Lipopolysaccharides of Rhizobium

Hot phenol-water extractions were carried out of cells from 12 strains of the fast-growing rhizobia Rhizobium leguminosarum, Rhizobium phaseoli, Rhizobium trifolii and Rhizobium meliloti. Purified lipopolysaccharide preparations contain neutral sugars, hexosamines, 2-keto-3-deoxyoctonate and uronic acids. Glucose, galactose, mannose, rhamnose and fucose are present in the majority of the LPS-preparations, but in varying proportions. Heptose was only found in some of them. O-methylated sugars are present in small amounts is most preparations, the kind of sugar being characteristic for lipopolysaccharides from different species. The lipid A part of lipopolysaccharides from all strains examined has identical patterns of fatty acids, namely -OH-C_14:0, -OH-C_15:0 (anteiso branched), -OH-C_16:0 and -OH-C_18:0. Comparison of the total compositions of Rhizobium lipopolysaccharides shows many differences among different species as among strains of a single species. Nearly identical lipopolysaccharide compositions also exist among certain strains, which constitute the same chemotype and which are also immunologically related. In view of a possible role of surface carbohydrates of Rhizobium in the root nodule symbiosis, the specificity of the binding of legume lectins with exo- and lipopolysaccharides of Rhizobium is discussed.Non-Standard Abbreviations LPS lipopolysaccharide(s) - EPS exopolysaccharides(s) - cetavlon cetyltrimethylammoniumbromide - KDO 2-keto-3-deoxyoctonate - ECL equivalent chain length Part II on Surface Carbohydrates of Rhizobium     

The Isolation and Partial Characterization of the Lipopolysaccharides from Several Rhizobium trifolii Mutants Affected in Root Hair Infection

The lipopolysaccharides (LPSs) from Rhizobium trifolii ANU843 and several transposon (Tn5) symbiotic mutants derived from ANU843 were isolated and partially characterized. The mutant strains are unable to induce normal root hair curling (Hac- phenotype) or nodulation (Nod-phenotype) in clover plants. The LPSs from the parent and mutants are very similar in composition. Analysis by PAGE shows that the LPSs consist of higher and lower molecular weight forms. The higher molecular weight form of the LPSs exists in several aggregation states when PAGE is done in 0.1% SDS but collapses into a single band when PAGE is done in 0.5% SDS. Mild acid hydrolysis of all the LPSs releases two polysaccharides, PS1 and PS2. Immunoblots of the PAGE gels and enzyme linked immunosorbant assay inhibition assays show that the PS1 fractions contain the immunodominant sites of the LPSs and that these sites are present in the higher molecular weight form of the LPSs. All the PS1 fractions contain methylated sugars, 2-amino-2,6-dideoxyhexose, heptose, glucuronic acid, and 2-keto-3-deoxyoctonic acid (KDO). All the PS2 fractions contain galacturonic acid, mannose, galactose, and KDO. The PS2 fractions have a molecular weight of about 700. The KDO is present at the reducing end of both the PS1 and the PS2 fractions. The PS1 and PS2 fractions from the mutants contain more glucose than these fractions from the parent. The LPS from a deletion mutant contains less acyl groups than the other LPSs. Immunoblots of the LPSs show that the parent and nod A mutant LPSs contain an additional antigenic band which is not observed in the other LPSs.     

Lipopolysaccharides of Vibrio cholerae: III. Biological functions

This review presents the salient features of the biological functions including the (i) endotoxic activities, (ii) antigenic properties, (iii) immunological responses to and (iv) phage receptor activities of the Vibrio cholerae lipopolysaccharides (LPS). The biological functions of the capsular polysaccharide (CPS) of V. cholerae have also been discussed briefly as a relevant topic. The roles of LPS and other extracellular polysaccharides in the (i) intestinal adherence and virulence of the vibrios and (ii) the biofilm formation by the organisms have been analysed on the basis of the available data. Every effort has been made to bring out, wherever applicable, the lacunae in our knowledge. The need for the continuous serogroup surveillance and monitoring of the environmental waters and the role of LPS in the designing of newer cholera vaccines has been discussed briefly in conclusion.     

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.     

乳链菌肽的分离纯化和部分生物学性质

用乳酸链球菌ＳＭ５２６进行乳链菌肽的发酵生产,产量为４０～５０ｍｇ／ｌ．经中空纤维超滤器超滤,非极性大孔吸附树脂ＸＡＤ－２层析,ＣＭ－ＳｅｐｈａｄｅｘＣ－２５层析和ＳｅｐｈａｄｅｘＧ－５０层析纯化了该肽。ＳＤＳ－ＰＡＧＥ表明达均一,ＲＰ－ＨＰＬＣ表明其纯度不低于９５％。ＳＤＳ－ＰＡＧＥ测其Ｍｒ约为３６００,用ＩＥＦ测其等电点为９．５．酸性条件下稳定且抗热;对胰蛋白酶、胃蛋白酶和木瓜蛋白酶不敏感,但对α－胰凝乳酶和蛋白酶Ｋ敏感。乳链菌肽对多种革兰氏阳性菌有强烈的抑制作用;以枯草杆菌和金黄色葡萄球菌为指示菌,其作用方式是杀菌。     

Rat Kidney Histamine N-Methyltransferase: Purification and Partial Characterization

Abstract

Histamine N-methyltransferase (HMT, EC 2.1.1.8) was purified 8,420-fold In 44% yield from rat kidney. The basic steps in the purification included differential centrlfugation, calcium phosphate adsorption, DEAE cellulose chromatography, and affinity chromatography on an S-adenosylhomocysteine-agarose matrix. The resulting protein was homogeneous as determined by gel electrophoresis and was stable for at least five months at ?80°C. The apparent molecular weight of the enzyme was found to be 31,500 as determined by gel filtration through Sephadex G-100 and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The Isoelectric point of the enzyme was determined to be 5.4. The K_m's for histamine and S-adenosyl-L-methionine were 12.4 ± 1.3 μM and 10.2 ±0.5 μM, respectively. When S-adenosyl-L-methionlne was the variable substrate, the K₁'s for S-adenosyl-L-homocysteine and S-adenosyl-D-homocys-teine were 31.9 ± 3.4 μM and 32.0 ± 3.5 μM, respectively. When histamine was the variable substrate, the K₁ for S-adenosyl-L-homocysteine was 11.8 ± 0.6 μM. Comparison of physico-chemical and catalytic properties of the rat kidney and the guinea pig enzymes suggest that these proteins have similar structural and catalytic characteristics.     

香灰菌凝集素的纯化及其部分性质

香灰菌菌丝体经磷酸缓冲液抽提、20%-70%饱和浓度的硫酸铵沉淀、DEAE-Cellulose和SephadexG-100柱层析纯化得到香灰菌凝集素(Hypoxylonsp.lectin,简称HSL)。HSL经PAGE检测为单一蛋白条带,SDS-PAGE测得其亚基分子量为15.9kD。过碘酸-Schiff染色法表明HSL为一种糖蛋白,糖基的含量为15.5%,β-消去反应测得其糖和蛋白质的连接键为O-型糖肽键。HSL能凝集多种动物红细胞和人的红细胞,在所测试的红细胞中,对兔红细胞的凝集作用最强。HSL对热较敏感,经50°C处理10min,其凝集活性明显降低,其在碱性环境中较稳定,而在酸性环境中较不稳定。HSL的凝集活性受Al3+、Fe3+、Ca2+和Zn2+等阳离子的影响。对鼠红细胞的凝集作用可被半乳糖和乳糖所抑制。     

Guinea Pig Brain Histamine N-Methyltransferase: Purification and Partial Characterization

Abstract: Histamine N-methyltransferase (EC 2.1.1.8) was purified 4400–fold in 12% yield from guinea pig brain. The basic steps in the purification included differential centrifugation, calcium phosphate adsorption, DEAE-cel-lulose chromatography, and affinity chromatography on an S-adenosylhomocysteine-agarose matrix. The resulting protein was homogeneous by gel electrophoresis and was stable for at least 3 months at 80°C. It had an apparent molecular weight of 29 ,000 ± 1000 as determined by both gel filtration through Sephadex G-100 and by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. The isoelectric point of the protein was found to be 5.3. The pH optima for methylation of histamine were determined to be 7.5 and 9.0; the K_ms for histamine and S-adenosyl-l-methionine were 13.57 ± 0.74 μM and 6.1 ± 0.12 μM, respectively; the K_i for S-adenosyl-l-homocysteine was 24.5 ± 1.45 μM.     

Partial Purification and Characterization of Feline Gamma-Like Interferon

Feline interferon (IFN) was produced from spleen cells stimulated by Staphylococcus enterotoxin A (SEA). The IFN was purified by a three-step procedure using controlled pore glass adsorption chromatography, concanavalin A (ConA)-agarose column chromatography and gel filtration. The final product of these procedures which had activity of an IFN appeared as a single peak of activity with molecular weight of approximately 50, 000. It was sensitive to acid and heat, suggesting that the isolated material was a gamma IFN.

The total recovery of feline gamma IFN was 8.2%. Specific activity was 2.95 × 10⁴ unit/μg protein and was concentrated 2.8 × 10⁴ times. This preparation of purified feline gamma IFN was destroyed completely by 0.1 % sodium dodecyl sulfate within 20 min. As an inducer of feline gamma IFN, SEA appeared to produce a more uniform IFN product than did ConA. Further, the presence of 10 7. ethylene glycol in the sample applied to ConA-agarose column as well as its absence in the elution buffer was effective in reducing contaminating acid- and heat-resistant INFs in the preparation.     

Characterization and Partial Purification of a Ganglioside-Associated Mitogen

A nonganglioside factor(s) present in Sigma types II and III mixed bovine brain ganglioside preparations synergises with suboptimal amounts of serum to induce proliferation specifically in nondividing B 103 neuroblastoma cultures. The active substance is nondialysable and soluble in water as well as in chloroform-methanol mixtures of 1:1-4:1 (vol/vol). It is completely insoluble in ether and acetone at room temperature. Biological activity survives heating to 70 degrees C in the presence of 0.1 M HCl for 1 h as well as boiling at neutral pH. Loss of activity occurs on heating to 70 degrees C for 1 h with 1 M HCl or 1 M NaOH. The activity is insensitive to digestion with neuraminidase, trypsin, pronase, and phospholipases A2 and C. The factor cochromatographs with gangliosides on Dowex AG 50W and Sephadex G100 and is partially recovered with GM1 on DEAE-Sepharose, but may be isolated in a ganglioside-free fraction by sequential chromatography on Sephadex LH20 and silicic acid columns. The substance(s) has the properties of a water-soluble proteolipid protein, the amino acid composition being reported. It is not immunologically cross-reactive with antibodies to GM1 ganglioside or the major proteolipid protein of myelin.     

Purification and Partial Characterization of Tomato Extensin Peroxidase

Early plant defense response is characterized by elevation of activity of peroxidases and enhanced insolubilization of hydroxyproline-rich glycoproteins, such as extensin, in the cell wall. The insolubilization process (cross-linking between soluble extensin precursor molecules) is catalyzed by extensin peroxidases. We have ionically eluted extensin peroxidases from intact water-washed suspension-cultured tomato (hybrid of Lycopersicon esculentum Mill. and Lycopersicon peruvianum L. [Mill.]) cells and purified them to homogeneity by molecular sieve and cation-exchange chromatography. Four ionic forms of peroxidase (PI,PII,EPIII, and EPIV) were resolved; only the latter two cross-linked tomato soluble extensin. The molecular weight (34,000-37,000), amino acid composition, and isoelectric point (9.0) of the extensin peroxidases were determined. Substrate specificities of the enzymes were investigated: soluble extensin and potato lectin (a hydroxyproline-rich glycoprotein with a domain that strongly resembles extensin) were cross-linked by only two forms of the enzyme, whereas bovine serum albumin, aldolase, insulin, a number of other marker proteins, and proteins eluted from tomato cells (except extensin) could not be cross-linked. We have also isolated a yeast elicitor that enhances total peroxidase activity and extensin insolubilization within 1 h of challenge in cultured cells of tomato. A highly sensitive enzyme-linked immunosorbent assay technique using polyclonal antiserum raised against soluble tomato extensin was used to demonstrate extensin insolubilization in vivo. A tomato cell-wall peroxidase that cross-links extensin has been purified and may have a role in plant defense.     

Host-Pathogen Interactions: XIV. Isolation and Partial Characterization of an Elicitor from Yeast Extract

An elicitor of glyceollin accumulation in soybeans (Glycine max L.) has been isolated from a commercially available extract of brewers' yeast. Yeast is not a known pathogen of plants. The elicitor was isolated by precipitation in 80% (v/v) ethanol followed by column chromatography on DEAE-cellulose, sulfopropyl-Sephadex, and concanavalin A-Sepharose. Compositional and structural analysis showed the elicitor to be a glucan containing terminal, 3-, 6-, and 3,6-linked glucosyl residues. The yeast elicitor stimulates the accumulation of glyceollin in the cotyledons and hypocotyls of soybeans when as little as 15 nanograms or 100 nanograms of the elicitor is applied to the respective tissues. The yeast elicitor is very similar in both structure and absolute elicitor activity to an elicitor isolated from the mycelial walls of Phytophthora megasperma var. sojae, a pathogen of soybeans. These and other results of this laboratory suggest that plants are able to respond to the presence of a wide range of fungi by recognizing, as foreign to the plant, structural polysaccharides of the mycelial walls of the fungi.     

The Bark Lectin of Robinia pseudoacacia: Purification and Partial Characterization

A lectin was purified from the bark of Robinia pseudoacaciaby sequential ion-exchange chromatography on DEAE-Sepharoseand CM-Toyopearl. The purified lectin was estimated to havea molecular weight of 106 kDa and to be a homotetramer of subunitswith a molecular weight of 29 kDa. Antibodies raised againstthe bark lectin cross-reacted with a 29-kDa polypeptide duringWestern blot analysis, showing that the antibodies are specificfor the bark lectin. The antibodies against the lectin fromRobinia bark cross-reacted with polypeptides in extracts ofthe seeds and bark of Sophora japonica, indicating that thelectin from Robinia bark is immunologically related to the lectinsof Sophora. However, the antibodies did not cross-react withproteins from Robinia seeds and leaves. The first twenty aminoacid residues from the N-terminus of the lectin from Robiniabark were determined and compared with those of the Sophoralectins. (Received July 13, 1991; Accepted December 12, 1991)     

重组E．coli胆固醇氧化酶的分离纯化及其酶学性质研究

对重组E.coli产生的胆固醇氧化酶采用70%硫酸铵盐析、CM Sepharose FF离子层析、Phenyl Sepharose 6 Fast疏水层析、Sephadex G-75凝胶过滤,得到的胆固醇氧化酶在SDS-PAGE上呈单一蛋白质条带,酶的纯化倍数为93,收率为21%.部分酶学性质表明:酶的最适反应温度为37℃,最适反应pH7.5,热稳定范围在40℃以下,酶的pH稳定范围为6～9,分子量分别为50 kD和52 kD.酶动力学参数Km值及Vmax分别为8.2×10-5 mol/L和0.21 mmol/(L.min).     

Isolation and Partial Characterization of DNA in Capsular Preparations of Rhizobium trifolii

The capsular polysaccharides from thymidine-(methyl-³H) labeled cultures of Rhizobium trifolii; strain 162S7 (Nitragin Co.) were centrifuged from bacterial cells and collected by ethanol precipitation. Following the addition of unlabeled carrier nucleic acids, labeled DNA, termed cap-DNA, was isolated from the capsular polysaccharides. Cap-DNA absorbed maximally at H-260 nm and was DNase sensitive. Approximately 11 μg of ³H-cap-DNA were consistently isolated per liter of 48 h cultures. Cap-DNA production was generally synchronized with the synthesis of the capsular polysaccharide and bacterial growth, attaining maximum recoverable amounts in 48 h cultures. By five days of culture growth, significant decreases in the amount of recoverable cap-DNA were noted. The presence of label in the cap-DNA demonstrated that the cap-DNA originated via de novo synthesis by the Rhizobium cells rather than from an anomalous source. The cap-DNA and intracellular Rhizobium DNA had similar buoyant densities of p= 1.719, indicating that cap-DNA arose specifically from the intracellular DNA. In 48-h cultures the specific activity of the cap-DNA was about one-third that of the intracellular DNA. This implies intracellular DNA was released during early growth with a relatively low specific activity which diluted the isotopic label of DNA released later. The evidence suggests lysogeny was the principal release mechanism.     

Partial Purification and Characterization of a Thermostable Actinomycete beta-Amylase

A thermostable amylase, possibly a beta-amylase from Thermoactinomyces sp. no. 2 isolated from soil, is reported. The enzyme was purified 36-fold by acetone precipitation, ion-exchange chromatography, and Sephadex G-200 gel filtration, and the molecular weight was estimated at 31,600. The enzyme was characterized by demonstration of optimum activity at 60 degrees C and pH 7 and by retention of 70% activity at 70 degrees C (30 min). It was stimulated by Mn and Fe but strongly inhibited by Hg. Maltose was the only detectable product of hydrolysis of starches and was quantitatively highest in plantain starch hydrolysate.     

Purification and Partial Characterization of Human C1-Esterase

C1-Esterase was purified from the euglobulin fraction of human plasma by successive column chromatography on DEAE-cellulose, hydroxylapatite and TEAE-cellulose. The final product, purified 3500-fold with respect to serum, hydrolyzed 1,155 μmoles of N^α-acetyl-l-tyrosine ethyl ester per milligram of protein at pH 7.4 and 37°C in 15 min. The homogeneity of the purified C1-esterase was confirmed by ultracentrifugation and disc-electrophoresis. Its s_20,w value was 4.3 and its molecular weight was determined as 113,000 by gel filtration on Sephadex G–200.

Cl-Esterase possesses esterolytic activity for both N^α-acetyl-l-tyrosine ethyl ester and N^α-tosyl-l-arginine methyl ester, and acts on human kininogen I and II releasing kinin very slowly.     

Purification and Partial Characterization of Polysaccharides From Coconut Milk

Ethanol-precipitated polysaccharides of the liquid endospermof coconut, Cocos nucifera L., were composed predominantly ofgalactose and arabinose with minor amounts of mannose and glucose.Gel filtration chromatography on Bio-Gel A-0.5 m revealed asingle major peak (Peak A) at the void volume and a minor peak(Peak B) partially included in the column volume. Peak A containedsome uronosyl residues, but was not susceptible to cleavageby endopolygalacturonase, indicating that it does not containsignificant amounts of polygalacturonic acid. Neutral glycosylresidue composition analysis of Peak A showed that it consistedof 72% galactose and 24% arabinose with minor amounts of glucoseand rhamnose. Coconut milk, Cocos nuciferaL, polysaccharides, glycosyl composition     

Purification and Partial Characterization of Bacillus subtilis Flagellar Hooks

A method for preparing bacterial flagellar hook structures is described. The method involves isolating intact flagella from a mutant which makes thermally labile flagellar filaments and heat-treating them to disaggregate the filament preferentially. The resulting hook preparation can be separated and purified by velocity and isopycnic centrifugation. The purified hooks sediment at a relative S value of 77. On acrylamide gel electrophoresis in sodium dodecyl sulfate, they show one major and a number of minor protein bands. The purified hooks can be used to immunize rabbits, and the resulting antiserum is hook-specific. These results support the notion that hooks are composed of a protein that differs from flagellin.     

Characterization of the Lipopolysaccharides and Capsules of Shewanella spp.

Electron microscopy, sodium dodecyl sulfate-polyacrylamide gel electrophoresis with silver staining and ¹H, ¹³C, and ³¹P-nuclear magnetic resonance (NMR) were used to detect and characterize the lipopolysaccharides (LPSs) of several Shewanella species. Many expressed only rough LPS; however, approximately one-half produced smooth LPS (and/or capsular polysaccharides). Some LPSs were affected by growth temperature with increased chain length observed below 25°C. Maximum LPS heterogeneity was found at 15 to 20°C. Thin sections of freeze-substituted cells revealed that Shewanella oneidensis, S. algae, S. frigidimarina, and Shewanella sp. strain MR-4 possessed either O-side chains or capsular fringes ranging from 20 to 130 nm in thickness depending on the species. NMR detected unusual sugars in S. putrefaciens CN32 and S. algae BrY^DL. It is possible that the ability of Shewanella to adhere to solid mineral phases (such as iron oxides) could be affected by the composition and length of surface polysaccharide polymers. These same polymers in S. algae may also contribute to this opportunistic pathogen's ability to promote infection.