耐热碱性磷酸酶(FD-TAP)的结构模型研究

 以大肠杆菌碱性磷酸酶 (BAP)为主要结构模板 ,用计算机同源结构模拟方法构建了耐热碱性磷酸酶 (FD TAP)的三维结构 ,对它们的结构特征进行了分析比较 ,并用Profile 3D和Ramachand ran图等方法分析了结构的合理性 .在此基础上 ,又构建了FD TAP 3个突变体的结构 ,用CHARMM能量计算法研究了FD TAP及其 3个突变体的能量与酶蛋白热稳定性之间的关系 ,得到了与实验完全一致的结果 .结果说明 ,如果氨基酸残基置换使蛋白质分子的总能量降低 ,疏水性增高和柔韧性减小 ,往往使蛋白质的耐热性增加     

牙鲆碱性磷酸酶cDNA序列分析与蛋白质高级结构预测

为研究碱性磷酸酶(EC 3.1.3.1; alkaline phosphatase,ALP)在牙鲆(Paralichthys Olivaceus)发育和变态中的作用,采用RACE的方法克隆了牙鲆ALP基因cDNA全长,通过生物信息学分析了核苷酸序列并进行蛋白结构预测. 结果表明,牙鲆ALP cDNA全长为1 811bp,能编码476个氨基酸的蛋白质,分子量为52 293.1,等电点为7.67. 编码区核苷酸GC含量在ALP同源基因中差异比较大,脊椎动物明显高于非脊椎动物和细菌. 分子系统分析显示,牙鲆ALP和青黑斑河豚(Tetraodon nigroviridis)、斑马鱼（Danio rerio）的组织非特异性ALP有较高的同源性,分子进化树和物种进化树是一致的. 在蛋白序列中的一些重要的功能位点,包括金属离子结合位点、N糖基化位点和丝氨酸磷酸化位点等表现了较高的保守性. 牙鲆ALP和人胎盘ALP(PALP)在蛋白序列上有43%的相似性,其3D结构非常接近.通过氨基酸空间位置比较发现,牙鲆ALP中141和203位半胱氨酸对应于人PALP的121和183位半胱氨酸,推测能形成一个二硫键. 在两者酶活性中心,3个金属离子结合的氨基酸残基非常保守,Zn离子周围的9个氨基酸中有2个不同;Mg离子周围的7个氨基酸也只有2个不同,包括一对类似的丝氨酸155和苏氨酸175.     

大凉疣螈碱性磷酸酶的分离纯化及部分性质

碱性磷酸酶 (alkaline phosphatase,AKP)在生物界的分布很广 ,动物、植物、微生物中均广泛存在 .提纯的 AKP常被应用于对核酸等的研究 ,是基因工程常用的工具酶 ,也是酶标免疫测定技术的常用工具酶之一 .人类血清中的 AKP在不同疾病状态下有显著变化 ,临床上将血清 AKP变化指标作为诊断某些疾病的依据 .对于细菌和高等动物的 AKP已有广泛的研究 ,但国内外对两栖爬行类动物 AKP的研究报导却很少 ,仅有蛇毒中 AKP的研究报导 [1,2 ] .本文对大凉疣螈皮肤的 AKP进行了分离纯化 ,并对其部分性质进行了初步研究 .1　材料和方法1 .1　材…     

碱性磷酸酶在文昌鱼胚胎和幼体中表达图式的研究(英文)

研究组织特异性酶在胚胎发育中的表达图式对于研究细胞、组织和器官的分化具有重要意义。本文以 ＢＣＩＰ为底物研究了碱性磷酸酶在文昌鱼胚胎和幼体中的表达图式。在囊胚、原肠胚和神经胚早期（１２～１３小时）未检测到碱性磷酸酶的特异性表达;到神经胚中期（１５小时,约６个体节）,碱性磷酸酶在胚胎每一体节的中部开始表达,在胚胎中形成３～６个条纹（Ｆｉｇ．１）;在１８小时神经胚（９～１０体节）中,碱性磷酸酶在肌节中表达（Ｆｉｇ．２）;到２４小时在后部内胚层中也开始表达（Ｆｉｇ．３）;在３６～４８小时幼体中,碱性磷酸酶在消化道中强烈表达,但在咽鳃区不表达,同时在肌节中仍有弱的表达（Ｆｉｇ．４）。研究表明碱性磷酸酶可能在肌节的发育过程中具有一定作用。碱性磷酸酶在消化道中的表达可能与这一时期消化道开始行使功能有关。另外,Ｌ－苯丙氨酸只抑制３６、４８小时文昌鱼肠碱性磷酸酶活性,不抑制其体节肠碱性磷酸酶活性（Ｆｉｇ．５）、表明文昌鱼至少存在两种碱性磷酸酶：在消化道中表达的是一种肠型的,与脊椎动物碱性磷酸酶可能为同一类型;另一种主要在肌节中表达,可能是组织非特异性的破性磷酸酶。以上结果说明文昌鱼早期发育中碱性磷酸酶的表达与脊椎动物相似,具     

定向合成化学配基亲和层析纯化碱性磷酸酶

设计合成一种含有碱性磷酸酶抑制剂－对氨基苄基磷酸的化学配基并将其耦联到琼脂糖凝胶上,制备出新型的亲和层析介质,分离纯化从小牛肠中提取的碱性磷酸酶。经过对不同配基浓度吸附剂碱性酸酶吸附容量的考察,确定配基浓度为６μｍｏｌ／ｇ的吸附剂具有最高的酰选择性。     

耐热碱性磷酸酯酶的基因克隆及在大肠杆菌中的表达

以pUC118质粒为载体,以E.coil TGl为受体,构建了产耐热碱性磷酸酯酶(FD-TAP)的菌株Thermus sp.FD3041的染色体基因文库。在包含1.2万个转化子的文库中,90％的转化子插入有3～10kb的外源DNA片段。用菌落原位碱性磷酸酯酶显色法从文库中筛选到5个阳性克隆。对其中的1个克隆(pTAP362)所产的TAP进行了研究,发现克隆的TAP与出发菌株所产的TAP的热稳定性、最适反应温度和最适pH等性质都相同。通过做物理图谱和测定部分缺失的质粒的TAP活性,将TAP基因定位于2kb的BamHⅠ-HindⅢ DNA片段上。模拟PCR实验表明该酶可用于PCR扩增产物的检出。     

Molecular Evolution of the Tissue-nonspecific Alkaline Phosphatase Allows Prediction and Validation of Missense Mutations Responsible for Hypophosphatasia

ALPL encodes the tissue nonspecific alkaline phosphatase (TNSALP), which removes phosphate groups from various substrates. Its function is essential for bone and tooth mineralization. In humans, ALPL mutations lead to hypophosphatasia, a genetic disorder characterized by defective bone and/or tooth mineralization. To date, 275 ALPL mutations have been reported to cause hypophosphatasia, of which 204 were simple missense mutations. Molecular evolutionary analysis has proved to be an efficient method to highlight residues important for the protein function and to predict or validate sensitive positions for genetic disease. Here we analyzed 58 mammalian TNSALP to identify amino acids unchanged, or only substituted by residues sharing similar properties, through 220 millions years of mammalian evolution. We found 469 sensitive positions of the 524 residues of human TNSALP, which indicates a highly constrained protein. Any substitution occurring at one of these positions is predicted to lead to hypophosphatasia. We tested the 204 missense mutations resulting in hypophosphatasia against our predictive chart, and validated 99% of them. Most sensitive positions were located in functionally important regions of TNSALP (active site, homodimeric interface, crown domain, calcium site, …). However, some important positions are located in regions, the structure and/or biological function of which are still unknown. Our chart of sensitive positions in human TNSALP (i) enables to validate or invalidate at low cost any ALPL mutation, which would be suspected to be responsible for hypophosphatasia, by contrast with time consuming and expensive functional tests, and (ii) displays higher predictive power than in silico models of prediction.     

Inactivation of the pgsA Gene Affects Biosynthesis and Secretion of Alkaline Phosphatase in Escherichia coli

Inactivation of pgsA, which is responsible for biosynthesis of anionic phospholipid phosphatidyl-glycerol (PG), was shown to affect biosynthesis and secretion of alkaline phosphatase (PhoA) in Escherichia coli. A decrease in PG, but not in total anionic phospholipids, correlated with reduction of PhoA secretion, suggesting the role of PG in this process. A dramatic decrease in PG (from 18 to 3, but not 8, percent of the total phospholipids) inhibited not only secretion, but also synthesis of PhoA. In addition, pgsA inactivation expedited repression of PhoA synthesis by exogenous orthophosphate.     

Interaction of SecB and SecA with the N-Terminal Region of Mature Alkaline Phosphatase on Its Secretion in Escherichia coli

Export-specific chaperone SecB and translocational ATPase SecA catalyze the cytoplasmic steps of Sec-dependent secretion in Escherichia coli. Their effects on secretion of periplasmic alkaline phosphatase (PhoA) were shown to depend on the N-terminal region of the mature PhoA sequence contained in the PhoA precursor. Amino acid substitutions in the vicinity of the signal peptide (positions +2, +3) not only dramatically inhibited secretion, but they also reduced its dependence on SecB. Immunoprecipitation reported their impaired binding with mutant prePhoA. The results testified that SecB and SecA interact with the mature PhoA region located close to the signal peptide in prePhoA.     

绝经后女性血清瘦素、骨密度及血骨特异性碱性磷酸酶和Ⅰ型胶原交联氨基末端肽的测定及意义

目的:测定绝经后女性血清瘦素(leptin)与骨密度及及血清骨特异性碱性磷酸酶(BAP)和Ⅰ型胶原交联氨基末端肽(NTx)并探讨其关系。方法:用酶联免疫吸附试验测定287名40-80岁健康绝经后女性血清leptin以及血清骨特异性碱性磷酸酶(BAP)和Ⅰ型胶原交联氨基末端肽(NTx);用双能X线骨密度扫描仪测定总体、腰椎正位、总髋部骨密度以及体脂、瘦体重;分析它们之间的关系。结果:Leptin与髋部总体BMD呈正相关(r=0.162,P<0.05),校正年龄和体脂后,Leptin与髋部总体BMD相关性消失,Leptin与BAP相关无统计学意义;与NTX呈负相关(r=-0.119,P<0.05),校正年龄和体脂后,相关无统计学意义。BAP与总体骨密度、腰椎骨密度、髋部总体骨密度均呈负相关(r=-0.210,r=-0.236,r=-0.223,P<0.05),校正年龄和体质指数后,相关性都依然存在(r=-0.168,r=-0.187,r=-0.169,P<0.05)。NTx与总体骨密度、腰椎骨密度、髋部总体骨密度均呈负相关(r=-0.238,r=-0.232,r=-0.239,P<0.05),校正年龄和体质指数后...     

长穗偃麦草酸性磷酸酶与碱性磷酸酶编码基因的染色体定位

以一套中国春-长穗偃麦草二体异附加系与二体异代换系为材料,用等电聚焦(IEF)研究长穗偃麦草基因组中酸性磷酸酶(AcPh)与碱性磷酸酶(APH)编码基因的染色体定位。结果表明,AcPh大多聚焦于pH5～7范围内,其编码基因位于3E染色体,而APH编码基因则位于4E染色体。由于5E染色体的附加,AcPh活性带强度显著减弱。     

Thermostable Alkaline Phosphatase of Bacterium Meiothermus ruber: Gene Cloning,Expression in Escherichia coli,and Biochemical Characterization of the Recombinant Protein

The Meiothermus ruber alkaline phosphatase gene was cloned, expressed in Escherichia coli cells, and sequenced. The enzyme precursor, including the putative signal peptide, was shown to consist of 503 residues (deduced molecular mass 54,229 Da). The recombinant enzyme showed the maximal activity at 60–65°C, pH 11.0, K _M = 0.055 mM with p-nitrophenyl phosphate. The enzyme proved to be moderately thermostable, retaining 50% activity after 6 h incubation at 60°C and being completely inactivated in 2 h at 80°C. In substrate specificity assays, the highest activity was observed with p-nitrophenyl phosphate and dATP. Vanadate, inorganic phosphate, and SDS were inhibitory, while thiol-reducing agents had virtually no effect. The enzyme activity strongly depended on exogenous Mg²⁺ and declined in the presence of EDTA.     

Molecular Cloning and Characterization of L-Galactose-1-phosphate Phosphatase from Tobacco (Nicotiana tabacum)

L-Galactose-1-phosphate phosphatase (GPPase) is an enzyme involved in ascorbate biosynthesis in higher plants. We isolated a cDNA encoding GPPase from tobacco, and named it NtGPPase. The putative amino acid sequence of NtGPPase contained inositol monophosphatase motifs and metal binding sites. Recombinant NtGPPase hydrolyzed not only L-galactose-1-phosphate, but also myo-inositol-1-phosphate. The optimum pH for the GPPase activity of NtGPPase was 7.5. Its enzyme activity required Mg²⁺, and was inhibited by Li⁺ and Ca²⁺. Its fluorescence, fused with green fluorescence protein in onion cells and protoplasts of tobacco BY-2 cells, was observed in both the cytosol and nucleus. The expression of NtGPPase mRNA and protein was clearly correlated with L-ascorbic acid (AsA) contents of BY-2 cells during culture. The AsA contents of NtGPPase over expression lines were higher than those of empty lines at 13 d after subculture. This suggests that NtGPPase contributes slightly to AsA biosynthesis.     

The role of the proteasome in generating cytotoxic T-cell epitopes: insights obtained from improved predictions of proteasomal cleavage

Cytotoxic T cells (CTLs) perceive the world through small peptides that are eight to ten amino acids long. These peptides (epitopes) are initially generated by the proteasome, a multi-subunit protease that is responsible for the majority of intra-cellular protein degradation. The proteasome generates the exact C-terminal of CTL epitopes, and the N-terminal with a possible extension. CTL responses may diminish if the epitopes are destroyed by the proteasomes. Therefore, the prediction of the proteasome cleavage sites is important to identify potential immunogenic regions in the proteomes of pathogenic microorganisms (or humans). We have recently shown that NetChop, a neural network-based prediction method, is the best method available at the moment to do such predictions; however, its performance is still lower than desired. Here, we use novel sequence encoding methods and show that the new version of NetChop predicts approximately 10% more of the cleavage sites correctly while lowering the number of false positives with close to 15%. With this more reliable prediction tool, we study two important questions concerning the function of the proteasome. First, we estimate the N-terminal extension of epitopes after proteasomal cleavage and find that the average extension is relatively short. However, more than 30% of the peptides have N-terminal extensions of three amino acids or more, and thus, N-terminal trimming might play an important role in the presentation of a substantial fraction of the epitopes. Second, we show that good TAP ligands have an increased chance of being cleaved by the proteasome, i.e., the specificity of TAP has evolved to fit the specificity of the proteasome. This evolutionary relationship allows for a more efficient antigen presentation.The new version of NetChop (NetChop 3.0) is available at .     

Novel HLA-B27-restricted Epitopes from Chlamydia trachomatis Generated upon Endogenous Processing of Bacterial Proteins Suggest a Role of Molecular Mimicry in Reactive Arthritis

Reactive arthritis (ReA) is an HLA-B27-associated spondyloarthropathy that is triggered by diverse bacteria, including Chlamydia trachomatis, a frequent intracellular parasite. HLA-B27-restricted T-cell responses are elicited against this bacterium in ReA patients, but their pathogenetic significance, autoimmune potential, and relevant epitopes are unknown. High resolution and sensitivity mass spectrometry was used to identify HLA-B27 ligands endogenously processed and presented by HLA-B27 from three chlamydial proteins for which T-cell epitopes were predicted. Fusion protein constructs of ClpC, Na⁺-translocating NADH-quinone reductase subunit A, and DNA primase were expressed in HLA-B27⁺ cells, and their HLA-B27-bound peptidomes were searched for endogenous bacterial ligands. A non-predicted peptide, distinct from the predicted T-cell epitope, was identified from ClpC. A peptide recognized by T-cells in vitro, NQRA(330–338), was detected from the reductase subunit. This is the second HLA-B27-restricted T-cell epitope from C. trachomatis with relevance in ReA demonstrated to be processed and presented in live cells. A novel peptide from the DNA primase, DNAP(211–223), was also found. This was a larger variant of a known epitope and was highly homologous to a self-derived natural ligand of HLA-B27. All three bacterial peptides showed high homology with human sequences containing the binding motif of HLA-B27. Molecular dynamics simulations further showed a striking conformational similarity between DNAP(211–223) and its homologous and much more flexible human-derived HLA-B27 ligand. The results suggest that molecular mimicry between HLA-B27-restricted bacterial and self-derived epitopes is frequent and may play a role in ReA.     

Interdependent Effects of the Charge of the N-Terminal Region of the Signal Peptide,SecA, and SecB on Secretion of Alkaline Phosphatase in Escherichia coli

The cytoplasmic step of posttranslational secretion in Escherichia coli is catalyzed by export-specific chaperone SecB and translocational ATPase SecA. In addition, the efficiency of secretion depends on the charge of the signal peptide (SP). Replacement of positively charged Lys(–20) with uncharged Ala or negatively charged Glu in the N-terminal region of SP of the alkaline phosphatase precursor (prePhoA) was shown to decrease the PhoA secretion in the periplasm. The effect on secretion increased in the absence of SecB and was especially high on SecA inactivation. A change in SP charge strengthened the SecA and SecB dependences of secretion. On evidence of immunoprecipitation, the charge of the N-terminal region of SP had no effect on prePhoA interaction with the cytoplasmic secretion factors, suggesting no direct binding between this region and SecA or SecB. Yet the charge of the N-terminal region proved to affect the functions of SP as an intramolecular chaperone and a factor of prePhoA targeting to the membrane in cooperation with SecA and SecB.     

血清睾酮联合红细胞分布宽度、白蛋白碱性磷酸酶比值对腹腔镜前列腺癌根治术后生化复发的预测价值

摘要 目的：分析血清睾酮联合红细胞分布宽度（RDW）、白蛋白碱性磷酸酶比值（AAPR）对腹腔镜前列腺癌根治术（LRP）后生化复发的预测价值。方法：选取2017年1月～2019年1月海南省人民医院收治的90例接受LRP治疗的前列腺癌（PCa）患者,根据LRP后是否生化复发将分为生化复发组（n=34）和无生化复发组（n=56）。收集PCa患者基础资料,并检测两组术前血清睾酮、RDW、AAPR水平。采用多因素Cox回归分析LRP后生化复发的影响因素,受试者工作特征（ROC）曲线分析血清睾酮、RDW、AAPR对LRP后生化复发的预测价值。结果：随访3年,90例PCa患者LRP后生化复发34例,生化复发率为37.78％。单因素分析显示,生化复发组与年龄、PSA、Gleason评分、病理分期、睾酮、AAPR、RDW有关（P＜0.05）。多因素Cox回归分析显示,年龄增加、PSA＞20 ng/mL、Gleason评分8分、病理分期pT₃期、RDW升高为LRP后生化复发的独立危险因素,睾酮升高、AAPR升高为独立保护因素（P＜0.05）。ROC曲线分析显示,血清睾酮、RDW、AAPR单独与联合预测LRP后生化复发的曲线下面积（AUC）分别为0.771、0.754、0.772、0.922,三项联合预测LRP后生化复发的AUC大于单独预测（P＜0.05）。结论：RDW升高是LRP后生化复发的危险因素,血清睾酮、AAPR升高是保护因素,可作为LRP后生化复发的辅助预测指标,血清睾酮、RDW、AAPR联合预测LRP后生化复发的价值较高。     

Purification of alkaline phosphatase from chicken intestine by three-phase partitioning and use of phenyl-Sepharose 6B in the batch mode

Alkaline phosphatase from chicken intestine was purified from the crude preparation employing three-phase partitioning and by the use of phenyl Sepharose-6B in the batch mode. TPP uses a combination of ammonium sulphate and t-butanol to precipitate proteins from crude aqueous extracts. The precipitated protein forms interface between lower aqueous phase and upper organic solvent phase. The fold purification of the finally purified enzyme was 80 and the activity recovery was 61%. The sodium dodecyl sulphate-polyacrylamide gel electrophoresis analysis of enzyme showed considerable purification and its molecular weight was found to be around 67 kDa.     

Prediction and characterization of T-cell epitopes for epitope vaccine design from outer membrane protein of Neisseria meningitidis serogroup B

Neisseria meningitidis serogroup B (MC58) is a leading cause of meningitis and septicaemia, principally infects the infants and adolescents. No vaccine is available for the prevention of these infections because the serogroup B capsular polysaccharide is unable to stimulate an immune response, due to its similarity with polysialic acid. To overcome these obstacles, we proposed to develop a peptide based epitope vaccine from outer membrane protein contained in outer membrane vesicles (OMV) based on our computational analysis. In OMV a total of 236 proteins were identified, only 15 (6.4%) of which were predicted to be located in outer membrane. The major requirement is the identification and selection of T-cell epitopes that act as a vaccine target. We have selected 13 out of 15 outer membrane proteins from OMV proteins. Due to similarity of the fkpA and omp85 with the human FKBP2 and SAMM50 protein, we removed these two sequences from the analysis as their presence in the vaccine is likely to elicit an autoimmune response. ProPred and ProPred1 were used to predict promiscuous helper T Lymphocytes (HTL) and cytotoxic T Lymphocytes (CTL) epitopes and MHCPred for their binding affinity in N. meningitidis serogroup B (MC58), respectively. Binding peptides (epitopes) are distinguished from nonbinding peptides in properties such as amino acid preference on the basis of amino acid composition. By using this dataset, we compared physico-chemical and structural properties at amino acid level through amino acid composition, computed from ProtParam server. Results indicate that porA, porB, opc, rmpM, mtrE and nspA are more suitable vaccine candidates. The predicted peptides are expected to be useful in the design of multi-epitope vaccines without compromising the human population coverage.