Subunit MW Research Articles

Cross protectivity analysis of 49.8 kDa pili sub units of Shigella flexneri against Vibrio cholerae infection: In vivo approach

Background: Candidates for the cholera and shigellosis vaccine have similarity characteristics as a molecule of hemagglutinin and adhesin. But there is a difference in the molecular weight of the two bacteria, which is 37.8 kDa for V. cholerae and 49.8 kDa S. flexneri. We investigate whether there is a similarity between the results of the immune response between infection with S.flexneri and V. cholerae. The form of anti-hemagglutination properties and protection against vaccinated with S.flexneri adhesion molecules are conducted as well. Methods and materials: The first step was an anti-hemagglutination test using antibody anti adhesion molecle S. flexneri (AMS). The second step is to find out the immune response of the protectivity. The research model in the second step used Mice Ligated Ileal Loop (MLIL), with the subjects mice balb /c. Mice separated into four groups. The frts group was negative control group. The second group infected with S. flexneri. The third group immunized before affected by Shigella flexneri. The last group same by the third but infected with V. cholerae. The immune response calculates the weight and counts the bacterial colonies. The last step find out of the others respone immune AMS which calculated β-defensin and s-IgA levels measured using ELISA. After that the cellular immune response measured the number of Th-17 and T-reg immune effector cells using the flow cytometry method. The pro-inflammatory cytokines IL-17A uses the ELISA. Data analysis used ANOVA, Independent T-test, Kruskal Walis, and Mann Whitney. Results: The results found cross-immune response anti-hemagglutination, intestinal weight, and bacterial colony count. Other findings were found to be significantly (p < 0.05) for levels of β-defensin, s-IgA, Th17, Treg, IL-17A. Conclusion: Protein subunit MW 49.8 kDa adhesion molecule of S. flexneri may act as the candidate for shigellosis and cholera in the future.

C5a induces caspase-dependent apoptosis in brain vascular endothelial cells in experimental lupus.

Blood-brain barrier (BBB) dysfunction complicates central nervous system lupus, an important aspect of systemic lupus erythematosus. To gain insight into the underlying mechanism, vascular corrosion casts of brain were generated from the lupus mouse model, MRL/lpr mice and the MRL/MpJ congenic controls. Scanning electron microscopy of the casts showed loss of vascular endothelial cells in lupus mice compared with controls. Immunostaining revealed a significant increase in caspase 3 expression in the brain vascular endothelial cells, which suggests that apoptosis could be an important mechanism causing cell loss, and thereby loss of BBB integrity. Complement activation occurs in lupus resulting in increased generation of circulating C5a, which caused the endothelial layer to become 'leaky'. In this study, we show that C5a and lupus serum induced apoptosis in cultured human brain microvascular endothelial cells (HBMVECs), whereas selective C5a receptor 1 (C5aR1) antagonist reduced apoptosis in these cells, demonstrating C5a/C5aR1-dependence. Gene expression of initiator caspases, caspase 1 and caspase 8, and pro-apoptotic proteins death-associated protein kinase 1, Fas-associated protein (FADD), cell death-inducing DNA fragmentation factor 45 000 MW subunit A-like effector B (CIDEB) and BCL2-associated X protein were increased in HBMVECs treated with lupus serum or C5a, indicating that both the intrinsic and extrinsic apoptotic pathways could be critical mediators of brain endothelial cell apoptosis in this setting. Overall, our findings suggest that C5a/C5aR1 signalling induces apoptosis through activation of FADD, caspase 8/3 and CIDEB in brain endothelial cells in lupus. Further elucidation of the underlying apoptotic mechanisms mediating the reduced endothelial cell number is important in establishing the potential therapeutic effectiveness of C5aR1 inhibition that could prevent and/or reduce BBB alterations and preserve the physiological function of BBB in central nervous system lupus.

Effects of the specific mechanical energy on the physicochemical properties of texturized soy protein during high-moisture extrusion cooking

Effects of the extrusion specific mechanical energy (SME) on the physicochemical properties including color parameters (L*, a*, b* and ΔE), textural properties (tensile strength, hardness, chewiness and degree of texturization), on-line viscosity at die, protein solubility, and the molecular weight distribution of texturized soy protein were investigated. A higher SME resulted in extrudates with lower L* but higher a*, ΔE, higher tensile strength, and hardness. The viscosity of dough at die decreased from 997.17 to 867.29Pas when SME increased from 819.70 to 1258.70kJ/kg. The increasing SME also caused an increase of protein solubility in phosphate buffer solution by 15.30%. Increasing the SME resulted in higher content of low Mw subunits as well, which was evidenced by SDS–PAGE. It was concluded that increasing SME increased the proportion of smaller fractions, and led to darker and higher tensile strength products.

Structure determination of theO-methyltransferase NovP using the `free lunch algorithm' as implemented inSHELXE

NovP is an S-adenosyl-L-methionine-dependent O-methyltransferase from Streptomyces spheroides (subunit MW = 29 967 Da). Recombinant N-terminally His-tagged NovP crystallizes in space group P2, with approximate unit-cell parameters a = 51.81, b = 46.04, c = 61.22 A, beta = 105.0 degrees , giving a solvent content of 44% for a single copy of the His-tagged protomer per asymmetric unit. Native synchrotron data to a resolution of 1.35 A were combined with three other native data sets collected at lower resolution (both in-house and at the synchrotron) for the sake of completeness and better scaling. Data to 2.45 A resolution were subsequently recorded in-house from a single mercury derivative. Three partial mercury sites could be located with SHELXD, but the resulting phases had a mean error of about 81 degrees and in our hands did not yield an interpretable map using standard automated software. Nevertheless, the structure of NovP could be solved by first tracing a small part of the structure by hand and then extrapolating within and beyond the experimental resolution limit using the ;free lunch algorithm' in SHELXE. The resulting phases have a mean phase error of 17 degrees relative to a refined model.

Purification and biochemical characterization of a superoxide dismutase from the soluble fraction of the cyanobacterium, Spirulina platensis

A superoxide dismutase (SOD) was purified from Spirulina platensis sonicate. The SOD was purified to homogeneity (48-fold and 0.24% yield) through ammonium sulphate precipitation and DEAE-52 anion exchange chromatography. The SOD from S. platensis appeared to be a homodimer with a molecular weight of 30kDa and a subunit MW of 15kDa as determined by both native polyacrylamide gel electrophoresis and mass spectrometry. The enzyme activity was stable at pH 6.5-10.0 and 50°C. Using group-specific chemical modifying reagents, the amino acids arginine, histidine, tryptophan, tyrosine and aspartic acid were identified to be essential for S. platensis SOD activity. The amino acid composition was found to lack methionine and cysteine. The inhibition of activity by H2O2 suggests that the enzyme may be an iron containing SOD.

Functional expression in Escherichia coli of the tyrosine-inducible tyrosine ammonia-lyase enzyme from yeast Trichosporon cutaneum for production of p-hydroxycinnamic acid

Deamination of aromatic amino acids phenylalanine or tyrosine is accomplished by the phenylalanine/tyrosine ammonia-lyase (PAL/TAL) enzyme. TAL enzyme is of interest since deamination of tyrosine produces p-hydroxycinnamic acid (pHCA), which has potential for a variety of applications. Among nine microorganisms tested for their ability to produce tyrosine-inducible TAL activity, the yeast, Trichosporon cutaneum showed the highest TAL catalytic activity and the lowest PAL/TAL catalytic efficiency ratio (0.8). The enzyme was purified to near homogeneity and its kinetics studied. The native enzyme appears to be a homo-tetramer with a calculated MW of 294 kDa, subunit MW of 73.5 kDa, and a p I of 5.8. When phenylalanine was used as substrate, the V max, K cat and K m were ∼4.0 ± 0.2 μg/min/mg purified enzyme), 588 ± 29 per min and 4.9 ± 0.9 mM, respectively. However, when tyrosine served as the substrate the V max and K cat were 0.59 ± 0.02 μg/min/mg purified enzyme), and 86.7 ± 29 per min, and substrate binding was apparently cooperative (nH ∼ 2.6 ± 0.4), with S 0.5 ∼ 0.6 mM. This is the first reported positive cooperativity for a TAL enzyme. Based on the NH 2-terminal and partial internal peptide sequences, the cDNA encoding the enzyme was cloned. Sequence analysis of TcTAL showed 56–62% similarity to other fungal PAL/TAL enzymes. High-level expression (∼30% of total soluble protein, based on SDS-PAGE analysis) of the cDNA in Escherichia coli was achieved using the arabinose inducible araB promoter. The recombinant enzyme possessed both PAL and TAL activities, as evident from the presence of both pHCA and CA in the culture medium.

Crystallization and preliminary X-ray analysis of the O-methyltransferase NovP from the novobiocin-biosynthetic cluster of Streptomyces spheroides.

Crystals of recombinant NovP (subunit MW = 29 967 Da; 262 amino acids), an S-adenosyl-L-methionine-dependent O-methyltransferase from Streptomyces spheroides, were grown by vapour diffusion. The protein crystallized in space group P2, with unit-cell parameters a = 51.81, b = 46.04, c = 61.22 A, beta = 104.97 degrees. Native data to a maximum resolution of 1.4 A were collected from a single crystal at the synchrotron. NovP is involved in the biosynthesis of the aminocoumarin antibiotic novobiocin that targets the essential bacterial enzyme DNA gyrase.

Crystallization and preliminary X-ray analysis of AbsC, a novel regulator of antibiotic production in Streptomyces coelicolor.

Crystals of recombinant AbsC (subunit MW = 18 313 Da; 158 amino acids), a novel regulator of antibiotic production from Streptomyces coelicolor, were grown by vapour diffusion. The protein crystallizes in space group P2(1)2(1)2(1), with unit-cell parameters a = 43.53, b = 121.30, c = 143.75 A. Native data to a resolution of 2.25 A were recorded at station PX 14.1 (Daresbury) from a single crystal. Preliminary analysis of these data suggests that the asymmetric unit contains four copies of the AbsC monomer, giving an estimated solvent content of 47.0%. AbsC belongs to the MarR family of proteins that mediate ligand-responsive transcriptional control.

Molecular replacement in the `twilight zone': structure determination of the non-haem iron oxygenase NovR fromStreptomyces spheroidesthrough repeated density modification of a poor molecular-replacement solution

Crystals of recombinant NovR (subunit MW = 29 924 Da; 270 amino acids), a non-haem iron oxygenase from Streptomyces spheroides, were grown by vapour diffusion. The protein crystallized in space group C2, with unit-cell parameters a = 86.69, b = 139.38, c = 100.82 A, beta = 101.18 degrees . Native data were collected to a resolution of 2.1 A from a single crystal at a synchrotron and a molecular-replacement solution was obtained using the program AMoRe. The starting phase information was very poor and did not permit model building. Phases were subsequently improved using a combination of fourfold averaging and very gradual phase extension in the program DM to yield an interpretable map. NovR belongs to a novel class of non-haem iron oxygenases that share sequence similarity with class II aldolases. It is predicted to perform two consecutive oxidative decarboxylation steps in the biosynthesis of the prenylated hydroxybenzoic acid moiety of the aminocoumarin antibiotic novobiocin.

Overexpression, purification and localization of apoptosis related protein from Plasmodium falciparum

A growing body of evidence has ascertained that apoptosis is not only restricted to metazoans but also exists in unicellular parasites. In Plasmodium falciparum, the presence of a putative gene having sequence homology with apoptosis related protein (PfARP) (Gene ID PFI0450c) has raised enormous interest to unravel the function of this unique protein in cell death of malaria parasite. To characterize this protein, the PfARP gene has been amplified from the P. falciparum transcriptome by RT-PCR and the amplified gene has been successfully cloned, over-expressed and purified to homogeneity. The purified PfARP exhibits minimum subunit MW of ∼24 kDa as evident from SDS–PAGE. CD analysis reveals that the α and β content of the recombinant PfARP are 61% and 15%, respectively. Semiquantitative RT-PCR analysis indicates the expression of PfARP at both metabolically less active ring and highly active trophozoite stages of malaria parasite. Immunofluorescence microscopy further supports that PfARP expresses stage specifically with the highest expression at trophozite stage and very little in the schizont stage. PfARP is a cytosolic protein as evident from immunofluorescence microscopy. The role of this protein in P. falciparum cell death and stage progression is not yet known. The identification, purification and characterization would certainly be a step to initiate work on this protein to evaluate its role in P. falciparum growth, multiplication and stage progression.

Crystallization and preliminary X-ray analysis of the aromatic prenyltransferase CloQ from the clorobiocin biosynthetic cluster of Streptomyces roseochromogenes.

Crystals of recombinant CloQ (subunit MW = 35 626 Da; 324 amino acids), an aromatic prenyltransferase from Streptomyces roseochromogenes, were grown by vapour diffusion. The protein crystallizes in space group I4(1)22, with unit-cell parameters a = b = 135.19, c = 98.13 A. Native data from a single crystal were recorded to a resolution of 2.2 A in-house. Preliminary analysis of these data indicated that the asymmetric unit corresponds to a monomer, giving an estimated solvent content of 60.6%. CloQ is involved in the biosynthesis of the aminocoumarin antibiotic clorobiocin, which targets the essential bacterial enzyme DNA gyrase.

Purification and characterization of ascorbic acid 2-kinase from Flavobacterium devorans ATCC 10829

Maximum activity for phosphorylating C 2–OH of the ascorbic acid was observed at the time of 16 h incubation from the culture of Flavobacterium devorans ATCC 10829. The enzyme was purified 1.178-fold, via ammonium sulfate fractionation, Fast Q anion exchange, and phenyl agarose chromatography. Gel chromatography and SDS-polyacrylamide electrophoresis experiments showed that the enzyme is a tetramer with subunit MW of 29 kDa. Among available second substrates, pyrophosphate showed the highest activity. Optimum temperature and pH were 45 °C and 5.5, respectively. The enzyme was chemically modified only by diethylpyrocarbonate and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), indicating that histidine and carboxylate are in the active site. pH studies showed that two histidines are involved in the binding of the substrates and a carboxylate in catalysis. Therefore, the chemical mechanism of the enzyme is likely that two histidines bind to pyrophosphate and carboxylate, respectively, and a carboxylate acts as a general base.

Crystallization and preliminary X-ray studies on the putative dTDP sugar epimerase NovW from the novobiocin biosynthetic cluster of Streptomyces spheroides.

Crystals of recombinant NovW (subunit MW = 22 289 Da), a putative dTDP sugar epimerase from Streptomyces spheroides, were grown by vapour diffusion. The protein crystallizes in space group P4(3)2(1)2, with unit-cell parameters a = b = 59.20, c = 109.23 A. Native data to a resolution of 2.0 A were collected from a single crystal at 100 K on a rotating-anode X-ray generator. Preliminary analysis of these data indicated that the asymmetric unit corresponded to a monomer, whilst dynamic light scattering (DLS) suggested that NovW was a dimer in solution. NovW is involved in the biosynthesis of the aminocoumarin antibiotic novobiocin, which targets the bacterial enzyme DNA gyrase, and represents the first enzyme to be crystallized from this biosynthetic pathway.

Crystallization and preliminary X-ray analysis of the E. coli hypothetical protein TdcF.

Crystals of the hypothetical protein TdcF (subunit MW = 14 007) from Escherichia coli were grown by vapour diffusion. The protein crystallizes in space group P2(1)2(1)2, with unit-cell parameters a = 72.67, b = 86.22, c = 62.62 A. Native data to a resolution of 2.35 A were collected from a single crystal at 100 K on a rotating-anode X-ray generator. Preliminary analysis of these data indicated that the asymmetric unit corresponded to a trimer, which was supported by a convincing molecular-replacement solution using the YjgF trimer as the probe structure.

Thymidylate synthase inhibitors.

Thymidylate synthase (TS, EC 2.1.1.45) is an ?#1 essential enzyme in pyrimidine biosynthesis. TS catalyses the methylation of deoxyuridine monophosphate (dUMP) to form deoxythymidine monophosphate (dTMP), using 5,10-methylenetetrahydrofolate as a co-substrate to donate methyl groups. TS is a cytoplasmic enzyme which consists of a dimer of two identical subunits of MW 38.5 kDa [1]. Two binding sites on the enzyme are recognized — the pyrimidine binding site and a pocket of the surface of the enzyme in which the folate co-substrate binds. The enzyme transfers a methyl group from the bound folate to the pyrimidine to form dTMP, followed by release of both products into the cytoplasm. This reaction provides the only endogenous source of dTMP available to cells and is the rate-limiting enzyme for the de novo synthesis of thymine nucleosides and nucleotides. Thus, TS plays an essential role in the synthesis and repair of DNA. The TS gene is located on chromosome 18p11.32 and consists of seven exons and eight introns. The expression of this gene is cell cycle dependent, in that the level increases sharply as cells enter S-phase, but it may also be proliferation dependent [2]. Thus, tumor cells have TS activities much above the level found in nonmalignant cells (see below). A high percentage of monosomic loss of the TS gene has been shown in some cancers, most notably colon carcinoma [3]. Because of its pivotal role in DNA metabolism TS, has long been a target for anticancer chemotherapy. Inhibition of TS leads to major disturbances in pyrimidine and folate metabolism. The desire to exploit this axis has led to the development of a number of TS inhibitors over several decades, which are either analogues of the pyrimidine substrate dUMP, or analogues of the folate cofactor. 5-Fluorouracil (5FU) is the classical pyrimidine analogue, which for several decades has been the mainstay of the treatment of various types of cancer including gastrointestinal (GI), breast and head and neck cancers [4]. Increasing investigation and development of these agents has occurred in recent years, which has maintained this group of drugs in the forefront of anticancer drug pharmacology and therapeutics. There are now a large range of TS inhibitors undergoing evaluation and clinical development (Figs. 1, 2 and 5). This chapter reviews recent developments in the understanding and clinical applications of FP and folate-based TS inhibitors. Readers requiring more background and historical data are referred to previous reviews of these agents [4–6].

Cooperative Binding of γ-Glutamyl Substrate to Human Glutathione Synthetase

Human glutathione synthetase is responsible for catalyzing the final step in glutathione biosynthesis. It is a homodimer with a monomer subunit MW of 52 kDa. Kinetic analysis reveals a departure from linearity of the Lineweaver–Burk double reciprocal plot for the binding of γ-glutamyl substrate, indicating cooperative binding. The measured apparent Km values for γ-glutamyl-α-aminobutyrate (an analog of γ-glutamyl-α-aminobutyrate) are 63 and 164 μM, respectively. Neither ATP (Km of 248 μM) nor glycine (Km of 452 μM) exhibits such cooperative binding behavior. Although ATP is proposed to play a key role in the sequential binding of γ-glutamyl substrate to the enzyme, the cooperative binding of the γ-glutamyl substrate is not affected by alterations of ATP concentration. Quantitative analysis of the kinetic results for γ-glutamyl substrate binding gives a Hill coefficient (h) of 0.75, indicating negative cooperativity. Our studies, for the first time, show that human glutathione synthetase is an allosteric enzyme with cooperative binding for γ-glutamyl substrate.

Carbonic Anhydrase from Vicia Canencens Leaves

ABSTRACT Carbonic anhydrases (CA: Carbonate hydrolase; E, C,4.2.1.1) from leaves Vicia canencens were purified and were characterized. The purification level of enzyme was 76-fold. The optimum temperature of this enzyme was 70°C. pH optimum was 9.2. Each of the enzyme molecules was a decamer having MW of 262,000 and the subunit MW was 26,400.

The Purification and Crystallisation of Phosphoglycerate Kinase from the Hyperthermophilic Eubacterium Thermotoga Maritima

Two phosphoglycerate kinase enzymes have been purified from the hyperthermophilic eubacterium Thermotoga maritima. One has been shown to be monomeric with a MW of 41 kDa, whilst the other, in contrast to other phosphoglycerate kinases that have been studied, is seen to be tetrameric by gel filtration, with a subunit MW of 70 kDa by SDS PAGE. The tetrameric PGK is a fusion protein with triose phosphate isomerase and has been crystallised from ammonium sulphate in the presence of the triose sugar substrate 3-phosphoglycerate

Evidence for a cytosolic NADP-malic enzyme in tomato

The similarity and cellular location of NADP +-malic enzyme (NADP-ME, EC 1.1.1.40) in developing fruit and other parts of the tomato ( Lycopersicon esculentum Mill.) plant were investigated in order to clarify the role of the enzyme in metabolism. There appeared to be a single enzyme (native NW 260 kD, subunit MW 66 kD) in mature green tomato fruit; this was purified to a specific activity of 42.5 μmol mg −1 min −1 and apparent homogeneity on SDS-PAGE. Polyclonal antibodies raised against this protein achieved 90% precipitation of enzyme activity, but required purification in order to detect specifically the 66 kD protein on Western blots. The purified antibodies recognized a similar protein in tomato leaves, roots and stems. The specific activity of NADP-ME was at least nine times higher in supernatant than in chloroplast fractions in tomato fruit and leaf, consistent with the distribution of a cytoplasmic marker (alcohol dehydrogenase), but in contrast to a chloroplast marker (rubisco). The purified NADP-ME antibodies did not detect a 66 kD protein in chloroplast preparations. A partial NADP-ME cDNA isolated from a fruit library specifically hybridized with a 2.1 kb transcript in RNA preparations from tomato leaves and fruits at different stages of development. The existence of a minor, possibly chloroplastidic NADP-ME in tomato cannot be excluded; the major NADP-ME is a cytosolic protein which is present in all plant organs analysed.

Alliin lyase (Alliinase) from garlic (Allium sativum). Biochemical characterization and cDNA cloning.

The garlic plant (Allium sativum) alliinase (EC 4.4.1.4), which catalyzes the synthesis of allicin, was purified to homogeneity from bulbs using various steps, including hydrophobic chromatography. Molecular and biochemical studies showed that the enzyme is a dimer of two subunits of MW 51.5 kDa each. Its Km using synthetic S-allylcysteine sulfoxide (+ isomer) as substrate was 1.1 mM, its pH optimum 6.5, and its isoelectric point 6.35. The enzyme is a glycoprotein containing 6% carbohydrate. N-terminal sequences of the intact polypeptide chain as well as of a number of peptides obtained after cyanogen bromide cleavage were obtained. Cloning of the cDNAs encoding alliinase was performed by a two-step strategy. In the first, a cDNA fragment (pAli-1-450 bp) was obtained by PCR using a mixed oligonucleotide primer synthesized according to a 6-amino acid segment near the N-terminal of the intact polypeptide. The second step involved screening of garlic lambda gt11 and lambda ZAPII cDNA libraries with pAli-1, which yielded two clones; one was nearly full length and the second was full length. These clones exhibited some degree of DNA sequence divergence, especially in their 3' noncoding regions, suggesting that they were encoded by separate genes. The nearly full length cDNA was fused in frame to a DNA encoding a signal peptide from alpha wheat gliadin, and expressed in Xenopus oocytes. This yielded a 50 kDa protein that interacted with the antibodies against natural bulb alliinase. Northern and Western blot analyses showed that the bulb alliinase was highly expressed in bulbs, whereas a lower expression level was found in leaves, and no expression was detected in roots. Strikingly, the roots exhibited an abundant alliinase activity, suggesting that this tissue expressed a distinct alliinase isozyme with very low homology to the bulb enzyme.