Biochemical Characterization and Distribution of Glutathione S-Transferases in Leaping Mullet (Liza saliens)

In this study, feral leaping mullet (Liza saliens) liver cytosolic glutathione S-transferases (GSTs) were investigated and characterized using 1-chloro-2,4-dinitrobenzene (CDNB) and ethacrynic acid (EA) as substrates. The average GST activities towards CDNB and EA were found to be 1365 +/- 41 and 140 +/- 20 nmol/min per mg protein, respectively. The effects of cytosolic protein amount and temperature ranging from 4 to 70 degrees C on enzyme activities were examined. While both activities towards CDNB and EA showed similar dependence on protein amount, temperature optima were found as 37 and 42 degrees C, respectively. In addition, the effects of pH on GST-CDNB and -EA activities were studied and different pH activity profiles were observed. For both substrates, GST activities were found to obey Michaelis-Menten kinetics with apparent V(max) and K(m) values of 1661 nmol/min per mg protein and 0.24 mM and 157 nmol/min per mg protein and 0.056 mM for CDNB and EA, respectively. Distribution of GST in Liza saliens tissues was investigated and compared with other fish species. Very high GST activities were measured in tissues from Liza saliens such as liver, kidney, testis, proximal intestine, and gills. Moreover, our results suggested that GST activities from Liza saliens would be a valuable biomarker for aquatic pollution.     

The Association Between Polymorphic Genotypes of Glutathione S-Transferases and COPD in the Turkish Population

Although smoking is regarded as the most important causal factor in chronic obstructive pulmonary disease (COPD), only 10–20% of smokers develop symptomatic COPD, which indicates the presence of genetic predisposing factors in its pathogenesis. This study investigates the association between gene polymorphysims of glutathione S-transferases (GSTs) and COPD. Blood samples were taken from 149 patients and 150 healthy controls. Polymorphisms of GSTT1, GSTM1, and GSTP1 were genotyped using Real-Time PCR. Multivariate logistic regression was used to calculate odds ratios (ORs) and 95% confidence intervals between specific genotypes and COPD. There was no difference in the frequencies of the genotypes of GSTM1 and GSTT1 between the groups, but the GSTP1 Ile/Ile genotype was significantly higher in the patients than in the controls (61.1% vs. 38%). GSTP1 Ile/Val and Val/Val genotypes were associated with a decreased risk of COPD when compared to the Ile/Ile genotype (2.12-fold and 4-fold, respectively). Thus we suggest that the Val allele of GSTP1 may have a protective effect for development of COPD. Furthermore, when we evaluated the association between GSTP1 genes and smoking status, smokers with the GSTP1 Ile allele had an increased risk for the development of COPD. Among the combinations of the genotypes, the combination of GSTM1, GSTT1 null, and GSTP1 Val/Val was associated with the maximal increased risk (12-fold) of COPD. Thus to explain the ethiopathogenesis of COPD, investigation of a single gene family is inadequate. Based on our results and the previous data, further studies should be focused on the GSTP1 gene and the interactions with other genes such as polymorphisms of N-acetyltransferases, GSTM1 and GSTT1, microsomal epoxide hydrolase, and allelic variants of cytochrome P450.     

拟南芥谷胱甘肽S-转移酶Zeta类进化酶的获得及其特性分析

拟南芥谷胱甘肽S-转移酶Zeta类(AtGSTZ)是一种与细胞代谢和环境净化密切相关的多功能酶.应用易错PCR和多轮DNA洗牌技术构建了AtGSTZ随机突变文库;再利用pH指示剂颜色改变法对突变文库进行筛选,获得了9个二氯乙酸脱氯活性提高的突变子.其中,NN23含25个氨基酸突变,比活力提高120%,NN20含24个氨基酸突变,比活力提高102%,EC1含2个氨基酸突变,比活力提高47%,其他6个为单点突变,比活力分别提高9%～60%.酶学分析显示,所有进化酶对底物二氯乙酸的催化效率和对谷胱甘肽的亲和力以及个别进化酶的复性能力都得到不同程度的提高,但热稳定性均没有明显改善.同时,对一系列与AtGSTZ空间折叠及催化活性相关位点进行了讨论.     

Proteomic Analysis of Glutathione S-Transferases of Arabidopsis thaliana Reveals Differential Salicylic Acid-Induced Expression of the Plant-Specific Phi and Tau Classes

Plant glutathione S -transferases (GSTs) are a large group of multifunctional proteins that are induced by diverse stimuli. Using proteomic approaches we identified 20 GSTs at the protein level in Arabidopsis cell culture with a combination of GST antibody detection, LC-MS/MS analysis of 23-30 kDa proteins and glutathione-affinity chromatography. GSTs identified were from phi, tau, theta, zeta and DHAR sub-sections of the GST superfamily of 53 members. We have uncovered preliminary evidence for post-translational modifications of plant GSTs and show that phosphorylation is unlikely to be responsible. Detailed analysis of GST expression in response to treatment with 0.01-1 mM of the plant defence signal salicylic acid (SA) uncovered some interesting features. Firstly, GSTs appear to display class-specific concentration-dependent SA induction profiles highlighting differences between the large, plant specific phi and tau classes. Secondly, different members of the same class, while sharing similar SA dose responses, may display differences in terms of magnitude and timing of induction, further highlighting the breadth of GST gene regulation. Thirdly, closely related members of the same class ( GSTF6 and GSTF7 ), arising via tandem duplication, may be regulated differently in terms of basal expression levels and also magnitude of induction raising questions about the role of subfunctionalisation within this family. Our results reveal that GSTs exhibit class specific responses to SA treatment suggesting that several mechanisms are acting to induce GSTs upon SA treatment and hinting at class-specific functions for this large and important, yet still relatively elusive gene family.     

蚯蚓两种抗菌肽的分离纯化及部分性质

经硫酸铵沉淀、超滤、阳离子交换分离和反相快速蛋白质液相色谱（FPLC）分析,得到了两种新的蚯蚓抗菌肽F-1与F-2,经电喷雾离子源质谱（ESI-MS）测定,其相对分子质量为535.27和519.27.串联质谱（MS/MS）数据表明F-1的肽序列为Ac-Ala-Met-Val-Ser-Ser,F-2的肽序列为Ac-Ala-Met-Val-Gly-Thr.最小抑菌浓度（MIC）实验表明,F-1与F-2对鹑鸡肠球菌(Enterococcus gallinarum)、绿脓杆菌(Pseudomonas pyocyanea)、鲍氏不动杆菌(Acinetobacter baumanii)、土生克雷伯氏菌（Klebsiella terrigena）的最小抑菌浓度分别为11.4 mg/L和12.85 mg/L,对粪肠球菌(Enterococcus faecalis)的最小抑菌浓度分别为22.8 mg/L和25.68 mg/L.对真菌白色念株菌（Candida albicans）没有表现为完全的抑制作用.     

Purification and Characterization of Kininogens from Sheep Plasma

High molecular weight kininogen (HMWK) and low molecular weight kininogen (LMWK) have been purified from sheep ( Avis Arias) plasma in three steps involving ammonium sulphate precipitation, column chromatography on Sephacryl-300HR and ion exchange chromatography on DEAE cellulose. HMWK gave a single band on native and SDS-PAGE with a molecular weight corresponding to 280 kDa. Under reducing conditions purified HMWK was again resolved to a single band with molecular weight corresponding to 140 kDa indicative of its dimeric nature. LMWK was resolved into two isoforms named as LMWK1 and LMWK2, with an apparent molecular weight of 68 kDa. The yield of HMWK, LMWK1 and 2 was about 8.1, 5.63 and 10.65 respectively. HMWK, LMWK1 and 2 strongly inhibited activities of ficin and papain but not of trypsin, chymotrypsin and bromelain. K_i values estimated for HMWK with papain and ficin was 0.8 and 0.6 nM respectively. K_i values estimated for LMWK1 and 2 with papain were 2.40 and 2.00 nM respectively. Binding of HMWK, LMWK1 and 2 to activated papain were accompanied by pronounced changes in secondary and tertiary structure that are compatible with perturbations of environment of aromatic residues.     

Purification and Characterisation of Hepatic Glutathione Transferases from a Herbivorous Marsupial, the Brushtail Possum (Trichosurus vulpecula)

A single glutathione transferase isoenzyme was purified from hepatic cytosol of the brushtail possum and shown to represent 3.6 ± 0.3% of the total cytosolic protein. Characterisation of the enzyme, termed Possum GST 1–1, indicated that it possessed similar catalytic activity and structural homology with isoenzymes belonging to the alpha class of glutathione transferases. This homodimeric GST exhibited a single band with an apparent molecular mass of 25.4 kDa on sodium dodecyl sulphate-polyacrylamide gels and an apparent pI of 9.8. Inhibition studies demonstrated that Possum GST 1–1 displays binding affinity for a range of inhibitors similar to that shown by alpha class GSTs purified from other mammals. Immunoblot analysis demonstrated immuno-cross reactivity between Possum GST 1–1 and antisera raised against human alpha GST, while this GST did not cross-react with antisera raised against human mu and pi GST. N-terminal sequencing of purified Possum GST 1–1 revealed that the N-terminus of the protein is chemically blocked. Sequence analysis of three internal peptide sequences demonstrated homology with mammalian alpha GSTs. Of particular interest is the significant substrate specificity that Possum GST 1–1 displays with both organic and inorganic hydroperoxides. It is proposed that this substrate specificity is an evolutionary adaptation to a diet high in potentially toxic plant allelochemicals.     

Evaluation of the physiological value of porcine luteal cells isolated in various stages of the luteal phase: Tissue culture approach

Porcine luteal cells were collected from corpora lutea in four different stages of the luteal phase and cultured as monolayers. Progesterone (P₄) secretion was assayed using radioimmunoassays (Gregoraszczuk, 1991). Luteal cells cultured from porcine corpora lutea collected in the early luteal phase maintained steroidogenic capacity for 6 days in culture until the time comparable with midluteal corpora lutea. Luteal cells collected from mature and regressing corpora lutea did not dedifferentiate during 2 days of culture. After this time secretion of progesterone decreased to undetectable amounts characteristic of old corpora lutea. The regression in the culture progressed. The results demonstrate that the degree of the decline of progesterone depends on the type of corpus luteum, which is connected to particular time intervals of the luteal phase. Before starting experiments it is necessary to take into consideration the stage of the luteal phase from which the material is collected for culture. This study provides evidence that long term culture is useful for investigating a variety of aspects of luteal function only if cells are collected in the early luteal phase. Short term culture is suitable for investigation of cells collected from mid and late luteal phase. Regulation of luteal function is dependent on stage of the luteal phase.     

Identification of Encephalitozoon cuniculi genotype III and two novel genotypes of Enterocytozoon bieneusi in swine

Samples of intestinal content from thirty fattened pigs of six farms slaughtered at an abattoir in North-Western Germany, and faecal samples of four pigs kept as laboratory animals at the Federal Institute for Risk Assessment (BfR, Berlin, Germany) were investigated for the occurrence of microsporidia by light microscopy, PCR and sequencing. A modified Webers trichrome staining and the immunohistochemistry (the Avidin-Biotin-Peroxidase-Complex technique with a polyclonal anti-Encephalitozoon cuniculi-serum and monoclonal antibodies against Encephalitozoon intestinalis and Enterocytozoon bieneusi) was used as a screening method for the light microscopical detection of these pathogenic eukaryotes. By this light microscopically methods microsporidia suspected organisms were found in all samples (100%). By the use of PCR, microsporidia were identified in fourteen samples (41.2%). The prevalence of microsporidia infections among the farms diversifies from 0 to 80% as considered by PCR. E. bieneusi was the most prevalent species and was identified in twelve fattened pigs (40%) from five of the six tested farms (83.3%) and in two of the four laboratory animals (50%). Three of the E. bieneusi species belonged to the genotype O, one to the genotype E, and one to the genotype F. Two isolates were identified as novel genotypes and two samples showed a mixed infection of different genotypes. In three faecal samples of the pigs from two farms E. cuniculi genotype III was identified. One sample contained both microsporidia species. To our knowledge, this is the first time that the genotype III of E. cuniculi was identified in swine.     

间羟苯甲酸4－羟化酶纯化及部分性质研究

经超声破碎、硫酸铵分级沉淀、凝胶过滤、磷酸钙胶层析和离子交换层析等步骤, 从Comamonas testosteroni菌株中获得了SDS-PAGE单一条带, 相对分子质量为62×10³的间羟苯甲酸4-羟化酶比活提高21倍, 产率为30%.此酶为FAD加单氧酶, 催化间羟苯甲酸转变为3,4二羟苯甲酸.     

Purification and Characterization of a Lectin from Crotalaria paulina Seeds

A lectin was purified from Crotalaria paulina seeds by ion-exchange and FPLC molecular exclusion chromatography. CrpL had an apparent molecular mass of 30 kDa, as determined by SDS-PAGE under non-reducing and reducing conditions. CrpL effectively agglutinated human and cow erythrocytes, and this activity was not affected by 20 mM EDTA, showing no dependence of divalent cations. Hemagglutination was inhibited by N-acetyl- D-galactosamine, D-galactose and was also inhibited by glycoproteins, fetuin and asialofetuin. The N-terminal amino acid sequence of CrpL was identical to those of other lectins from the genus Crotalaria, and amino acid composition showed high amounts of Asx and Glx, and was rich in Gly, Ala and Ser, as also reported for lectins from other Crotalaria species. CrpL inhibited the growth of Xanthomonas axonopodis pv. phaseoli and Xanthomonas axonopodis pv. passiflorae, suggesting a role of this lectin in the defense of seeds against bacterial infections.     

Purification and characterization of fungal and mammalian phosphomannose isomerases

Phosphomannose isomerase (PMI) is essential for the production of yeast cell walls. An inhibitor which inhibits the fungal enzyme without altering the activity of the mammalian enzyme would be a potential fungicidal agent, increasingly important in view of the increasing mortality from visceral mycoses in immunosuppressed patients. We have purified human, porcine, andCandida albicans enzymes 29,000-fold to homogeneity, and characterized their physical properties, as well as their kinetic parameters, inhibition constants, andpH dependences. Surprisingly, in view of the large differences betweenPseudomonas aerugenosa andSaccharomyces cerevisiae PMI, the human andC. albicans enzymes are almost identical. We suggest therefore that species-selective inhibition of the fungal rather than mammalian enzyme may require molecules which bind away from the substrate binding pocket of the enzyme.Abbreviations PMI phosphomannose isomerase - Tris/HCl tris(hydroxymethyl)aminomethane - Hepes 4-(2-hydroxyethyl)-piperazine-1-ethanesulfonic acid - Mes 2-(N-morphilino) ethanesulfonic acid     

Pilot-Scale Fermentation and Purification of the Recombinant Allophycocyanin Over-Expressed in <Emphasis Type="Italic">Escherichia coli</Emphasis>

A recombinant allophycocyanin (rAPC), used for treatment of tumors, has been expressed in E. coli which was grown in glucose fed-batch culture in a 30 l fermentor. Recombinant allophycocyanin was purified from soluble E. coli cell lysate using hydrophobic interaction chromatography followed by chromatography using amylose affinity column. The purity of product was greater than 98% and yielded an average of 5.5 g kg⁻¹ dry cells. Recombinant allophycocyanin significantly inhibited H₂₂ hepatoma (p ( 0.01) in mice with inhibition rates ranging from 36% to 62% with doses from 6.25 to 50 mg kg⁻¹ d⁻¹.     

Purification and Characterization of S-Adenosyl-L-Methionine Nicotinic Acid-N-Methyltransferase from Leaves of Glycine max

Trigonelline (TRG), which act as a cell cycle regulator and a compatible solute in response to salinity and water-stress, is the N-methyl conjugate of nicotinic acid the formation of which is catalyzed by S-adenosyl-L-methionine nicotinic acid-N-methyltransferase. The enzyme was purified 2650-fold from soybean (Glycine max L.) leaves with a recovery of 4 %. The purification procedure included ammonium sulfate (45 – 60 %) precipitation, linear gradient DEAE-Sepharose chromatography, adenosine-agarose affinity chromatography, hydroxyapatite chromatography and gel filtration (Sephacryl-S-200). The purified enzyme preparation showed a major band with a molecular mass of 41.5 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis that is related to the enzyme activity. The native enzyme had a molecular mass of about 85 kDa as estimated by gel filtration. The K_m values for S-adenosyl-L-methionine and nicotinic acid were 31 and 12.5 M, respectively. The purified enzyme showed optimum activity at pH 6.5 and temperature of 40 – 45 °C. High concentration of dithiothreitol (10 mM) and glycerol (20 %) stabilize the enzyme during purification and storage. Hg²⁺ strongly inhibits enzyme activity.     

Morganella morganii J-8羰基不对称还原酶的分离纯化及性质研究

由本实验室筛选得到的摩尔摩根氏菌J-8菌株可将底物1-苯基-2-甲氨基丙酮专一性地转化为d-伪麻黄碱。以M. morganii J-8为出发菌株,菌体超声破碎后,经硫酸铵沉淀、Phenyl Superose疏水柱层析、DEAD阴离子柱层析和非变性凝胶电泳四步纯化获得电泳纯羰基不对称还原酶。亚基分子质量为42.5 kD,高效液相色谱分析酶的分子质量约为84.1 kD,初步认为该酶为二聚体蛋白。对所得到的部分纯化酶的酶学性质做了初步研究,纯酶进行基质辅助激光解析电离-飞行质谱分析,比对结果显示为与亮氨酸脱氢酶蛋白有很高相似性。     

Morganella morganii J-8羰基不对称还原酶的分离纯化及性质研究

由本实验室筛选得到的摩尔摩根氏菌J-8菌株可将底物1-苯基-2-甲氨基丙酮专一性地转化为d-伪麻黄碱。以M. morganii J-8为出发菌株,菌体超声破碎后,经硫酸铵沉淀、Phenyl Superose疏水柱层析、DEAD阴离子柱层析和非变性凝胶电泳四步纯化获得电泳纯羰基不对称还原酶。亚基分子质量为42.5 kD,高效液相色谱分析酶的分子质量约为84.1 kD,初步认为该酶为二聚体蛋白。对所得到的部分纯化酶的酶学性质做了初步研究,纯酶进行基质辅助激光解析电离-飞行质谱分析,比对结果显示为与亮氨酸脱氢酶蛋白有很高相似性。     

Purification and Characterization of a Xylanase Produced by Chaetomium thermophile NIBGE

Summary Xylanase was produced by growing Chaetomium thermophile NIBGE in a submerged liquid culture using wheat straw and urea as carbon and nitrogen sources respectively. The xylanase was purified to electrophoretic homogeneity after ammonium sulphate precipitation, anion exchange chromatography by FPLC and gel filtration. The molecular mass of this xylanase BII was 50 kDa. The pH and temperature optima were 6.5 and 70 °C respectively. The xylanase BII showed reasonable stability at high pH and 65 °C temperature. Some metal ions and EDTA caused little inhibition at low concentrations but complete inhibition was observed at concentrations higher than 2 mM. The K_m and V_max values with oat spelt xylan as the substrate were found to be 12.5 mg/ml and 83.3 IU/mg protein, respectively. Liberation of reducing sugars from commercial paper pulp samples suggest the feasibility of a biopulping process using this xylanase.     

Partial Purification and Characterization of a 37 kDa Extracellular Proteinase from Trichophyton vanbreuseghemii

An exocellular proteinase synthesized by the geophilic dermatophyte Trichophyton vanbreuseghemii has been purified and characterized. The fungus obtained from soil in Iran was cultivated in modified Czapek–Dox liquid medium containing 0.1% bacteriological peptone and 1% glucose as the nitrogen and carbon sources. Partial purification of the proteinase was accomplished by (NH₄)₂SO₄ precipitation, followed by ion exchange chromatography. Analysis of the enzyme by SDS-PAGE revealed a single polypeptide chain with an apparent molecular mass of 37 kDa. Proteinase activity was optimum at pH 8, but remained high in the range of pH 7–11. Moreover, the partially purified enzyme presented a keratinolytic activity as evidenced by the keratin azure test. The inhibition profile and the good activity of the enzyme towards the synthetic substrate N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide suggested that it belonged to the chymotrypsin/subtilisin group of serine proteinases. The keratinolytic properties of T. vanbreuseghemii suggest that this fungus may be an alternative for the recycling of industrial keratinic wastes.     

Immunohistochemical identification of Purkinje fibers and transitional cells in a terminal portion of the impulse-conducting system of porcine heart

Summary The ultrastructure of porcine ventricular tissue was studied by electron microscopy and immunocytochemical techniques. Electron-dense specific granules were found in both Purkinje fibers and transitional cells in the ventricular walls, and were positively stained by the immunogold staining method using an antiserum against atrial natriuretic polypeptide (ANP). This suggests that both the Purkinje fibers and transitional cells display the same specific granules as atrial cardiocytes containing ANP. These results demonstrate that Purkinje fibers and two types of transitional cells, in addition to the ordinary ventricular cardiocytes, can be identified in porcine ventricular wall tissue.