Gel Native PAGE Research Articles

Molecular identification of hyaluronate lyase, not hyaluronidase, as an intrinsic hyaluronan-degrading enzyme in Clostridium perfringens strain ATCC 13124.

Clostridium perfringens, an opportunistic pathogen, produces mu-toxin hyaluronidases including endo-β-N-acetylglucosaminidases (Nags) as a virulence invasion factor. To clarify an intrinsic factor for degradation of host extracellular hyaluronan, we focused on hyaluronate lyase (HysA), distinct from endo-β-N-acetylglucosaminidases. C. perfringens strain ATCC 13124 was found to assimilate host-derived extracellular mucosubstances, hyaluronan and mucin, which induced expression of the hyaluronan-related genetic cluster, including hyaluronate lyase gene (hysA), but repressed endo-β-N-acetylglucosaminidase genes. This genetic cluster is conserved in some strains of C. perfringens. The recombinant strain ATCC 13124 hyaluronate lyase HysA showed an hyaluronan-degrading activity through β-elimination reaction. The hyaluronan-degrading enzyme in the culture supernatant of strain ATCC 13124 exhibited the lyase activity and was identical to the recombinant hyaluronate lyase on the native-PAGE gel, followed by activity straining. These results demonstrated that the intrinsic hyaluronan-degrading enzyme of C. perfringens strain ATCC 13124 is hyaluronate lyase HysA, but not hyaluronidases NagH, NagJ, and NagK.

Determination of ROS Generated by Arabidopsis Xanthine Dehydrogenase1 (AtXDH1) Using Nitroblue Tetrazolium (NBT) and 3,3'-Diaminobenzidine (DAP).

Plants generate reactive oxygen species (ROS) during different metabolic processes, which play an essential role in coordinating growth and response. ROS levels are sensitive to environmental stresses and are often used as a marker for stress in plants. While various methods can detect ROS changes, histochemical staining with nitroblue tetrazolium (NBT) and 3,3'-diaminobenzidine (DAB) is a popular method, though it has faced criticism. This staining method is advantageous as it enables both the quantification and localization of ROS and the identification of the enzymatic origin of ROS in plants, cellular compartments, or gels. In this protocol, we describe the use of NBT and DAP staining to detect ROS generation under different stresses such as nitrogen starvation, wounding, or UV-C. Additionally, we describe the use of NBT staining for detecting enzymatic generation of ROS in native and native SDS PAGE gels. Our protocol also outlines the separation and comparison of the origin of ROS generated by xanthine dehydrogenase1 (XDH1) using different substrates.

The Secondary Structure of Potato Spindle Tuber Viroid Determines Its Infectivity in Nicotiana benthamiana.

The function of RNAs is determined by their structure. However, studying the relationship between RNA structure and function often requires altering RNA sequences to modify the structures, which leads to the neglect of the importance of RNA sequences themselves. In our research, we utilized potato spindle tuber viroid (PSTVd), a circular-form non-coding infectious RNA, as a model with which to investigate the role of a specific rod-like structure in RNA function. By generating linear RNA transcripts with different start sites, we established 12 PSTVd forms with different secondary structures while maintaining the same sequence. The RNA secondary structures were predicted using the mfold tool and validated through native PAGE gel electrophoresis after in vitro RNA folding. Analysis using plant infection assays revealed that the formation of a correct rod-like structure is crucial for the successful infection of PSTVd. Interestingly, the inability of PSTVd forms with non-rod-like structures to infect plants could be partially compensated by increasing the amount of linear viroid RNA transcripts, suggesting the existence of additional RNA secondary structures, such as the correct rod-like structure, alongside the dominant structure in the RNA inoculum of these forms. Our study demonstrates the critical role of RNA secondary structures in determining the function of infectious RNAs.

Acid and Alkaline Phosphatases Specific activities and Expression as Biomarkers in Brain Tumor Grading and Correlation of Anaplastic Oligodendroglioma G-III to Meningiomas G-I among Brain Tumors and their primary culture

BackgroundAcid Phosphatase (ACP) and Alkaline Phosphatases (ALP) are hydrolases that remove phosphate groups from protein and nucleic acid respectively for regulation of cell function from ACP as lysosomal defence function and ALP membrane-bound as a barrier of the cell. The ACP and ALP-specific activities of Meningiomas (n = 75) and gliomas (n = 81) were compared among brain tumors, normal brain, and derived primary cell culture. MethodsTotal Protein and Phosphatases assays estimated by Spectrophotometer and Native PAGE Gel Electrophoresis. Brain tumor and primary explant lysosome studies were performed with an electron microscope. ResultsAverage ACP specific activity exhibited 9.32617 ± 4.1144 for meningiomas (n = 55) and 5.91 ± 5.8305 for gliomas (n = 60) respectively as compared to normal brain 7.104 ± 1.33 (n = 120) nm/min/mg of protein. Average ALP exhibited 37.1862 ± 39.91 (n = 36) for meningiomas and 5.91 ± 5.83 (n = 60) for gliomas respectively as compared to normal brain (n = 117) 2.463 ± 1.01 nm/min/mg of protein.ACP and ALP exhibited higher activities for meningiomas but not for gliomas as compared to normal brain, in contrast, both expressed more activities in the majority of glioma cell lines and lower in meningioma cell lines. Interestingly gliomas exhibited similar average specific activities for ACP and ALP. While GBM IV exhibits lower ALP activities due to cell migration and higher ACP activity correlate too many storage lysosomes from Electron microscopic observation as compared to meningiomas. ConclusionsHigher ALP activities can be surrogate markers from meningiomas G-I, G-II to G-III respectively. However meningiomas G-III are similar to gliomas excluding Anaplastic Oligodendroglioma G- III which is similar to Meningiomas G-I even for cells growth patterns. Therefore, an ALP level in meningiomas indicates complementary diagnosis as antibody-ALP conjugates with anticancer drugs for efficiency in targeting brain tumor reduction.

The effect of thermal pasteurization, freeze-drying, and gamma irradiation on donor human milk

The availability of donor human milk (DHM) is currently limited by the volumes that can be thermally pasteurized and kept in long-term cold storage. This study assesses the application of freeze-drying followed by low-dose gamma irradiation of DHM for simplified, safe long-term storage. Solid-phase microextraction (SPME) GC–MS, SDS and native PAGE gel electrophoresis demonstrated that the overall changes in volatile and protein profiles in Holder pasteurized and freeze-dried DHM was negligible compared to the natural variations in DHM. Freeze-dried DHM samples (moisture < 2.2 %) processed with 2 kGy gamma irradiation did not show any significant lipid oxidation end-products and variation in protein profile. Therefore, freeze-drying followed by in-packaging gamma irradiation could be a safe method for pasteurization, convenient storage and delivery of DHM at ambient temperature. These methods may generate a means to create a reserve stock of DHM for emergencies and humanitarian aid.

Production, partial purification and characterization of ligninolytic enzymes from selected basidiomycetes mushroom fungi.

In recent years, many research on the quantity of lignocellulosic waste have been developed. The production, partial purification, and characterisation of ligninolytic enzymes from various fungi are described in this work. On the 21st day of incubation in Potato Dextrose (PD) broth, Hypsizygus ulmarius developed the most laccase (14.83 × 10−6 IU/ml) and manganese peroxidase (24.11 × 10−6 IU/ml), while Pleurotus florida produced the most lignin peroxidase (19.56 × −6 IU/ml). Laccase (Lac), lignin peroxidase (LiP), and manganese peroxidase (MnP), all generated by selected basidiomycetes mushroom fungi, were largely isolated using ammonium sulphate precipitation followed by dialysis. Laccase, lignin peroxidase, and manganese peroxidase purification findings indicated 1.83, 2.13, and 1.77 fold purity enhancements, respectively. Specific activity of purified laccase enzyme preparations ranged from 305.80 to 376.85 IU/mg, purified lignin peroxidase from 258.51 to 336.95 IU/mg, and purified manganese peroxidase from 253.45 to 529.34 IU/mg. H. ulmarius laccase (376.85 IU/mg) with 1.83 fold purification had the highest specific activity of all the ligninolytic enzymes studied, followed by 2.13 fold purification in lignin peroxidase (350.57 IU/mg) and manganese peroxidase (529.34 IU/mg) with 1.77-fold purification. Three notable bands with molecular weights ranging from 43 to 68 kDa and a single prominent band with a molecular weight of 97.4 kDa were identified on a Native PAGE gel from mycelial proteins of selected mushroom fungus. The SDS PAGE profiles of the mycelial proteins from the selected mushroom fungus were similar to the native PAGE. All three partially purified ligninolytic isozymes display three bands in native gel electrophoresis, with only one prominent band in enzyme activity staining. The 43 kDa, 55 kDa, and 68 kDa protein bands correspond to laccase, lignin peroxidase, and manganese peroxidase, respectively.

Enzymological characteristics of pepsinogens and pepsins purified from lizardfish (Saurida micropectoralis) stomach

One major pepsinogen, PG-I, and two minor pepsinogens, PG-II and PG-III were purified from lizardfish stomach by ammonium sulfate precipitation and two chromatographic columns. The three purified PGs migrated as single bands in native-PAGE gels with molecular weights (MW) ranging from 36 to 38 kDa. Each PG was converted to pepsin (P) at pH 2.0, and the MW were determined as 32 kDa (for P-I), 31 kDa (for P-II) and 30 kDa (for P-III). The optimum pH and temperature of pepsins were 2.0–3.5, and 40–50 °C. All 3 pepsins were strongly inhibited by pepstatin A. Divalent cations slightly stimulated the pepsin activities, but ATP had no effect on the pepsins. Purified pepsins were effective in the hydrolysis of various proteins. Km and kcat of the three pepsins for hemoglobin hydrolysis were 107.64–276.61 µM and 18.30–32.68 s−1, respectively. The new pepsins have potential for use in protein food procession and modification.

Reductions of copper ion-mediated low-density lipoprotein (LDL) oxidations of trypsin inhibitors, the sweet potato root major proteins, and LDL binding capacities

BackgroundThe root major proteins of sweet potato trypsin inhibitors (SPTIs) or named sporamin, estimated for 60 to 80% water-soluble proteins, exhibited many biological activities. The human low-density lipoprotein (LDL) showed to form in vivo complex with endogenous oxidized alpha-1-antitrypsin. Little is known concerning the interactions between SPTIs and LDL in vitro.ResultsThe thiobarbituric-acid-reactive-substance (TBARS) assays were used to monitor 0.1 mM Cu2+-mediated low-density lipoprotein (LDL) oxidations during 24-h reactions with or without SPTIs additions. The protein stains in native PAGE gels were used to identify the bindings between native or reduced forms of SPTIs or soybean TIs and LDL, or oxidized LDL (oxLDL). It was found that the SPTIs additions showed to reduce LDL oxidations in the first 6-h and then gradually decreased the capacities of anti-LDL oxidations. The protein stains in native PAGE gels showed more intense LDL bands in the presence of SPTIs, and 0.5-h and 1-h reached the highest one. The SPTIs also bound to the oxLDL, and low pH condition (pH 2.0) might break the interactions revealed by HPLC. The LDL or oxLDL adsorbed onto self-prepared SPTIs-affinity column and some components were eluted by 0.2 M KCl (pH 2.0). The native or reduced SPTIs or soybean TIs showed different binding capacities toward LDL and oxLDL in vitro.ConclusionThe SPTIs might be useful in developing functional foods as antioxidant and nutrient supplements, and the physiological roles of SPTIs-LDL and SPTIs-oxLDL complex in vivo will investigate further using animal models.

Plant-Derived Trimeric CO-26K-Equivalent Epitope Induced Neutralizing Antibodies Against Porcine Epidemic Diarrhea Virus.

Porcine epidemic diarrhea virus (PEDV) is a causative agent of a highly infectious disease with a high mortality rate, especially in newborn piglets in Asian countries resulting in serious economic loss. The development of a rapid, safe, effective and cost-efficient vaccine is crucial to protect pigs against PEDV infection. The COE antigen is regarded to be a major target for subunit vaccine development against PEDV infection. The naturally assembled COE protein forms a homotrimeric structure. In the present study, we successfully produced a trimeric COE protein as a native structure by fusion with the C-terminal isoleucine zipper trimerization (GCN4pII) motif in Nicotiana benthamiana, with a high expression level shown via semi-quantified Western blots. Trimeric COE protein was purified via immobilized metal affinity chromatography (IMAC), and its trimeric structure was successfully demonstrated by a cross-linking reaction, and a native PAGE gel. A crude extract containing the COE trimer was used for evaluating immunogenicity in mice. After 1 and 2 booster immunizations, the crude extract containing trimeric COE elicited elevated PEDV-specific humoral responses, as demonstrated by ELISA and Western blot analyses. Notably, a virus-neutralizing antibody assay indicated that the neutralization activities of sera of mice vaccinated with the crude extract containing COE-GCN4pII were similar to those of mice vaccinated with a commercial vaccine. These results suggest that crude extract containing trimeric COE is a promising plant-based subunit vaccine candidate for PEDV prevention.

Targeting folate receptors (α1) to internalize the bleomycin loaded DNA-nanotubes into prostate cancer xenograft CWR22R cells

DNA-nanotechnology based on DNA scaffolding technique is an established approach to formulate water-miscible nano-structural frameworks. We have designed the hydrophilic DNA-nanotubes (D-NTs) as a (water-soluble) vehicle to encapsulate a water-insoluble DNA-intercalating anticancer drug. Bleomycin (BM) is a model hydrophobic anticancer drug used in this study capable to bind with the D-NTs to formulate BM loaded D-NTs. This BM@D-NTs system was evaluated for the improvement of the in vitro anti-cancer effects on the resistant prostate cancer xenograft CWR22R cells, over-expressed with the folate receptors/alpha (FRα). D-NTs were functionalized with the FRα-targeting antibodies to interact with the FRα (receptors) highly expressed on the resistant prostate cancer xenograft CWR22R cells. D-NTs not only increased the aqueous miscibility/dispersibility of BM but also enhanced the therapeutic efficiency as a targeted (site-specific) drug delivery system. D-NTs synthesis was achieved by sticky ends cohesions of DNA triangular tiles. DNA triangles were self-assembled from a freshly circularized short scaffold chain (84-NT) by annealing with the various staple strands. The polymerization of the triangular tiles gave rise to DNA-nanosheet lattices which underwent morphological transition guided by the twists in the DNA duplexes. This DNA double helix curvature caused self-coiling of the DNA 2D nano-sheets to condense into D-NTs morphology. Native-Page gel experiment showed decreased electrophoretic mobility down the gel confirming the successful execution of the polymerized lattices via sticky ends cohesion of the DNA-triangles. The final morphology and self-coiling of 2D DNA nano-sheets were confirmed through atomic force microscopy (AFM) showed the successful synthesis of D-NTs having diameter 3 to 5 μm and length 200 to 600 nm. BM was loaded onto D-NTs via incubation of hydro-alcoholic solution of the BM with the aqueous solution of D-NTs followed by the evaporation of alcohol and intercalation of the BM onto D-NTs. The intercalation of BM onto D-NTs was confirmed by UV-shift analysis. The targeted cytotoxicity of the BM@D-NTs for the CWR22R (resistant prostate cancer xenograft) cells was confirmed through MTT assay and the flow cytometry compared to the highly compatible empty D-NTs. Confocal microscopy revealed the time-dependent transfection of BM@D-NTs into CWR22R cells compared to the control cell line.

Visualized characterization of bacterial penicillin G acylase for the hydrolysis of β-lactams using an activatable NIR fluorescent probe

Penicillin G acylase (PGA) identified from bacteria, is used as industrial biocatalyst for the hydrolysis of penicillins along with the production of 6-aminopenicillanic acid, a key intermediate of the semi-synthetic β-lactam antibiotics. Due to wide usage of PGA, some novel homology bioactive enzymes are investigated in various bacteria, and visualized analysis technique would be necessary and helpful for the development and utilization of PGA function in living systems. In the present work, an activated off-on near-infrared (NIR) fluorescent probe (DDAP) deduced from a novel fluorophore has been designed and developed for sensitively and selectively detecting endogenous PGA in real-time. The hydrolysis mechanism has been elucidated using in silico docking analysis, which confirmed DDAP as the specific substrate of PGA. Using DDAP as the fluorescent probe, the endogenous bacterial PGA was imaged in living systems successfully, which could be applied to identify bacteria with PGA expression from complex microorganism samples. Furthermore, using DDAP as the staining agent, the endogenous PGA in various bacteria has been profiled visually on native-PAGE gel, and the PGA activity in bacterial lysates could be effectively determined. The PAGE-gel for PGA analysis stained by DDAP was more sensitive than generally used silver and Coomassie Blue dyes. Finally, a series of β-lactams were hydrolyzed by recombinant PGA to further confirm its hydrolase property, corresponding to the hydrolysis of DDAP.

Small Molecule Inhibitors of the BfrB-Bfd Interaction Decrease Pseudomonas aeruginosa Fitness and Potentiate Fluoroquinolone Activity.

The iron storage protein bacterioferritin (BfrB) is central to bacterial iron homeostasis. The mobilization of iron from BfrB, which requires binding by a cognate ferredoxin (Bfd), is essential to the regulation of cytosolic iron levels in P. aeruginosa. This paper describes the structure-guided development of small molecule inhibitors of the BfrB–Bfd protein–protein interaction. The process was initiated by screening a fragment library and followed by obtaining the structure of a fragment hit bound to BfrB. The structural insights were used to develop a series of 4-(benzylamino)- and 4-((3-phenylpropyl)amino)-isoindoline-1,3-dione analogs that selectively bind BfrB at the Bfd binding site. Challenging P. aeruginosa cells with the 4-substituted isoindoline analogs revealed a dose-dependent growth phenotype. Further investigation determined that the analogs elicit a pyoverdin hyperproduction phenotype that is consistent with blockade of the BfrB–Bfd interaction and ensuing irreversible accumulation of iron in BfrB, with concomitant depletion of iron in the cytosol. The irreversible accumulation of iron in BfrB prompted by the 4-substituted isoindoline analogs was confirmed by visualization of BfrB-iron in P. aeruginosa cell lysates separated on native PAGE gels and stained for iron with Ferene S. Challenging P. aeruginosa cultures with a combination of commercial fluoroquinolone and our isoindoline analogs results in significantly lower cell survival relative to treatment with either antibiotic or analog alone. Collectively, these findings furnish proof of concept for the usefulness of small molecule probes designed to dysregulate bacterial iron homeostasis by targeting a protein–protein interaction pivotal for iron storage in the bacterial cell.

Spectroscopic, Gel Electrophoretic, and Surface Plasmon Resonance Characterization of G‐Triplex DNA Formation

G‐triplex DNA is a recently discovered class of non‐canonical DNA structures related to G‐quadruplex DNA (G4). Originally identified as a folding intermediate in the formation of certain G4, G‐triplex DNA structures can be formed by oligonucleotides corresponding to truncated G4 sequences. Here we present a study of the sequence and environmental effects on G‐triplex formation using spectroscopic, gel electrophoretic, and surface plasmon resonance methods.We examined 16 different variants of the G≥2T1–4G≥2T1–4G≥2 sequence as well as truncated versions of the TBA and human telomeric G4 DNA in both forward and reverse permutations. CD spectroscopy, UV thermal‐difference spectroscopy, CD and UV derived melting temperature measurements, SPR, agarose gel electrophoresis, and native PAGE gels were employed to characterize the topology and thermal stability of these structures in Li+, Na+, K+, Mg2+ and Ca2+ containing buffers. All of these sequences can adopt G‐triplex structures. However, we note that the number of nucleotides in the loop and sequence direction affect G‐triplex topology. Sequences with longer G‐tracks tend to form parallel G‐triplex, as do sequences with fewer loop residues. Permutation of the direction of the truncated G4 DNA sequence also affect G‐triplex folding topology. Environmental effects on topology were noted, with divalent metal ions (Mg2+ and Ca2+) favoring parallel topologies. Using thermal‐difference spectroscopy, we have identified two families of spectra that correlate to G‐triplexes with a parallel topology. Analysis by native PAGE indicates that some sequences may form multiple G‐triplex topologies under certain environmental conditions. Temperature melts show that sequences with longer G‐runs (GGGG &gt; GGG &gt; GG) and shorter T‐loops (TT &gt;TTT &gt; TTTT) form G‐triplex structures that are more thermally stable. Direction of sequence also effects the stability. Environmental conditions such as the presence of different cations have an effect on stability (Ca2+ &gt; K+ &gt; Mg2+ ≥ Na+). Analysis by agarose gel electrophoresis has suggested that some sequences of G‐triplex forming DNA fold into monomeric intramolecular structures, while sequences with long G‐runs form highly oligomeric species. SPR has also been performed in order to probe G‐triplex formation. Immobilized G‐triplex‐forming sequences were probed with short, complementary C‐rich oligonucleotides and the kinetics of hybridization under different buffer conditions provides further insight into the stability and kinetics of G‐triplex formation.The biological relevance of G‐triplex DNA is still not entirely understood. Understanding how sequence effects and environmental conditions affect G‐triplex structure and stability is essential for better studying G‐quadruplex DNA and the biological relevance of G‐triplex DNA.Support or Funding InformationCRPIT HIHR Grant RP160852 and Texas State UniversityThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Formation of Disulfide Bonds in Proinsulin is Dependent on Both the Substrate Chemical Form and the Conditions Used for Purification and Folding

Misfolding of proinsulin (PI), the biosynthetic precursor to insulin, is believed to be the underlying cause of some forms of both type 1 and type 2 diabetes mellitus. It has been proposed that the ER stress response plays a role in recognizing and responding to PI misfolding in the cell. My laboratory is developing an in vitro model system to examine the roles of PI, protein disulfide isomerase (PDI) and the redox co‐factor glutathione in the folding reaction.Both PI and PDI were expressed as N‐terminal poly‐His‐tagged proteins in bacteria. The PI fusion protein was recovered from inclusion bodies and the poly‐His tag was removed by cyanogen bromide cleavage. Liberated PI was recovered by rotary evaporation and subjected to further chemical treatment. PDI was purified from the soluble fraction of bacteria using a combination of IMAC and gel filtration chromatography.Two forms of PI were studied: sulfonated and reduced (PI‐SO4 and PI‐red, respectively). Both versions of PI were subjected to gel filtration chromatography at pH 7.2. PI‐SO4 eluted within the included volume of the column while PI‐red eluted at the void volume, suggesting that the latter version had undergone significant aggregation. Subsequent analysis using non‐reducing SDS‐PAGE indicated that both forms of PI migrated through the gel as monomers. Reaction of PI‐red with iodoacetic acid prior to non‐reducing SDS‐PAGE resulted in a mobility shift, reinforcing the hypothesis that the aggregation event was not due to the formation of incorrect disulfide cross‐links between PI molecules.Folding of both versions of PI (25 μM) was tested in the presence of reduced and oxidized glutathione (GSH/GSSG, 3 mM of each), PDI (5 μM) or a combination of GSH/GSSG and PDI. Previous studies had established that samples of PI‐red taken directly from the reduction reaction (performed in the presence of either 6 M guanidine or 8 M urea); obtained following buffer exchange without chaotropic agents at pH 10.6; or obtained after folding in the presence of GSH/GSSG at pH 10.6; could all be analyzed using RP‐HPLC. However, PI‐red that had been subjected to chromatography between pH 7 and 8 in the absence of chaotropic agents could not be analyzed using this method as no PI‐red peak could be detected and the HPLC column quickly became clogged. As an alternative, native PAGE was investigated as an alternative analytical method. Results from this study indicated that folding of PI‐SO4 at pH 7.2 can occur in the presence of either GSH/GSSG or a combination of GSH/GSSG and PDI, but not in the presence of PDI alone. In contrast, it was found that PI‐red does not enter the native PAGE gels under any of the conditions tested. This suggests that the non‐disulfide bonded, aggregated form of PI‐red could not be properly folded at pH 7.2 using concentrations of PI and GSH/GSSG similar to those that resulted in proper folding at pH 10.6.In conclusion, these results suggest that folding of PI in islet beta cells must take place under tightly controlled conditions and that mimicking these conditions in an in vitro assay will require further understanding of the biochemistry of the PI molecule.Support or Funding InformationSupported by the Creighton University Health Science Strategic Investment FundThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Isolation, purification and characterization of an extracellular L-asparaginase produced by a newly isolated Bacillus megaterium strain MG1 from the water bodies of Moraghat forest, Jalpaiguri, India.

An extracellular L-asparaginase was isolated and purified from Bacillus megaterium MG1 to apparent homogeneity. The purification procedure involved a combination of ammonium sulfate precipitation, ion-exchange chromatography, and gel filtration techniques, resulting in a purification factor of 31.52 fold with a specific activity of 215 U mg-1. The molecular mass of the purified enzyme was approximately 47 kDa on SDS-PAGE and 185 kDa on native PAGE gel as well as in gel filtration column chromatography, revealing that the enzyme was a homotetramer. The Km and Vmax values of the purified enzyme were calculated to be 2.0 ⅹ 10-4 M and 1.198 mM s-1. Maximum enzyme activity was observed over a wide range of temperature and pH values with an optimum temperature of 37°C and pH 8.5. SDS and metal ions such as Fe2+, Cu2+, Mg2+, Co2+, Mn2+, and Ca2+ decreased the enzyme activity remarkably, whereas the addition of Na+ and K+ led to an increase in activity. The insensitivity of the protein in the presence of EDTA suggested that the enzyme might not essentially be a metalloprotein. Its marked stability and activity in organic solvents and reducing agents suggest that this asparaginase is highly suitable as a biotechnological tool with industrial applications.

Towards efficient enzymatic conversion of D-galactose to D-tagatose: purification and characterization of L-arabinose isomerase from Lactobacillus brevis.

L-arabinose isomerase (L-AI) (EC 5. 3. 1. 4. L-AI) that mediates the isomerization of D-galactose to D-tagatose was isolated from Lactobacillus brevis (MF 465792), and was further purified and characterized. Pure enzyme with molecular weight of 60.1kDa was successfully obtained after the purification using Native-PAGE gel extraction method, which was a monomer in solution. The L-AI was found to be stable at 45-75°C, and at pH 7.0-9.0. Its optimum temperature and pH was determined as 65°C and 7.0, respectively. Besides, we found that Ca2+, Cu2+, and Ba2+ ions inhibited the enzyme activity, whereas the enzyme activity was significantly enhanced in the presence of Mg2+, Mn2+, or Co2+ ions. The optimum concentration of Mn2+ and Co2+ was determined to be 1mM. Furthermore, we characterized the kinetic parameters for L-AI and determined the Km (129mM) and the Vmax (0.045mM min- 1) values. Notably, L. brevisL-AI exhibited a high bioconversion yield of 43% from D-galactose to D-tagatose under the optimal condition, and appeared to be a more efficient catalyst compared with other L-AIs from various organisms.

A Simple Method for On-Gel Detection of Myrosinase Activity

Myrosinase is an enzyme present in many functional foods and spices, particularly in Cruciferous vegetables. It hydrolyses glucosinolates which thereafter rearrange into bioactive volatile constituents (isothiocyanates, nitriles). We aimed to develop a simple reversible method for on-gel detection of myrosinase. Reagent composition and application parameters for native PAGE and SDS-PAGE gels were optimized. The proposed method was successfully applied to detect myrosinase (or sulfatase) on-gel: the detection solution contains methyl red which gives intensive red bands where the HSO4− is enzymatically released from the glucosinolates. Subsequently, myrosinase was successfully distinguished from sulfatase by incubating gel bands in a derivatization solution and examination by LC-ESI-MS: myrosinase produced allyl isothiocyanate (detected in conjugate form) while desulfo-sinigrin was released by sulfatase, as expected. After separation of 80 µg protein of crude extracts of Cruciferous vegetables, intensive color develops within 10 min. On-gel detection was found to be linear between 0.031–0.25 U (pure Sinapis alba myrosinase, R2 = 0.997). The method was successfully applied to detection of myrosinase isoenzymes from horseradish, Cruciferous vegetables and endophytic fungi of horseradish as well. The method was shown to be very simple, rapid and efficient. It enables detection and partial characterization of glucosinolate decomposing enzymes without protein purification.

Influence of Proline Priming on Antioxidative Potential and Ionic Distribution and its Relationship with salt Tolerance of Wheat

Mechanisms involved in salt tolerance urge exploration and investigation of genotypic variation to assist future breeding programs. Comparative examination of ten wheat cultivars for salt tolerance and their response towards proline-seed-priming was performed. Exposure of wheat seedlings to salinity resulted in prominent reduction in root and shoot growth attributes of all cultivars. Furthermore, decrease in the chlorophyll contents was evident although this varied among cultivars. Wheat seedlings grown from proline pre-treated seeds exhibited improved photosynthetic pigments, besides this response was also cultivar and concentration dependent. Generally, salt stressed plants exhibited higher antioxidant enzyme activities. Proline priming significantly influenced antioxidant activities, however, its magnitude varied. The peroxidase activity varied among wheat cultivars that were evident from the analysis of POD activity on Native-PAGE gel. Salinity caused the accumulation of Na+ in the roots and the magnitude of Na+ translocation to the shoot was cultivar dependent. Similarly, K+ uptake and its distribution among root and shoot varied. Priming treatments affected ion distribution of Na+ and K+ but inter-cultivar variations were evident. Conclusively, all the cultivars investigated exhibited differential response to salinity and proline seed pre-treatments. However, the proline-priming mediated improvements in growth and antioxidant enzyme activities contributed to stress tolerance which partly relied on the ability of the plant to uptake sodium and its partitioning in the roots. Of the cultivars tested, Faisalabad-08 and Bhakhar-2002 were ranked as relatively salt tolerant and the cvs. AARI-10, MH-97 and Auqab-2000 as relatively salt sensitive.

A novel signal sequence negative multimeric glycosomal protein required for cell cycle progression of Leishmania donovani parasites

Expression of the intracellular form amastigote specific genes in the Leishmania donovani parasite plays a major role in parasite replication in the macrophage. In the current work, we have characterized a novel hypothetical gene, Ld30b that is specifically transcribed in the intracellular stage of the parasite. The recombinant Ld30b protein exists as a pentamer in solution as identified by native-PAGE and size exclusion gel chromatography. Structural analysis using circular dichroism and molecular modeling indicate that Ld30b belongs to family of cAMP-dependent protein kinase type I-alpha regulatory subunit. Co-localization immunofluorescence microscopy and western blot analyses (using anti-Ld30b antibody and anti-hypoxanthine-guanine phosphoribosyl transferase, a glycosome marker) on the isolated parasite glycosome organelle fractions show that Ld30b is localized in glycosome, though lacked a glycosome targeting PTS1/2 signal in the protein sequence. Episomal expression of Ld30b in the parasite caused the arrest of promastigotes and amastigotes growth in vitro. Cell cycle analysis using flow cytometry indicates that these parasites are arrested in ‘sub G0/G1’ phase of the cell cycle. Single allele knockout of Ld30b in the parasite similarly attenuated its growth by accumulation of cells in the S phase of cell cycle, thus confirming the probable importance of appropriate level of protein in the cells. Studying such intracellular stage expressing genes might unravel novel regulatory pathways for the development of drugs or vaccine candidates against leishmaniasis.

Tuberculosis serine hydrolase variable expression, isolation, and characterization under hypoxia conditions

Tuberculosis (TB), caused by infection by Mycobacterium tuberculosis, is the deadliest infectious disease worldwide. Complicating the treatment of TB is its ability to transition between an intransient dormant state and an active infectious state. One class of enzymes required for the maintenance of this dormant state and the transition into the active state is serine hydrolases. Our research goal was to globally characterize serine hydrolase activity in actively growing M. smegmatis and under hypoxia conditions that mimic dormant growth. To measure hydrolase activity, lysates were prepared under different growth conditions and then separated by native‐PAGE. Native‐PAGE gels were incubated with a library of fluorogenic ester substrates and hydrolase activity quantitated based on fluorogenic substrate activation. By following the shift in fluorogenic activation, we were able to measure changes in hydrolase protein expression versus fluorogenic substrate and in relation to induction of dormant growth conditions. Reactive hydrolases were excised from the gel and identified by mass spectrometry. Our results provided a global map of mycobacterial serine hydrolase activity and identified starting points for finding novel drug targets for dormant TB.Support or Funding InformationThis work was supported by a grant from the National Institutes of Health (NIH 1 R15 GM110641‐01A1)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.