patulolide C to patulolide A. The molar ratio for patulolides C, A and H2O2 was determined to be 1:1:1. The optimum pH and temperature were determined to be 7 and 35-40 degrees C, respectively, and the enzymes were stable at pH 6-9 and 4-40 degrees C. The oxidases showed characteristic absorption at 345 and 450 nm, indicating the presence of flavin as coenzyme. Among several analogues of patulolide C tested, the oxidases showed very narrow substrate-specificity; only patulolide C was oxidized to patulolide A. No enzyme activity for the reverse reaction, i.e. from patulolide A to patulolide C, was present in the cell-free extract of P. urticae S11R59. Patulolide C oxidases therefore play a key role in the biosynthesis of patulolides.     

Separation and characterization of venom components in Loxosceles reclusa—III. Hydrolytic enzyme activity

Fractions 3, 4, 5, 6, 8 and 9 of the prep-disc electrophoretic separation of Loxosceles reclusa spider venom showed enzymic activity toward the hydrolysis of indoxyl-acetate and were therefore considered as hydrolytic enzyme components in the spider venom. Fraction 5 was designated as a lipase due to its higher affinity for the longer chain synthetic substrates. Fraction 6 was designated as a nonspecific esterase due to its action on alkyl and phosphate esters. Fraction 9 was designated as the primary alkaline phosphatase activity in the venom. Electrophoretic assays revealed two venom protein bands exhibiting alkaline phosphatase and three venom proteins exhibiting esterase activity. Two of the bands were arylesterases and one was an aliesterase; acetylcholinesterase was not detected.     

Tacrine protection of acetylcholinesterase from inactivation by diisopropylfluorophosphate: a circular dichroism study

Tacrine (1,2,3,4-tetrahydro-9-aminoacridine) showed an apparent noncompetitive inhibition of Torpedo acetylcholinesterase (AChE) with a dissociation constant, Ki, of 8.5 nM. It altered the CD bands of AChE in the near-UV region, which monitor the local conformation of aromatic side groups, but not those in the far-UV region, which measure the secondary structure. An extrinsic CD band was induced at 348 nm, with a molar ellipticity of 35,000 deg cm2 dmol-1 (bases on tacrine), when each AChE subunit (Mr = 67,000) was saturated with one tacrine (mol/mol). With this band as a probe, the bound tacrine could be displaced by edrophonium or decamethonium, both of which are known to bind to the anionic site at the active center of AChE, but not by propidium, which binds to the peripheral site of the enzyme. Tacrine protected AChE from inactivation by diisopropylfluorophosphate (DFP). AChE completely lost its enzymatic activity when 1 mol of DFP was bound per mol of subunit upon incubation of 7 microM AChE (subunit) with 100 microM DFP for 40 min, but tacrine-treated AChE retained 60% of its activity and bound only 0.2 mol of DFP per mol of subunit under similar conditions. The corresponding CD, at 348 nm, of the AChE-tacrine-DFP complex increased or decreased gradually, depending on the order of addition of tacrine and DFP, and reached an equilibrium value (80% of its original) after 2 days. The difference absorption spectrum of the AChE-tacrine-DFP complex was the same as that of the AChE-tacrine complex. These results suggest that the protective effect of tacrine may be due to steric hindrance at the esteratic site of the enzyme.     

角果木树皮来源放线菌多样性及生物活性初探

摘要：勘探红树植物角果木树皮来源放线菌多样性,挖掘具有高效广谱抗农用真菌、高产多种功能酶和一定耐药性的放线 菌,为开发农用生防菌肥提供参考依据。应用可培养技术和16S rRNA基因序列的系统发育分析研究红树植物角果木中可培养放 线菌的多样性,并对其进行抑制植物病原菌、功能酶活性及其基因组DNA的聚酮合酶(PKS)基因与非核糖体肽合成酶(NRPS)基 因筛选;同时对抑菌活性菌株开展有机磷农药耐受试验。结果表明,从角果木树皮中共分离到30种放线菌,隶属于10科12属; 从中筛选出10株放线菌在至少1个抗菌活性检测中显示阳性,且对多种有机磷农药均表现出一定的耐受性;此外,其中24株放 线菌具有至少一种功能酶活性,及18株放线菌的基因组DNA扩增出抗生素生物合成基因。综上所述,海南东寨港红树林保护区 内红树植物角果木树皮来源放线菌具有丰富的物种多样性和多种显著的生物活性。     

Inflammatory action of 8-methoxypsoralen-spermine photoproduct (8-MOP-Spm-P(GFC)) and effects of various drugs on rat paw edema induced by 8-MOP-Spm-P(GFC)

The photoproducts produced by irradiating 8-methoxypsoralen (8-MOP) in the presence of spermine (Spm) were fractionated using gel filtration chromatography (GFC) on a Sephadex G-25 column. As a result, two bands which were characterized by the effects on hyaluronidase activity were obtained. The first band strongly activated the hyaluronidase, but a second band did not exhibit any effect on the enzyme activity. The first and second bands contained photoproducts with molecular weights (MW)>2700 and MW<728, respectively, determined by the GFC method. The photoproducts, 8-MOP-Spm-P(GFC) obtained from the first band, but not the photoproducts with lower MW from the second band, showed enzyme activating action. 8-MOP-Spm-P(GFC) induced paw edema, which was stronger in the first phase than the second one in rats, differing from that induced by carrageenin. This photoproduct was a substance with lower cell toxicity because it did not cause hemolysis on red blood cells or the release of lactic dehydrogenase from mast cells in rats. The effects of various drugs on 8-MOP-Spm-P(GFC)-induced edema were investigated. As a result, edema formation was inhibited by drugs with an anti-histaminic action, such as alimemazine, dl-chlorpheniramine, promethazine, ketotifen and azelastine, and with anti-serotonin action such as cyproheptadine. On the other hand, tranilast did not show significant inhibition and indomethacin showed a tendency to increase its formation. These results suggested that 8-MOP-Spm-P(GFC) is a new inflammatory substance and is very useful as an agent to develop new anti-inflammatory drugs without cyclooxygenase inhibitory action.     

Inhibition of eel enzymatic activities by cadmium

The aim of the present work was to study the in vitro effect of cadmium on enzymes, such as intestinal and branchial carbonic anhydrase (CA) and Na(+)-K(+)-ATPase which play a key role in salt- and osmoregulation and acid-base balance in the teleost fish, Anguilla anguilla. Carbonic anhydrase activities in gill and intestinal homogenates were significantly inhibited by CdCl(2), the gill CA being more sensitive to the heavy metal (IC(50) for the branchial CA=9.97+/-1.03x10(-6) M, IC(50) for the intestinal CA=3.64+/-1.03x10(-5) M, P<0.01). With regards to the intestinal CA activity, it has been shown in a previous study (Maffia et al., 1996) that two isoforms exist, a cytosolic and a brush-border membrane bound. These two isoforms show a different sensitivity to cadmium, with the membrane-bound enzyme less sensitive with respect to the cytosolic one, since it showed still an incomplete inhibition at the highest cadmium concentration tested. The inhibition of all the CA activity tested revealed a time-dependence since it required at least 10 min (1 h for the membrane-bound isoform) preincubation with the heavy metal to appear. Na(+)-K(+)-ATPase enzymatic activities, measured in intestinal and branchial homogenates, were inhibited by cadmium in a dose-dependent manner, with the branchial activity being more sensitive to the action of the heavy metal than the intestinal one (IC(50) for the branchial enzyme=1.38+/-0.09x10(-7) M, IC(50) for the intestinal enzyme=2.86+/-0.02x10(-7) M, P<0.01). The most of inhibition of the enzyme appeared without any preincubation with the heavy metal. Mg(2+)-ATPase activity was not significantly altered by the in vitro cadmium exposure either in the gills or in the intestine. These findings observed in vitro could be useful in the understanding of the toxic effects that cadmium elicits on aquatic organisms in vivo. In fact, the impairment of the activity of enzymes which carry out key physiological roles could cause alterations of the physiology of the whole organism.     

Solubilization and separation of ethacrynic acid (EA) highly sensitive and EA less sensitive Mg2+-ATPases in the rat brain

Rat brain microsomal Mg2+-ATPases with two distinct activities: ethacrynic acid (EA) highly sensitive and EA less sensitive Mg2+-ATPase activities were solubilized by the combined treatment with 10 mM 3-(3-chlolamidopropyl)-dimethylammonio-1-propane-sulfate (CHAPS) and 30 mM octyl-beta-D-glucoside. The solubilized enzymes had properties similar to those of the membrane-bound enzyme in microsomes with respect to the sensitivity to EA and Cl-, although the optimal pH and the affinity to ATP were slightly altered after the solubilization. Fast protein liquid chromatography of the solubilized enzymes on an anion-exchanger (Mono Q) column with a linear NaCl gradient (0-1.0 M) yielded separate peaks for EA highly sensitive and EA less sensitive Mg2+-ATPase activities at 0.1 and 0.35 M NaCl, respectively. Polyacrylamide gradient gel electrophoresis of the samples from the peak-fractions of EA highly sensitive and EA less sensitive Mg2+-ATPase activities yielded prominent bands at 600 and 70 kDa, respectively. These results indicate that EA highly sensitive Mg2+-ATPase is solubilized and separated from EA less sensitive Mg2+-ATPase as a large enzyme molecule with anion-sensitive sites.     

Soluble epoxide hydrolase in rat inflammatory cells is indistinguishable from soluble epoxide hydrolase in rat liver.

Soluble epoxide hydrolase (sEH) is a ubiquitous mammalian enzyme for which liver and kidney are reported to have the highest activity. We have shown that the soluble epoxide hydrolase (sEH) activity present in rat neutrophils and macrophages is kinetically, immunologically, and physically indistinguishable from rat liver cytosolic sEH. Cytosol from rat liver or inflammatory cells and recombinant rat sEH were incubated with trans-diphenylpropene oxide (tDPPO), a selective substrate for sEH. The tDPPO hydration activity we observed in inflammatory cell cytosol was lower than that from liver. The Km for tDPPO hydration observed in rat inflammatory cell cytosol was the same as the Km for rat liver cytosol (10 microM). Recombinant rat sEH and cytosol from rat liver or inflammatory cells were incubated with the sEH inhibitors, chalcone oxide, 4-fluorochalcone oxide, and 4-phenylchalcone oxide. The IC50 values were 40, 8, and 0.4 microM, respectively, in all samples. Furthermore, sEH activity could be completely immunoprecipitated out of the samples, and the amount of antibody required to do so was apparently identical, regardless of the source of enzyme. SDS-polyacrylamide gel electrophoresis followed by Western blot analysis revealed a single sEH band with a molecular weight of 62 kDa. Isoelectric focusing followed by Western blot analysis revealed multiple bands containing tDPPO-hydrating activity. Although the inflammatory cell bands had the same pattern as those from liver cytosol, the recombinant sEH showed a different banding pattern. These multiple bands were not an artifact of the IEF gel selected. Furthermore, in a 2-dimensional IEF gel, the bands re-migrated to the same position. The presence of sEH in inflammatory cells suggests that this enzyme may have an important endogenous function.