Active nucleoprotein filaments of single-stranded binding protein and recA protein on single-stranded DNA have a regular repeating structure

When E. coli single-stranded DNA binding protein (SSB) coats single-stranded DNA (ssDNA) in the presence of 1 mM MgCl2 it inhibits the subsequent binding of recA protein, whereas SSB binding to ssDNA in 12 mM MgCl2 promotes the binding of recA protein. These two conditions correspond respectively to those which produce 'smooth' and 'beaded' forms of ssDNA-SSB filaments. By gel filtration and immunoprecipitation we observed active nucleoprotein filaments of recA protein and SSB on ssDNA that contained on average 1 monomer of recA protein per 4 nucleotides and 1 monomer of SSB per 20-22 nucleotides. Filaments in such a mixture, when digested with micrococcal nuclease produced a regular repeating pattern, approximately every 70-80 nucleotides, that differed from the pattern observed when only recA protein was bound to the ssDNA. We conclude that the beaded ssDNA-SSB nucleoprotein filament readily binds recA protein and forms an intermediate that is active in the formation of joint molecules and can retain substantially all of the SSB that was originally bound.

Cloning, overexpression, folding and purification of a biosynthetically derived three disulfide scorpion toxin (BTK-2) from Mesobuthus tamulus

BTK-2, a 32 residue scorpion toxin initially identified in the venom of red Indian scorpion Mesobuthus tamulus was cloned, overexpressed and purified using Cytochrome 155 fusion protein system developed in our laboratory. The synthetic gene coding for the peptide was designed taking into account optimal codon usage by Escherichia coli. High expression levels of the fusion protein enabled facile purification of this peptide. The presence of disulfide bonded isomers, occurring as distinctly populated states even in the fusion protein, were separated by gel filtration chromatography. The target peptide was liberated from the host protein by Tev protease cleavage and subsequent purification was achieved using RP-HPLC methods. Reverse phase HPLC clearly showed the presence of at least two isomeric forms of the peptide that were significantly populated. The oxidative folding of BTK-2 was achieved under ambient conditions during the course of purification. Structural characterization of the two forms, by solution homonuclear and heteronuclear NMR methods, has shown that these two forms exhibit significantly different structural properties, and represent the natively folded and a "misfolded" form of the peptide. The formation of properly folded BTK-2 as a major fraction without the use of in vitro oxidative refolding methods clearly indicate the versatility of the Cytochrome b(5) fusion protein system for the efficient production of peptides for high resolution NMR studies.

Numerical Simulation of Groundwater Flow with Gradually Increasing Heterogeneity due to Clogging

Well injection replenishes depleting water levels in a well field. Observation well water levels some distance away from the injection well are the indicators of the success of a well injection program. Simulation of the observation well response, located a few tens of meters from the injection well, is likely to be affected by the effects of nonhomogeneous medium, inclined initial water table, and aquifer clogging. Existing algorithms, such as the U.S. Geological Survey groundwater flow software MODFLOW, are capable of handling the first two conditions, whereas time-dependent clogging effects are yet to be introduced in the groundwater flow models. Elsewhere, aquifer clogging is extensively researched in theory of filtration; scope for its application in a well field is a potential research problem. In the present paper, coupling of one such filtration theory to MODFLOW is introduced. Simulation of clogging effects during “Hansol” well recharge in the parts of western India is found to be encouraging.

Role of histidine residues in EcoP15I DNA methyltransferase activity as probed by chemical modification and site-directed mutagenesis

Towards understanding the catalytic mechanism of M.EcoP15I [EcoP15I MTase (DNA methyltransferase); an adenine methyltransferase], we investigated the role of histidine residues in catalysis. M.EcoP15I, when incubated with DEPC (diethyl pyrocarbonate), a histidine-specific reagent, shows a time- and concentration-dependent inactivation of methylation of DNA containing its recognition sequence of 5'-CAGCAG-3'. The loss of enzyme activity was accompanied by an increase in absorbance at 240 nm. A difference spectrum of modified versus native enzyme shows the formation of N-carbethoxyhistidine that is diminished by hydroxylamine. This, along with other experiments, strongly suggests that the inactivation of the enzyme by DEPC was specific for histidine residues. Substrate protection experiments show that pre-incubating the methylase with DNA was able to protect the enzyme from DEPC inactivation. Site-directed mutagenesis experiments in which the 15 histidine residues in the enzyme were replaced individually with alanine corroborated the chemical modification studies and established the importance of His-335 in the methylase activity. No gross structural differences were detected between the native and H335A mutant MTases, as evident from CD spectra, native PAGE pattern or on gel filtration chromatography. Replacement of histidine with alanine residue at position 335 results in a mutant enzyme that is catalytically inactive and binds to DNA more tightly than the wild-type enzyme. Thus we have shown in the present study, through a combination of chemical modification and site-directed mutagenesis experiments, that His-335 plays an essential role in DNA methylation catalysed by M.EcoP15I.

Purification, characterization and molecular cloning of a monocot mannose-binding lectin from Remusatia vivipara with nematicidal activity

A mannose-binding lectin (RVL) was purified from the tubers of Remusatia vivipara, a monocot plant by single-step affinity chromatography on asialofetuin-Sepharose 4B. RVL agglutinated only rabbit erythrocytes and was inhibited by mucin, asialomucin, asialofetuin and thyroglobulin. Lectin activity was stable up to 80A degrees C and under wide range of pH (2.0-9.3). SDS-PAGE and gel filtration results showed the lectin is a homotetramer of Mr 49.5 kDa, but MALDI analysis showed two distinct peaks corresponding to subunit mass of 12 kDa and 12.7 kDa. Also the N-terminal sequencing gave two different sequences indicating presence of two polypeptide chains. Cloning of RVL gene indicated posttranslational cleavage of RVL precursor into two mature polypeptides of 116 and 117 amino-acid residues. Dynamic light scattering (DLS) and gel filtration studies together confirmed the homogeneity of the purified lectin and supported RVL as a dimer with Mr 49.5 kDa derived from single polypeptide precursor of 233 amino acids. Purified RVL exerts potent nematicidal activity on Meloidogyne incognita, a root knot nematode. Fluorescent confocal microscopic studies demonstrated the binding of RVL to specific regions of the alimentary-tract and exhibited a potent toxic effect on M. incognita. RVL-mucin complex failed to interact with the gut confirming the receptor mediated lectin interaction. Very high mortality (88%) rate was observed at lectin concentration as low as 30 A mu g/ml, suggesting its potential application in the development of nematode resistant transgenic-crops.

Physicochemical studies on the gelation of hen egg yolk; separation of gelling protein components from yolk plasma

A study of the component(s) in egg yolk responsible for gelation of yolk on freezing and thawing has shown that granule-free yolk plasma, obtained by high-speed centrifugation of yolk, has the capacity to gel. As with the whole yolk, gelation of yolk plasma on freezing and thawing could be inhibited by additives such as sugars, sodium chloride, proteolytic enzymes, and phospholipase-A. Phospholipase-C, which induces gelation of whole yolk at room temperature, has a similar effect on yolk plasma. Yolk plasma has been separated into aggregating (gelling) and soluble fractions by delipidation, using formic acid. Each of these fractions consists of three or four protein components, as observed by gel filtration, ultracentrifugation, and agar electrophoresis. The proteins are glycoproteins and contain bound hexoses, hexosamine, and sialic acid. The gelation of yolk has been attributed to the interactions between protein molecules following disruption of lipid-protein bonds.

Oxidation of catechol in plants : III. Purification and properties of the diphenylene dioxide 2,3-quinone-forming enzyme system from Tecoma leaves

In attempting to determine the nature of the enzyme system mediating the conversion of catechol to diphenylenedioxide 2,3-quinone, in Tecoma leaves, further purification of the enzyme was undertaken. The crude enzyme from Tecoma leaves was processed further by protamine sulfate precipitation, positive adsorption on tricalcium phosphate gel, and elution and chromatography on DEAE-Sephadex. This procedure yielded a 120-fold purified enzyme which stoichiometrically converted catechol to diphenylenedioxide 2,3-quinone. The purity of the enzyme system was assessed by polyacrylamide gel electrophoresis. The approximate molecular weight of the enzyme was assessed as 200,000 by gel filtration on Sephadex G-150. The enzyme functioned optimally at pH 7.1 and at 35 °C. The Km for catechol was determined as 4 × 10−4 Image . The enzyme did not oxidize o-dihydric phenols other than catechol and it did not exhibit any activity toward monohydric and trihydric phenols and flavonoids. Copper-chelating agents did not inhibit the enzyme activity. Copper could not be detected in the purified enzyme preparations. The purified enzyme was not affected by extensive dialysis against copper-complexing agents. It did not show any peroxidase activity and it was not inhibited by catalase. Hydrogen peroxide formation could not be detected during the catalytic reaction. The enzymatic conversion of catechol to diphenylenedioxide 2,3-quinone by the purified Tecoma leaf enzyme was suppressed by such reducing agents as GSH and cysteamine. The purified enzyme was not sensitive to carbon monoxide. It was not inhibited by thiol inhibitors. The Tecoma leaf was found to be localized in the soluble fraction of the cell. Treatment of the purified enzyme with acid, alkali, and urea led to the progressive denaturation of the enzyme.

Purification and characterization of two cellobiohydrolases from Chaetomium thermophile var. coprophile

Cellobiohydrolases I and II were purified to homogeneity from culture filtrates of a thermophilic fungus, Chaetomium thermophile var. coprophile, by using a combination of ion-exchange and gel filtration chromatographic procedures. The molecular weights of cellobiohydrolase I and II were estimated to be 60000 and 40000 and the enzymes were found to be glycoproteins containing 17 and 22.8% carbohydrate, respectively. The two forms differed in their amino-acid composition mainly with respect to threonine, alanine, methionine and arginine. Antibodies produced against either form of cellobiohydrolases failed to cross-react with the other. The tryptic maps of the two enzymes were found to be different. The temperature optima for cellobiohydrolase I and II were 75 and 70°C, and they were optimally active at pH 5.8 and 6.4, respectively. Both enzymes were stable at higher temperatures and were able to degrade crystalline cellulosic materals.

Biochemical characterization of the intracellular domain of the human guanylyl cyclase C receptor provides evidence for a catalytically active homotrimer

Guanylyl cyclase C (GCC) is the receptor for the family of guanylin peptides and bacterial heat-stable enterotoxins (ST). The receptor is composed of an extracellular, ligand-binding domain and an intracellular domain with a region of homology to protein kinases and a guanylyl cyclase catalytic domain. We have expressed the entire intracellular domain of GCC in insect cells and purified the recombinant protein, GCC-IDbac, to study its catalytic activity and regulation. Kinetic properties of the purified protein were similar to that of full-length GCC, and high activity was observed when MnGTP was used as the substrate. Nonionic detergents, which stimulate the guanylyl cyclase activity of membrane-associated GCC, did not appreciably increase the activity of GCC-IDbac, indicating that activation of the receptor by Lubrol involved conformational changes that required the transmembrane and/or the extracellular domain. The guanylyl cyclase activity of GCC-IDbac was inhibited by Zn2+, at concentrations shown to inhibit adenylyl cyclase, suggesting a structural homology between the two enzymes. Covalent crosslinking of GCC-IDbac indicated that the protein could associate as a dimer, but a large fraction was present as a trimer. Gel filtration analysis also showed that the major fraction of the protein eluted at a molecular size of a trimer, suggesting that the dimer detected by cross-linking represented subtle differences in the juxtaposition of the individual polypeptide chains. We therefore provide evidence that the trimeric state of GCC is catalytically active, and sequences required to generate the trimer are present in the intracellular domain of GCC.

Purification and properties of β-glucosidase from a thermophilic fungus Humicola lanuginosa (Griffon and Maublanc) Bunce

An extracellular β-glucosidase (EC 3.2.1.21) has been purified to homogeneity from the culture filtrate of a thermophilic fungus, Humicola lanuginosa (Griffon and Maublanc) Bunce, using duplicating paper as the carbon source. The enzyme was purified 82-fold with a 43% yield by ion-exchange chromatography and gel filtration. The molecular weight of the protein was estimated to be 135,000 by gel filtration and 110,000 by electrophoresis. The sedimentation coefficient was 10.5 S. It was an acidic protein containing high amounts of acidic amino acid residues. It was poor in sulphur-containing amino acids. It also contained 9% carbohydrate. The enzyme activity was optimum at pH 4.5 and at 60°C. The enzyme was stable in the pH range 6–9 for 24 h at 25°C. The enzyme had similar affinities towards cellobiose and p-nitrophenyl-β-d-glucoside with Km values of 0.44 mM and 0.50 mM, respectively. The enzyme was capable of hydrolysing larchwood xylan, xylobiose and p-nitrophenyl-β-d-xyloside, though to a lesser extent. The enzyme was specific for the β-configuration and glucose moiety in the substrate.

Sorghum proteinase inhibitors: purification and some biochemical properties

An investigation has been carried out on the proteinase inhibitors of grain sorghum (Sorghum bicolor (L.) Moench). One of the inhibitors has been isolated in a pure form and characterized. The proteinase inhibitor was extracted from the acetone-defatted sorghum meal and purified by selective thermal denaturation, ammonium sulfate fractionation, Sephadex gel filtration and DEAE-cellulose chromatography (DEAE-preparation II). This preparation was demonstrated to be a mixture of three inhibitor components by polyacrylamide disc gel electrophoresis. Further resolution of this mixture into Inhibitors I to III was achieved by QAE-Sephadex chromatography. Sorghum Inhibitor III was homogeneous by the criteria of disc gel electrophoresis and has been more fully characterized. A molecular weight of 25,000 was obtained for Inhibitor III by gel filtration and was in agreement with the value calculated from the amino acid composition of the inhibitor. The N-terminal amino acid residue of Inhibitor III, a single chain protein, was isoleucine. Sorghum proteinase inhibitors inhibit specifically the serine proteinases and are inactive towards the other classes of proteinases. Inhibitor III is primarily a chymotrypsin inhibitor, whereas Inhibitors I and II inhibit both trypsin and chymotrypsin.

Purification and characterization of endo-1,4-β-xylanase from Paecilomyces varioti Bainier

An endo-xylanase (1,4-β-d-xylanxylanohydrolase EC 3.2.1.8) was isolated from the culture filtrate of Paecilomyces varioti Bainier. The enzyme was purified 3.2 fold with a 60% yield by gel filtration and ion exchange chromatography. The purified enzyme had a molecular weight of 25,000 with a sedimentation coefficient of 2.2 S. The isoelectric point of the enzyme was 3.9. The enzyme was obtained in crystalline form. The optimum pH range was 5.5–7.0 and the temperature, 65°C. The Michaelis constant was 2.5 mg larchwood xylan/ml. The enzyme was found to degrade xylan by an endo mechanism producing arabinose, xylobiose, xylo- and arabinosylxylo-oligosaccharides, during the initial stages of hydrolysis. On prolonged incubation, xylotriose, arabinosylxylotriose and xylobiose were the major products with traces of xylotetraose, xylose and arabinose.

Purification and characterization of a beta-glucosidase and endocellulase from Humicola insolens

A .beta.-glucosidase and an endocellulase were purified from the culture filtrates of a thermophilic cellulolytic fungus Humicola insolens. Both the preparations were homogeneous by PAGE, ultracentrifugation and gel filtration (Mr 45,000). Ouchterlony immunodiffusion showed complete cross reactivity between the antibodies and the two enzyme antigens, indicating the presence of a common epitope on the two enzyme proteins. The two enzymes, however, differ in their amino acid composition and their substrate specificity. .beta.-Glucosidase acts on p-nitrophenyl .beta.-D-glucopyranoside and hydrolyses cellulose to release mainly glucose and small amounts of cellobiose from the non-reducing end. On the other hand, endocellulase hydrolyses cellulose to release cellopentaose, cellotetraose, cellotriose along with cellobiose and glucose and also hydrolyses larch wood xylan.

Characterization of coproporphyrinogen III oxidase in Plasmodium falciparum cytosol

A unique hybrid pathway has been proposed for de novo heme biosynthesis in Plasmodium falciparum involving three different compartments of the parasite, namely mitochondrion, apicoplast and cytosol. While parasite mitochondrion and apicoplast have been shown to harbor key enzymes of the pathway, there has been no experimental evidence for the involvement of parasite cytosol in heme biosynthesis. In this study, a recombinant P. falciparum coproporphyrinogen III oxidase (rPfCPO) was produced in E. coli and confirmed to be active under aerobic conditions. rPfCPO behaved as a monomer of 61 kDa molecular mass in gel filtration analysis. Immunofluorescence studies using antibodies to rPfCPO suggested that the enzyme was present in the parasite cytosol. These results were confirmed by detection of enzyme activity only in the parasite soluble fraction. Western blot analysis with anti-rPfCPO antibodies also revealed a 58 kDa protein only in this fraction and not in the membrane fraction. The cytosolic presence of PfCPO provides evidence for a hybrid heme-biosynthetic pathway in the malarial parasite. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

Purification and characterization of three toxins and two agglutinins from Abrus precatorius seed by using lactamyl-Sepharose affinity chromatography

Three toxins, abrin-I, -II, and -III, and two agglutinins, APA-I and -II, were purified from the seeds of Abrus precatorius by lactamyl-Sepharose affinity chromatography followed by gel filtration and DEAE-Sephacel column chromatography. abrin-I did not bind on DEAE-Sephacel column chromatography and the bound abrin-II, abrin-III, APA-I, and APA-II were eluted with a sodium acetate gradient. The identity of each protein was established by sodium dodecylsulfate-polyacrylamide gel electrophoresis and isoelectric focusing. The relative molecular weights are abrin-I, 64,000; abrin-II and abrin-III, 63,000 each: APA-I, 130,000; and APA-II, 128,000. Isoelectric focusing revealed microheterogeneity due to the presence of isoforms in each protein. Toxicity and binding studies further confirmed the differences among the lectins. The time course of inhibition of protein synthesis in thymocytes by the toxins showed lag times of 78, 61, and 72 min with Ki's of 0.55, 0.99, and 0.74 ms−1 at a 0.63 nImage concentration of each of abrin-I, -II, and -III, respectively. A Scatchard plot obtained from the equilibrium measurement for the lectins binding to lactamyl-Sepharose beads showed nonlinearity, indicating a cooperative mode of binding which was not observed for APA-I binding to Sepharose 4B beads. Further, by the criterion of the isoelectric focusing profile, it was shown that the least toxic abrin-I and the highly toxic abrin-II isolated by lactamyl-Sepharose chromatography were not retained on a low-affinity Sepharose 4B matrix, which signifies the necessity of using a high-affinity matrix for the purification of the lectins.