Expression of the vanadium-dependent bromoperoxidase gene from a marine macro-alga Corallina pilulifera in Saccharomyces cerevisiae and characterization of the recombinant enzyme

The vanadium-dependent bromoperoxidase from the marine macro-alga Corallina pilulifera was heterologously expressed in Saccharomyces cerevisiae. The enzyme was purified and crystals in "tear drop" form were obtained. The catalytic properties of the recombinant enzyme were studied and compared with those of the native enzyme purified from C. pilulifera. Differences in thermal stability and chloroperoxidase activity were observed. The recombinant enzyme retained full activity after preincubation at 65 degrees C for 20 min, but the native enzyme was completely inactivated under the same conditions. The chlorinating activity of the native enzyme was more than ten times higher than that of the recombinant enzyme. Other properties, such as K(m) values for KBr and H(2)O(2), and optimal temperature and pH, were similar for each source of C. pilulifera bromoperoxidase.     

水团花药材鉴定方法的研究

目的:建立中药水团花的药材鉴定方法.方法:通过对主产地六批药材的研究,从薄层定性检查、水份测定、浸出物检查和灰分测定4个方面,建立了水团花的药材鉴定方法.结果:运用TLC法,定性鉴别了水团花中的2种化学成分;药材的含水量不得过9.6%;药材的水溶性浸出物不得少于31%;药材的醇溶性浸出物不得少于35%;药材的总灰分不得过5.0%.结论:该方法可用于控制水团花的质量.     

Characterization of nonheme type bromoperoxidase in Corallina pilulifera

Bromoperoxidase was purified from the crude extract of Corallina pilulifera to be homogeneous upon polyacrylamide disc gel and sodium dodecyl sulfate-polyacrylamide gel electrophoreses according to the procedures previously reported (Itoh, N., Izumi, Y., and Yamada, H. (1985) Biochem. Biophys. Res. Commun. 131, 428-435). The enzyme had a molecular weight of approximately 790,000 and was composed of 12 subunits of identical molecular weights (Mr 64,000). Hexagonal molecular shapes of the enzyme were observed by electron microscopy. The isoelectric point of the enzyme was 3.0, and the predominance of acidic amino acids was revealed by amino acid analysis of the enzyme. The enzyme was specific for I- and Br- and inactive toward Cl- and F-. The optimum pH of the enzyme was 6.0, and the enzyme was stable in a range from pH 5.0 to 11.0. The enzyme had no hemeor flavin-like compounds as a prosthetic group. Plasma emission spectroscopy revealed that the enzyme contains 2.3 +/- 0.2 atoms of iron and 1.6 +/- 0.1 atoms of magnesium/molecule of protein. Hence, bromoperoxidase of C. pilulifera was distinct from other haloperoxidases and many peroxidases, which are hemoproteins.     

Vanadate activation of bromoperoxidase from Corallina officinalis

A nonheme bromoperoxidase has been purified to homogeneity from the red seaweed Corallina officinalis. Like the corresponding enzyme previously reported from C. pilulifera, this bromoperoxidase contains a significant amount of nonheme iron. However, it is vanadate ion and not iron that activates the enzyme, and maximal activity is achieved with stoichiometric vanadium incorporation. The absence of competition between vanadium and iron suggests that they occupy distinct binding sites in the protein. A correlation between vanadium content and catalytic activity indicates that less than 12 percent of the maximal activity of the enzyme can be derived from metals other than vanadium.     

Physiological function of bromoperoxidase in the red marine alga, Corallina pilulifera: production of bromoform as an allelochemical and the simultaneous elimination of hydrogen peroxide.

The physiological function of vanadium-bromoperoxidase (BPO) in the marine red alga, Corallina pilulifera, has been characterized from the viewpoint of allelochemical formation. The algae emit bromoform (CHBr3) depending on the enzyme activity level in vivo (Itoh, N., Shinya, M., 1994. Seasonal evolution of bromomethanes from coralline algae and its effect on atmospheric ozone. Marine Chemistry 45, 95-103). We demonstrated that bromoform produced by C. pilulifera played an important role in eliminating epiphytic organisms, especially microalgae on the surface. Such data suggest a strong relationship between the coralline algae and the coralline flat (deforested area in the marine environment: called isoyake in Japanese). Lithophyllum yessoense, the main inhabitant of coralline flats in Japan, produced a lower level of CHBr3 than C. pilulifera, and showed BPO activity. On the other hand, the seasonal change of BPO activity in C. pilulifera in vivo was in proportion to superoxide dismutase (SOD) activity and in inverse proportion to catalase activity. The phenomenon implies that BPO could be a potential substitute for catalase, because the enzyme catalyzes an efficient Br(-)-dependent catalase reaction.     

水团花黄酮类成分及其体外抗病毒活性

从水团花乙醇提物的乙酸乙酯部位分离到6个黄酮类化合物,根据光谱数据分别鉴定为柚皮素（1）、圣草酚（2）、槲皮素（3）、柚皮素-7-O-β-D-葡萄糖苷（4）、圣草酚-7-O-β-D-葡萄糖苷（5）、槲皮素-3-O-β-D-葡萄糖苷（6）,化合物1、2,4和5为首次从该植物中分离得到。采用细胞病变抑制法（CPEr eductionassay）和MTT法测定化合物的体外抗病毒活性,结果显示,三个黄酮苷元1、2和3均具有不同程度的体外抑制呼吸道合胞病毒（RSV）和柯萨奇B3型病毒（CVB3）活性,反之,3个黄酮苷均不显示活性。     

小珊瑚藻对赤潮异弯藻的克生效应及其 对UV-B辐射增强的响应

通过共培养方法,以细胞密度为主要测定指标,研究了小珊瑚藻对赤潮异弯藻的克生效应及其对UV-B辐射增强的响应.结果表明：小珊瑚藻的新鲜组织和水溶性抽提液对赤潮异弯藻的生长表现出显著的抑制作用(P＜0.05),说明小珊瑚藻对赤潮异弯藻具有克生效应;而小珊瑚藻干粉末和培养水过滤液对赤潮异弯藻生长则无明显影响(P>0.05).将小珊瑚藻用不同剂量的UV-B辐射预处理后,再与赤潮异弯藻共培养,除培养水过滤液外,其新鲜组织、干粉末和水溶性抽提液对赤潮异弯藻生长的作用效果均有所改变,高剂量(3.0 J·m^-2)UV-B辐射使小珊瑚藻对赤潮异弯藻的抑制作用减弱(P＜0.05),而低剂量(0.9 J·m^-2) UV-B辐射则使抑制作用增强(P＜0.05).     

小珊瑚藻对赤潮异弯藻的化感效应

研究了不同浓度的小珊瑚藻组织4种不同极性有机溶剂(甲醇、丙酮、乙醚、氯仿)提取物对赤潮异弯藻的生长抑制作用.结果表明：小珊瑚藻组织甲醇提取物对赤潮异弯藻的生长抑制活性最强,并且在较高浓度下能使赤潮异弯藻完全死亡,其他3种有机溶剂提取物对赤潮异弯藻的生长无明显影响.表明小珊瑚藻组织中含有的对赤潮异弯藻有抑制作用的活性物质具有较高的极性.对小珊瑚藻的甲醇提取物进行液液萃取,将其分离为石油醚相、乙酸乙酯相、正丁醇相和蒸馏水相, 并对赤潮异弯藻进行生物活性检测.结果发现石油醚相和乙酸乙酯相具有较强的杀藻活性,表明脂肪酸可能是小珊瑚藻组织内抑制赤潮异弯藻生长的化感物质的重要组成成分之一.     

Hypocrea/Trichoderma species with pachybasium-like conidiophores: teleomorphs for T. minutisporum and T. polysporum and their newly discovered relatives

We describe or redescribe species of Hypocrea/Trichoderma (Ascomycetes, Hypocreales) having hyaline ascospores and pachybasium-like conidiophores. Teleomorphs are reported for Trichoderma minutisporum (Hypocrea minutispora sp. nov.) and T. polysporum (H. pachybasioides). Hypocrea pilulifera/T. piluliferum is redescribed. Trichoderma croceum is synonymized with T. polysporum. The new species H. parapilulifera, H. stellata and H. lacuwombatensis are described. All of these species fall within the morphological concept of Trichoderma sect. Pachybasium and within the phylogenetic group pachybasium B5 of Kullnig-Gradinger et al (2002). Parsimony analysis of nucleotide sequences from three unlinked loci-ITS1 and 2, endochitinase (ech42) and translation elongation factor 1-alpha (tef1)-detects two distinct phylogenetic lineages within the group pachybasium B5. One comprises H. pachybasioides/T. polysporum, H. pilulifera/T. piluliferum, H. parapilulifera and H. stellata; this group, the "polysporum" lineage, is characterized by having conidia that are white in mass and is the only lineage within Hypocrea characterized by such conidia. The second group includes the green conidial T. minutisporum and H. lacuwombatensis. The partition homogeneity test reveals significant recombination within the "polysporum" lineage but not within the "minutisporum" lineage.     

Substrate specificity, regiospecificity and stereospecificity of halogenation reactions catalyzed by non-heme-type bromoperoxidase of Corallina pilulifera

Many organic compounds were found to be substrates for halogenation reactions catalyzed by the non-heme-type bromoperoxidase found in the red alga Corallina pilulifera. Anisole, 1-methoxynaphthalene and thiophene were converted to o and p-bromoanisoles, 1-methoxy-4-bromonaphthalene and 2-bromothiophene respectively. Regiospecificity of the enzymatic bromination of anisole was tested and found to be the same as in the chemical reaction with NaOBr. The enzyme also acted on substituted alkenes such as styrene, cyclohexene, trans-cinnamic acid, trans-cinnamyl alcohol and cis-propenylphosphonic acid, to give the respective bromohydrin compounds or decarboxylated bromo compound. These bromohydrin compounds were always mixtures of stereoisomers. In the light of the above findings together with the previous studies concerning the halogenation mechanism, the bromoperoxidase of C. pilulifera was considered to have no specific restriction site for these substrates.     

Expression of human cytochrome P450 2B6 in Escherichia coli: characterization of catalytic activity and expression levels in human liver

Expression of human cytochrome P450 (P450) 2B6 in Escherichia coli was achieved following supplementation of the expression medium with chloramphenicol. The recombinant protein was purified using Ni(2+)-nitrilotriacetate chromatography and was characterized with regard to its spectral properties and catalytic activities toward typical P450 substrates. The purified recombinant protein was also used to raise polyclonal antibodies in rabbits. Examination of a panel of human liver microsomal preparations revealed expression of P450 2B6 in most samples, with levels of <1 to 30 pmol 2B6/mg microsomal protein. Examination of purified P450 2B6 preparations revealed the presence of a protease-sensitive site located 126 residues away from the N-terminus. The identity of the cleavage boundary was verified by protein sequence analysis. Cleavage of P450 2B6 at that site results in the presence of a lower molecular weight fragment of approximately 35 kDa in purified preparations. An immunoreactive peptide of a similar molecular weight was consistently observed in some but not all human liver microsomal preparations suggesting cleavage at the same site. Examination of catalytic activities of the purified reconstituted protein indicated the potential utility of (S)-mephenytoin N-demethylation and testosterone 16beta-hydroxylation as markers for P450 2B6.     

Purification and properties of an acetylxylan esterase from Thermobifida fusca

An acetylxylan esterase from Thermobifida fusca NTU22 was purified 51-fold as measured by specific activity from crude culture filtrate by ultrafiltration concentration, Sepharose CL-6B and DEAE-Sepharose CL-6B column chromatography. The overall yield of the purified enzyme was 14.4%. The purified enzyme gave an apparent single protein band on an SDS-PAGE. The molecular mass of purified enzyme as estimated by SDS-PAGE and by gel filtration on Sepharose CL-6B was found to be 30 and 28kDa, respectively, indicating that the acetylxylan esterase from T. fusca NTU22 is a monomer. The pI value of the purified enzyme was estimated to be 6.55 by isoelectric focusing gel electrophoresis. The N-terminal amino acid sequence of the purified esterase was ANPYERGP. The optimum pH and temperature for the purified enzyme were 8.0 and 80°C, respectively. The Zn(2+), Hg(2+), PMSF and DIPF inhibited the enzyme activity. The K(m) value for p-nitrophenyl acetate and acetylxylan were 1.86μM and 0.15%, respectively. Co-operative enzymatic degradation of oat-spelt xylan by purified acetylxylan esterase and xylanase significantly increased the acetic acid liberation compared to the acetylxylan esterase action alone.     

Purification of the insulin receptor from human placental membranes

Insulin receptors were purified from human placental microsomal membranes by solubilisation with Triton X-100 followed by Sepharose 6B chromatography, phosphate gradient elution from hydroxyapatite and affinity chromatography on concanavalin A-Sepharose. 2000-fold purification was achieved with 63% overall recovery. The purified receptor gave a single band on 3.75% polyacrylamide (0.1% Triton X-100) gel electrophoresis. On sodium dodecyl sulphate-polyacrylamide gel electrophoresis there was a major band at 75,000 and a minor band at 80,000 daltons. The purified receptor rechromatographed on Sepharose 6B with an apparent molecular weight of 300,000.     

Modification of halogen specificity of a vanadium-dependent bromoperoxidase

The halide specificity of vanadium-dependent bromoperoxidase (BPO) from the marine algae, Corallina pilulifera, has been changed by a single amino acid substitution. The residue R397 has been substituted by the other 19 amino acids. The mutant enzymes R397W and R397F showed significant chloroperoxidase (CPO) activity as well as BPO activity. These mutant enzymes were purified and their properties were investigated. The maximal velocities of CPO activities of the R397W and R397F enzymes were 31.2 and 39.2 units/mg, and the K(m) values for Cl(-) were 780 mM and 670 mM, respectively. Unlike the native enzyme, both mutant enzymes were inhibited by NaN(3). In the case of the R397W enzyme, the incorporation rate of vanadate into the active site was low, compared with the R397F and the wild-type enzyme. These results supported the existence of a specific halogen binding site within the catalytic cleft of vanadium haloperoxidases.     

Red Algae of Peter the Great Bay as a Source of Arachidonic and Eicosapentaenoic Acids

In order to find new sources of arachidonic (AA) and eicosapentaenoic (EPA) acids, the composition of fatty acids was studied and lipid concentrations were determined in the thalluses of 32 species of red algae from Peter the Great Bay, Sea of Japan. The greatest level of EPA and a small concentration of AA were registered in the thalluses of Corallina pilulifera, Palmaria stenogona, Halosaccion yendoi, and Laurencia nipponica. Taking into consideration the level of the lipid concentrations in the algae, as well as their biomass and frequency of occurrence, the algae C. pilulifera, P. stenogona, L. nipponica, and Polysiphonia morrowii may be of interest as potential sources of EPA. Among the examined algae, only Gracilaria verrucosa showed a high level of AA.     

Purification of the rabbit small intestinal sucrase-isomaltase complex: Separation from other maltases

The rabbit intestinal sucrase-isomaltase complex has been purified to homogeneity after solubilization with Triton X 100 followed by chromatography on DEAE Sepharose CL 6B and a second solubilization with papain. After hydrophobic chromatography on Octyl Sepharose CL 6B, separation from other contaminating maltases was achieved by gel filtration on Ultrogel ACA 22. The final enzyme was purified 390 fold, with a specific activity of about 10 units per mg protein.     

Improved isolation method and some properties of soybean gamma-conglycinin

Soybean gamma-conglycinin was isolated by isoelectric precipitation and ammonium sulphate fractionation. The crude protein was purified by ion-exchange chromatography on DEAE-Sepharose CL-6B and gel filtration on Sepharose CL-6B. The purified gamma-conglycinin was homogeneous on two kinds of gel electrophoresis and an ultracentrifugal analysis. A subunit band, distinguishable from other subunit bands of beta-conglycinin and glycinin, was detected by sodium dodecyl sulphate electrophoresis. Amino acid composition was similar to those of the other storage proteins of soybean. Some physical properties were also studied.     

HIV induces IL-6 production by human B lymphocytes. Role of IL-4.

In vitro, normal B cells can produce TNF-alpha and IL-6 when activated with a first signal, and cytokines and B lymphocytes from some HIV-infected individuals spontaneously secrete TNF-alpha and IL-6, although the direct involvement of HIV has not been fully explored. In this study, we examined the effects of HIV (purified virus and a recombinant envelope protein) and various IL on TNF-alpha and IL-6 in vitro production by highly purified normal B cells. HIV alone did not induce IL-6 or TNF-alpha production by B cells from healthy subjects. HIV induced IL-6 production (500 to 1500 pg) in the presence of IL-4, with a slight production of TNF-alpha. IL-6 production occurred independently of the presence or absence of TNF-alpha in contrast with Staphylococcus aureus cowan + IL-2-activated B cells. Other IL, particularly IL-2, were unable to induce IL-6 secretion by HIV-activated B cells. In vivo-activated B cells from HIV-infected patients spontaneously produce moderate quantities of IL-6 and TNF-alpha. This secretion was markedly increased by HIV, suggesting that IL-6-secreting B cells contain anti-HIV antibody-producing B cells. However, contrary to normal B cells, IL-6 production by B cells from HIV-infected patients was not further enhanced by IL-4. Then HIV itself is able to induce an autocrine production of IL-6 upon interaction with IL-4, which can contribute to the hypergammaglobulinemia and to the global B cell dysfunction observed in HIV-infected patients.     

Purification and positional specificity of sn-glycerol-3-phosphate acyltransferase from Escherichia coli membranes.

SN-Glycerol-3-phosphate acyltransferase was solubilized from membranes of Escherichia coli B and K-12 and purified on an affinity column of Sepharose 4B coupled with 6-phosphogluconic acid. Phosphatidylglycerol was required for activation and stabilization of the purified enzyme. The acyl residues were exclusively transferred to the position 1 of sn-glycerol 3-phosphate by the enzyme, regardless of whether the acyl-CoA was saturated or unsaturated.     

对脆弱类杆菌β-内酰胺酶及其与肠道微生态学关系的初步探讨

本文利用β-内酰胺酶作为分类学研究的方法,探讨了脆弱类杆菌与肠道菌之间的微生态学关系。临床分离菌株脆弱类杆菌55的β-内酰胺酶经离子交换层析、凝胶过滤和制备型聚丙烯酰胺凝胶电泳进行纯化,纯酶与脂质体-CPS-K佐剂混合制备抗血清,并建立IgG-ELISA和Western blotting方法,其测定结果均表明脆弱类杆菌的β-内酰胺酶不同于其它类杆菌和肠道菌的β-内酰胺酶,具有种的特异性。