重组人核苷二磷酸激酶A的理化性质

对重组人核苷二磷酸激酶A（rhNDPK-A）进行纯化,并对重组产物的理化性质及在溶液中的聚合状态进行鉴定。NDPK-A工程菌发酵后的菌体高压匀浆,然后微孔过滤、超滤浓缩,所得样品经DEAE阴离子交换、Cibacron Blue亲和层析、分子筛层析三步纯化后,以SDS-PAGE和RP-HPLC分析纯化产物的纯度,RP-HPLC测定酶活性。合格制品以基质辅助激光解析飞行时间质谱测定相对分子质量（MW）;Edman降解法测定N末端序列;多角度激光散射法测定重组产物在溶液中的表观分子量。结果表明,rhNDPK-A纯化产物的SDS-PAGE纯度为97.3%,RP-HPLC纯度为99.2%;比活性为（900±100）u/mg;单体相对分子质量为17017,与NDPKA分子量理论值相差132。测序结果表明,rhNDPK-A N末端缺失Met残基,其理论分子量为17017,与飞行质谱测定结果完全一致。表观分子量测定结果表明,rhNDPK-A在溶液中形成六聚体,表观分子量为102kD。上述结果说明, NDPK-A重组产物具与天然产物相同的自发形成六聚体性质,这为NDPK-A新药开发和机理研究打下了良好基础。     

氧化还原对NDPK-A及突变体磷酸转移酶活性的影响

对核苷二磷酸激酶A(NDPK-A)及其4种半胱氨酸突变体进行诱导表达及纯化,测定它们在氧化还原条件及正常条件下的磷酸转移酶活性,研究氧化还原及二硫键异构对NDPK-A及突变体活性的影响。将实验室之前构建成功的野生型NDPK-A(PBV-NDPK-A)及4种突变型NDPK-A基因(PBV-NDPK-A C4S,PBV-NDPK-A C109S,PBV-NDPK-A C145S,PBV-NDPK-A C4/109/145S)在大肠杆菌中高效表达;以DEAE-sepharose Fast Flow离子交换层析与Cibacron Blue 3GA Sepharose CL-4B亲和层析技术纯化目的蛋白;HPLC法测定比较野生型NDPK-A及突变体在氧化还原和正常环境下磷酸转移酶活性。结果显示,NDPK-A及突变体在大肠杆菌中高效表达;经纯化分别获得了均一的NDPK-A蛋白及突变体蛋白,纯度均达到98%;在还原环境下NDPK-A及突变体的磷酸转移酶活性均高于正常环境下的活性,但是在氧化环境下的磷酸转移酶活性明显低于正常环境下。氧化还原环境对NDPK-A结构异构及磷酸转移酶活性有一定的影响,提示氧化还原环境可能调控NDPK-A二硫键的形成,影响蛋白的聚集状态,从而影响蛋白的磷酸转移酶活性,并且NDPK-A结构中可能有更为复杂的氧化还原调控酶活性机制。     

液相色谱-串联质谱分析化学合成多肽及其主要副产物(英文)

用与反相高效液相色谱偶联的串联质谱 (RPHPLC MS MS)分析一个固相化学合成的九肽H2 N Tyr Val Asn Val Asn Met Gly Leu Lys CONH2 及其 2个主要的副产物 .根据Fmoc化学 ,在Fmoc PAL PEG·PS树脂上从C端至N端手工操作逐步偶联合成九肽 .偶联完成后 ,用试剂R(90 %TFA ,5 %苯甲硫醚 ,3%二巯基乙烷 ,2 %苯甲醚 )室温 (2 0～ 2 5℃ )处理 2 5h ,进行多肽的去保护和切除树脂 .RP HPLC分析结果表明 ,合成粗品中含有 1个主要成分、2个次要成分及多个微量成分 .用RPHPLC MS MS分别对主要成分和 2个次要成分进行了鉴定 .结果证明 ,主要成分即为目标九肽 ;先于目标肽洗脱的副产物分子量比目标肽的分子量多 16 .MS MS证明 ,该副产物包括 2种九肽衍生物 :1种为其Tyr1氧化生成 ,另 1种为Met6氧化生成 ;后于目标肽洗脱的副产物为九肽的Tyr1残基增加 12所致 ,这种现象尚未见报道 .对副产物形成的可能机制进行了讨论     

叶绿体硫氧还蛋白系统的调节机制

硫氧还蛋白(Trx)属于巯基-二硫键氧化还原酶家族, 通过作用于底物蛋白侧链2个半胱氨酸残基之间的二硫键(还原、异构和转移)来调控胞内蛋白的结构和功能。叶绿体Trx系统包括Trx及Trx类似蛋白、铁氧还蛋白(Fd)依赖的硫氧还蛋白还原酶(FTR)和还原型烟酰腺嘌呤二核苷磷酸(NADPH)依赖的硫氧还蛋白还原酶C (NTRC)。除了基质蛋白酶类活性变化及叶绿体蛋白的转运受Trx系统调控之外, 在叶绿体中还存在1条跨类囊体膜的还原势传递途径, 把基质Trx的还原势经跨膜转运蛋白介导, 最终传递给类囊体腔蛋白。FTR和NTRC共同作用维持叶绿体的氧化还原平衡。该文对叶绿体硫氧还蛋白系统的调节机制进行了综述, 同时讨论了叶绿体硫氧还蛋白系统对维持植物光合效率的重要意义。     

N末端亲和标签可增进人硫氧还蛋白-1对氧化剂的敏感性

人细胞质硫氧还蛋白(hTrx1)在抗氧化和氧化还原调控中起重要作用.如果静脉注射重组hTrx1,动物抗氧化能力将增高.近年来,随着人们对氧化还原调控的关注,hTrx1需求不断增加.为了快速获得高纯度重组hTrx1,N末端亲和标签,如组氨酸标签(His-tag)和谷胱甘肽S-转移酶标签(GST-tag),被用于hTrx1亲和纯化.带N末端标签的hTrx1融合蛋白在实验中用的越来越多.但N末端延长是否会影响hTrx1特性尚不清楚.我们构建与优化了hTrx1原核表达质粒,在大肠杆菌中高效表达了含天然N末端、带His-tag或带GST-tag的3种重组hTrx1.纯化蛋白在SDS-PAGE上呈现1条带,对应的分子量分别为12kD、17kD及38kD.在无氧化剂存在时,它们催化胰岛素还原的能力不分仲伯.当有H2O2存在时,天然N末端hTrx1通过形成可逆二聚体,对H2O2表现出较强的耐受性;而N末端亲和标签有干扰二聚体形成,使hTrx1对H2O2耐受性降低的作用,其中GST-tag干扰作用明显大于His-tag.此外,体内重要的氧化还原对GSH/GSSG,有增进hTrx1及其还原酶催化NADPH氧化的作用,N末端亲和标签可明显扩大GSH/GSSG的这种作用.我们分析了N末端亲和标签对hTrx1活性影响的可能机理.     

酵母tRNA结合蛋白(43kD蛋白)羧端cDNA的克隆及其序列测定

为了研究酵母分子量为 4 3kD的tRNA结合蛋白的基因来源 ,通过溴化氰部分化学裂解此蛋白 ,产生的 18kD肽段经过蛋白质氨端序列测定 ,测得 18kD肽段氨端部分序列为AFTFKK .针对序列AFTFKK设计简并引物 ,利用简并PCR方法和cDNA的 3′末端的快速扩增方法 (3′RACE) ,成功克隆 4 3kD蛋白mRNA的 3′端序列 .DNA序列测定结果表明 ,该序列位于 3 磷酸甘油酸激酶 (PGK1)基因 10 0 3位— 170 0位 ,长度为 6 98bp ,编码 3 磷酸甘油酸激酶羧基端的 180氨基酸残基以及 3′端非翻译区 .结果证实 ,4 3kD蛋白的基因就是PGK1酶的基因     

NDPK-A C4/109/145 S突变体降低磷酸转移酶活性但增加DNase活性

核苷二磷酸激酶A( nucleoside diphosphate kinase A, NDPK-A）有广泛的生物学活性,在肿瘤转移和调控中起重要作用.单独抑制NDPK-A中任何1个Cys所形成的二硫键,不会降低NDPK-A的磷酸转移酶活性和DNase活性.本实验通过构建C4/109/145S突变体并研究其生物学效应,为NDPK A结构与功能的研究提供参考.用定点突变法将NDPK-A 的4位、109位和145位Cys突变为Ser,构建pBV220-NDPK-A C4/109/145S和pEGFP-NDPK-A C4/109/145S两种重组质粒.在大肠杆菌中高效表达NDPK-A C4/109/145S突变体,纯化后可获得均一的重组NDPK-A C4/109/145S突变体蛋白.HPLC法和DNA消化法测定发现,C4/109/145S突变体磷酸转移酶活性低于野生型NDPK-A,而DNase活性高于野生型NDPK-A.以A549细胞作为模式细胞的流式细胞仪周期检测表明,C4/109/145S突变体与野生型NDPK-A一致,均可将细胞周期延滞在S期和G2/M期.这些结果证实,NDPK A结构异构与其磷酸转移酶活性密切相关,其酶活性至少需要1个Cys残基存在,NDPK-A结构中的二硫键也可能是其DNase活性的负调控机制之一,胞内NDPK-A的氧化还原异构可能对细胞周期无显著影响.     

一个Ⅱ型甲烷氧化菌中甲烷单加氧酶羟基化酶的分离纯化和理化性质

甲烷氧化细菌能够催化甲烷和一系列小分子烃类化合物的羟基化反应,对控制全球变暖起着重要作用,在工业催化和生物除污中具有非凡的潜能。应用层析方法纯化了Ⅱ型甲烷氧化细菌MethylosinustrichosporiumIMV3011中甲烷单加氧酶的羟基化酶,并对其进行了表征。凝胶过滤法测定了该酶分子量为201.3kD;SDS-PAGE表明羟基化酶含有三个亚基(αβγ),分子量分别为58kD、36kD和23kD,比较两种方法证明该羟基化酶是一个同型二聚体构型(αβγ)2,总分子量为234kD。薄层等电聚焦测定该酶的等电点为5.2。酶的比活力为603.6nmol/(min.mg),活力回收为34.3%。HPLC法测定该酶的纯度在95%以上。原子吸收光谱显示每分子羟基化酶中含有3.02个Fe原子。羟基化酶的稳定性pH值为6.2～7.5,稳定性温度为低于35℃。菌株IMV3011的细胞表观构型呈现了长型、稍微弯曲的杆状形态。     

氧化还原作用对热休克转录因子1结构和功能的调控

为了评价半胱氨酸巯基氧化还原介导剂对人热休克转录因子 1(hHSF1)的氧化还原、结构和功能的作用 ,在体外用浓度为 0 .3～ 0 .5mmol/L的巯基氧化型介导剂二酰胺 (diamideDM )处理hHSF1;在体内用浓度为0 .1mmol/L的γ 谷氨酰半胱氨酸合成酶抑制剂丁硫堇处理HeLa细胞 ,都可形成一种致密的、分子内二硫键交联的氧化型hHSF1(ox hHSF1) ,使hHSF1三体形成和活化被阻断。二酰胺的这种作用呈剂量依赖 ;在电泳前加入浓度为 0 .4～ 0 .5mmol/L的巯基还原剂二硫苏糖醇 (DTT)到DM处理过的标本中再培育 ,能迅速和完全逆转这种作用。HSF1单体和三体功能域α 螺旋卷曲结构的计算机模型显示 ,在hHSF1单体N端和C端的疏水重复区中 ,半胱氨酸C1(第 15 3位氨基酸 )与半胱氨酸C4(第 373位氨基酸 )、C5(第 378位氨基酸 )非常接近 ,在合适的氧化作用下很容易形成二硫键 ,使HSF1单体形式较为稳定 ,不能形成三体并活化。结果表明 ,hHSF1的结构和功能与半胱氨酸上巯基的氧化还原化学性能相关 ;氧化作用和转录因子分子内巯基二硫键交联形成ox HSF1单体 ,可能是衰老细胞热体克转录反应呈渐减性的原因。     

关于巯基和Mn~(2+)介导豆壳过氧化物酶氧化藜芦醇的研究

藜芦醇作为非酚型木素模型物具有较高的氧化还原电位,豆壳过氧化物酶(soybeanhullperoxidase,SHP,EC.1.11.1.7)通过依赖于过氧化氢的正常过氧化物酶催化循环不能氧化藜芦醇,但在还原型谷胱甘肽、Mn2+和有机酸络合剂存在下却可以通过不依赖于过氧化氢的氧化酶反应途径完成对藜芦醇的氧化,产物为藜芦醛,反应最适pH为4.2。动力学研究表明该反应遵循顺规序列反应机制;对藜芦醇的表观KM值为4.3mmol/L,对谷胱甘肽的表观KM值为4.8mmol/L。巯基还原剂二硫苏糖醇、L-半胱氨酸和β-巯基乙醇亦可替代还原型谷胱甘肽促进藜芦醇氧化     

Oxidative modification of nucleoside diphosphate kinase and its identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Nucleoside diphosphate kinase (NDPK, Nm23) has been implicated as a multifunctional protein. However, the regulatory mechanism of NDPK is poorly understood. We have examined the modification of NDPK in oxidative stresses. We found that oxidative stresses including diamide and H(2)O(2) treatment cause disulfide cross-linking of NDPK inside cells. This cross-linking was reversible in response to mild oxidative stress, and irreversible to strong stress. This suggests that disulfide cross-linked NDPK may be a possible mechanism in the modification of cellular regulation. To confirm this idea, oxidative modification of NDPK has been performed in vitro using purified human NDPK H(2)O(2) inactivated the nucleoside diphosphate (NDP) kinase activity of NDPK by producing intermolecular disulfide bonds. Disulfide cross-linking of NDPK also dissociated the native hexameric structure into a dimeric form. The oxidation sites were identified by the analysis of tryptic peptides of oxidized NDPK, using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Intermolecular cross-linking between Cys109-Cys109, which is highly possible based on the X-ray crystal structure of NDPK-A, and oxidations of four methionine residues were identified in H(2)O(2)-treated NDPK. This cross-linkng was confirmed using mutant C109A (NDPK-A(C109A)) which had similar enzymatic activity as a wild NDPK-A. Mutant NDPK-A(C109A) was not cross-linked and was not easily denatured by the oxidant. Therefore, enzymatic activity and the quaternary structure of NDPK appear to be regulated by cross-linking with oxidant. These findings suggest one of the regulatory mechanisms of NDPK in various cellular processes.     

50L规模中试发酵重组核苷二磷酸激酶A工程菌

中试规模发酵重组人核苷二磷酸激酶A（rhNDPK-A）工程菌,并对表达产物进行纯化。摇瓶培养一级种子至合适密度,以10%比例接种二级种子培养基,在7L发酵罐中培养至OD600为9.6～10.5,然后转入80L发酵罐中进行补料分批培养,所得菌体裂解后,经离子交换层析和亲和层析两步纯化得重组蛋白制品。结果表明,50L培养液经过10h培养后,湿菌收量为1560 g/批,NDPK-A表达量为23.8%。另外,补料方式对发酵密度有明显影响。与单纯补加碳源相比,同时补加碳源和氮源可以显著提高菌体产量,但对目的蛋白表达量地提高不明显。在较优条件下,菌体产量为(2220.00±169.71) g/批,蛋白表达量为(22.00±0.42) %,纯化后重组蛋白得率为510mg/L。产物可溶、密度适中、工艺简便的中试发酵条件的建立为高得率、大规模制备重组rhNDPK-A奠定了基础。     

Determination of the glycosylation patterns, disulfide linkages, and protein heterogeneities of baculovirus-expressed mouse interleukin-3 by mass spectrometry.

The primary structure of mouse interleukin-3 (IL-3) expressed by recombinant baculovirus-infected silkworm (Bombyx mori) larvae was analyzed by subjecting isolated IL-3 derived peptides to liquid secondary ion mass spectrometry. Two species of IL-3 were isolated from the silkworm hemolymph by reverse-phase high-pressure liquid chromatography. The major component has M(r)20-22 x 10(3) as determined by SDS-PAGE. Liquid secondary ion mass spectrometric analysis was carried out on the reduced tryptic and endopeptidase lysyl-C peptides of glycosylated and deglycosylated IL-3. These studies provided evidence that (1) Asn-16 is heterogeneously glycosylated with four different oligosaccharides, (2) Asn-86 is either nonglycosylated or has attached to it one oligosaccharide, (3) the N-glycosylation sites Asn-44 and Asn-51 are not glycosylated, and (4) there is no O-glycosylation. Liquid secondary ion mass spectrometric analysis of the unreduced tryptic peptides provided evidence for disulfide linkages between Cys-140 and Cys-79 or Cys-80 and between Cys-17 and Cys-79 or Cys-80. In comparison to the major component, a minor IL-3 species (M(r) 17-19 x 10(3) by SDS-PAGE) isolated from the hemolymph showed no difference with respect to the glycosylation pattern or the disulfide linkages, but it was cleaved between Ala-127 and Ser-128, and only a disulfide linkage between Cys-140 and Cys-79 or Cys-80 held the molecule together.(ABSTRACT TRUNCATED AT 250 WORDS)     

Determination of disulfide array and subunit structure of taste-modifying protein, miraculin

The taste-modifying protein, miraculin (Theerasilp, S. et al. (1989) J. Biol. Chem. 264, 6655-6659) has seven cysteine residues in a molecule composed of 191 amino acid residues. The formation of three intrachain disulfide bridges at Cys-47-Cys-92, Cys-148-Cys-159 and Cys-152-Cys-155 and one interchain disulfide bridge at Cys-138 was determined by amino acid sequencing and composition analysis of cystine-containing peptides isolated by HPLC. The presence of an interchain disulfide bridge was also supported by the fact that the cystine peptide containing Cys-138 showed a negative color test for the free sulfhydryl group and a positive test after reduction with dithiothreitol. The molecular mass of non-reduced miraculin (43 kDa) in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was nearly twice the calculated molecular mass based on the amino acid sequence and the carbohydrate content of reduced miraculin (25 kDa). The molecular mass of native miraculin determined by low-angle laser light scattering was 90 kDa. Application of a crude extract of miraculin to a Sephadex G-75 column indicated that the taste-modifying activity appears at 52 kDa. It was concluded that native miraculin in pure form is a tetramer of the 25 kDa-peptide and native miraculin in crude state or denatured, non-reduced miraculin in pure form is a dimer of the peptide. Both tetramer miraculin and native dimer miraculin in crude state had the taste-modifying activity.     

Formation of albumin dimers induced by exposure to peroxides in human plasma: a possible biomarker for oxidative stress

Human serum albumin (HSA) has one free thiol residue at Cys-34 that is likely oxidized by various reactive oxygen species (ROS). We attempted to identify the oxidation product of Cys-34 of HSA following exposure of plasma to ROS. Oxidation induced by tert-butyl hydroperoxide (t-BuOOH) of this free cysteine residue in HSA was observed in detail. Analysis of oxidized albumin in a partially purified fraction obtained by affinity column chromatography clearly revealed the formation of albumin disulfide dimers following t-BuOOH exposure. Albumin disulfide dimer formation was observed in normal plasma following treatment with various peroxides, as well as in untreated plasma from patients on hemodialysis using SDS-PAGE and Western blot analysis. The present results indicate that albumin dimers are oxidative products derived from peroxides, and that their presence in plasma might be a marker of oxidative stress as secondary metabolites of peroxidation.     

猪血纤维蛋白溶酶原的纯化及性质研究

 用偶联有L-赖氨酸的Sepharose 4B亲和柱对猪血纤维蛋白溶酶原进行分离纯化,所得酶原在酸性及SDS-聚丙烯酰胺凝胶电泳中显示单一蛋白质区带,在碱性条件下电泳及等电聚焦电泳表现出较明显的不均一性。还原及非还原SDS-聚丙烯酰胺凝胶电泳测得酶原分子量为88kD,经尿激酶激活后,进行还原SDS-凝胶电泳,出现两条新的蛋白质区带,分子量分别为63kD和26kD。酶原含中性糖1.35％,N-末端为异亮氨酸。尿激酶激活后产生的血纤维蛋白溶酶以对甲苯磺酰-L-精氨酸甲酯为底物,测得Km为4.2m mol/L,V_(max)为13.5 IU。6-氨基已酸对酶活性有双重影响。此外,还观察了胰蛋白酶对猪血纤维蛋白溶酶原的激活。     

The underlying mechanism for the diversity of disulfide folding pathways

The disulfide folding pathway of bovine pancreatic trypsin inhibitor (BPTI) is characterized by the predominance of folding intermediates with native-like structures. Our laboratory has recently analyzed the folding pathway(s) of four 3-disulfide-containing proteins, including hirudin, potato carboxypeptidase inhibitor, epidermal growth factor, and tick anticoagulant peptide. Their folding mechanism(s) differ from that of BPTI by 1) a higher degree of heterogeneity of 1- and 2-disulfide intermediates and 2) the presence of 3-disulfide scrambled isomers as folding intermediates. To search for the underlying causes of these diversities, we conducted kinetic analyses of the reductive unfolding of these five proteins. The experiment of reductive unfolding was designed to evaluate the relative stability and interdependence of disulfide bonds in the native protein. It is demonstrated here that among these five proteins, there exists a striking correlation between the mechanism(s) of reductive unfolding and that of oxidative folding. Those proteins with their native disulfide bonds reduced in a collective and simultaneous manner exhibit both a high degree of heterogeneity of folding intermediates and the accumulation of scrambled isomers along the folding pathway. A sequential reduction of the native disulfide bonds is associated with the presence of predominant intermediates with native- like structures.