DEAE-cellulose Anion Exchange Column Research Articles

Characterization, antimicrobial and antitumor activity of superoxide dismutase extracted from Egyptian honeybee venom (Apis mellifera lamarckii)

BackgroundSuperoxide dismutase is an important antioxidative stress enzyme which is found in honeybee venom and has a wide pharmaceutical and medical applications. ResultsWe reported the purification and characterization of venom SOD from Egyptian honeybee Apis mellifera lamarckii and termed BVSOD. It was purified to homogeneity from the Egyptian honeybee venom. The purification procedures included crude extraction, DEAE-cellulose anion exchange column chromatography, and Sephacryl S-300 gel filtration column chromatography. The purified BVSOD is found to be homogeneous as investigated by native PAGE. It exhibited homodimeric structure with a molecular weight of native form of 32 kDa and subunits of 16.0 kDa. It displayed the maximum activity at pH 7.4. CuCl2, ZnCl2, and MgCl2 and elevated the activity of BVSOD, while CoCl2, FeCl2, and NiCl2 inhibited BVSOD activity. Potassium cyanide and hydrogen peroxide were most potent inhibitors for BVSOD activity suggesting that it is a Cu/Zn-SOD type. ConclusionsThe purified BVSOD is found to have antimicrobial and antitumor activities which can be used for various medical and clinical applications.

Structural characterization and immunomodulatory activity of an exopolysaccharide produced by Lactobacillus helveticus LZ-R-5

Exopolysaccharide (R-5-EPS) was isolated from the fermented milk of Lactobacillus helveticus LZ-R-5 and purified by DEAE-52 cellulose anion-exchange column, and characterization of the structure was conducted. Results showed that R-5-EPS was a heteropolysaccharide containing linear repeating units of →6)-β-D-Galp-(1→3)-β-D-Glcp-(1→3)-β-D-Glcp-(1→3)-β-D-Glcp-(1→3)-β-D-Glcp-(1→ with an average Mw of 5.41 × 105 Da. Furthermore, at a cellular level, R-5-EPS showed immunostimulatory activity due to its strong effect on increasing proliferation of RAW264.7 macrophages and enhancing phagocytosis, acid phosphatase activity, nitric oxide production and cytokines production in macrophages. These results suggest that R-5-EPS have a potent immunostimulatory activity and may be explored as a potential immunomodulatory agent.

Antioxidant and Hypolipidemic Activities of Acid-Depolymerised Exopolysaccharides by Termitomyces albuminosus.

The acid-depolymerised exopolysaccharides (ADES) of Termitomyces albuminosus were obtained, and the major fraction of ADES1 was isolated and purified by DEAE-52 cellulose anion-exchange column chromatography. Physicochemical characterizations showed that ADES1 was an α- and a β-configuration with the molecular weight of 2.43 kDa, containing (1→3, 4)-linked-Glcp, (1→4)-linked-D-Glcp, (1→3)-linked-D-Xylp, (1→4)-linked-D-Manp, T-Glcp, (1→6)-linked-D-Galp, and (1→4)-linked-L-Arap. The in vivo assays showed that ADES1 could reduce lipid levels in the serum and liver, decrease serum enzyme activities, and improve antioxidant enzyme activities and p-AMPKα expressions in hyperlipidemic mice, which were also confirmed by histopathological observations. These data indicated that ADES1 might be considered as a novel substance to treat and prevent hyperlipidemia and as a hepatoprotective agent.

Characterization, antioxidant, and anticancer activities of a neutral polysaccharide from Duchesnea indica (Andr.) Focke.

A neutral polysaccharide (DIP-1) from Duchesnea indica (Andr.) Focke was obtained by hot water extraction, ethanol precipitation and chromatographic separation (DEAE-52 cellulose anion-exchange column and Sephadex G-100 gel column). The physicochemical properties of DIP-1 were elucidated by gel permeation chromatography, monosaccharide composition, Fourier transform infrared spectrometry, UV-visible spectrophotometry, scanning electron microscope and Congo red test. The results indicated that DIP-1 was consisted of mannose, glucosamine, glucose, galactose and arabinose in a ratio of 1.00:0.42:18.36:14.17:0.81, and its molecular weight was 218.3kDa. Meanwhile, DIP-1 presented a straight hexahedron structure, but no triple-helical conformation. In antioxidant activity tests, DIP-1 exhibited powerful scavenging activities on hydroxyl, DPPH, ABTS radicals and reducing power in a dose-dependent manner. Especially, DIP-1 demonstrated high inhibitory activities against SKOV-3 and Hep-G2 cells in vitro, with IC50 values of 1.42 and 1.23mg/ml, respectively. PRACTICAL APPLICATIONS: D. indica has been used for a long time as a Chinese medicine for therapy of many diseases, including cancer, inflammation, leprosy, fever, bleeding and so on. At present, polysaccharides have attracted comprehensive attention because of a large range of pharmacological and biological properties, including antitumor, antidiabetic, antioxidant and immunomodulatory activity. In the present study, we purified and characterized a neutral polysaccharide from D. indica for the first time. Moreover, the neutral polysaccharide exhibits significant antioxidant and antitumor activities. Therefore, the present study laid a foundation for the high-value application of D. indica polysaccharides in functional food and pharmaceutical industries.

Inonotus obliquus polysaccharides protect against Alzheimer's disease by regulating Nrf2 signaling and exerting antioxidative and antiapoptotic effects.

Inonotus obliquus polysaccharide (IOPS) was initially separated and purified via precipitation from an aqueous extract with 80% alcohol, a DEAE-52 cellulose anion exchange column, and a Sephadex G-100 gel permeation chromatography system. IOPS was found to have a molecular weight of 111.9 kDa. In L-glutamic acid (L-Glu)-damaged HT22 cells, a 3-h pre-incubation with IOPS enhanced cell viability, inhibited apoptosis and caspase-3 activity, reduced the release of lactate dehydrogenase, restored the dissipated mitochondrial membrane potential, and suppressed the excess accumulation of intracellular reactive oxygen species. Compared with L-Glu-exposed cells, IOPS pre-treated cells exhibited reduced levels of Bcl-2 associated X protein (Bax) and Kelch-like ECH-associated protein 1 (Keap1) and enhanced levels of B-cell lymphoma-2 (Bcl-2), NF-E2p45-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), superoxide dismutase-1 (SOD-1), and cysteine ligase catalytic subunit. In amyloid precursor protein/presenilin 1 (APP/PS1) transgenic mice, an 8-week course of IOPS improved the pathological behaviors related to memory and cognition, reduced the deposition of β-amyloid peptides and neuronal fiber tangles induced by enhanced phosphor-Tau in the brain, and modulated the levels of anti- and pro-oxidative stress enzymes. Additionally, IOPS enhanced the expression levels of Nrf2 and its downstream proteins, including HO-1 and SOD-1, in the brains of APP/PS1 mice. The present study successfully demonstrated the protective effect of IOPS against AD and revealed the possible mechanism underlying the ability of IOPS to modulate oxidative stress, especially Nrf2 signaling, and mediate mitochondrial apoptosis.

Protective roles of Amanita caesarea polysaccharides against Alzheimer's disease via Nrf2 pathway

This study explores the neuro-protective effects of Amanita caesarea polysaccharides (ACPS), obtained by 80% alcohol precipitation of water extract and purified using a DEAE-52 cellulose anion exchange column, related to antioxidant activity. A 3-h pre-treatment of ACPS prior to l‑glutamic acid (l‑Glu) co-exposure reversed the decreased cell viability, inhibited apoptosis, suppressed the accumulation of intracellular reactive oxygen species and restored mitochondrial membrane potential in HT22 cells. Compared to l‑Glu-exposed cells, ACPS enhanced the nuclear levels of NF-E2p45-related factor 2 (Nrf2), reduced the cytoplasmic levels of Nrf2 and cytochrome C, suppressed the expression of Kelch-like ECH-associated protein 1, and enhanced the expression of heme oxygenase‑1, superoxide dismutase 1 and cysteine ligase catalytic subunit. In a d‑galactose and aluminum trichloride Alzheimer's disease (AD) mouse model, 42-day administration of ACPS improved the abnormal behaviors. ACPS suppressed the deposition of β‑amyloid peptide in the brain and ameliorated oxidative stress via modulating the levels of related enzymes. ACPS improved the functioning of the central cholinergic system, as indicated by an increase in acetylcholine and choline acetyltransferase concentrations, and reduced acetylcholine esterase levels in the serum, hypothalamus and cerebral cortex. Our data suggest that ACPS may be a promising candidate for the treatment of AD.

Studies on the purification of polysaccharides separated from Tremella fuciformis and their neuroprotective effect.

The present study aimed to investigate the protective effect of purified polysaccharides from Tremellafuciformis against glutamate-induced cytotoxicity in differentiated PC12 (DPC12) cells. The aqueous extract of Tremellafuciformis was purified using a DEAE-52 cellulose anion exchange column and a Sepharose G-100 column, respectively. A fraction termed TL04 with a 2,033kDa molecular weight was obtained. The backbone of TL04 is composed of (1→2)‑ and (1→4)-linked-mannose and (1→3)-linked-glucans. Results revealed that TL04 treatment improved cell viability and suppressed reactive oxygen species accumulation, lactose dehydrogenase release and caspase-3 activity, and ameliorated mitochondrial abnormal alteration caused by glutamate. TL04 pretreatment enhanced the level of B‑cell lymphoma2 (Bcl‑2), and suppressed Bax expression and cytochromec (CytoC) release in glutamate-treated cells. Exposure to glutamate strongly increased the activity of caspase‑8, caspase‑9 and caspase‑3, which were significantly reversed by TL04 pretreatment. The presence of Ac-DEVD-CHO (a caspase-3 inhibitor) markedly enhanced the potency of TL04 in improving the viability of glutamate-exposed DPC12 cells. Collectively, the results demonstrated that the purified polysaccharides separated from Tremella fuciformis (TL04) possess a neuroprotective effect against glutamate-induced DPC12 cell damage predominantly through the caspase-dependent mitochondrial pathway. The present study provides an experimental foundation supporting purified TL04 as a potential therapeutic agent for neurodegenerative diseases.

Mitochondria Related Pathway Is Essential for Polysaccharides Purified from Sparassis crispa Mediated Neuro-Protection against Glutamate-Induced Toxicity in Differentiated PC12 Cells.

The present study aims to explore the neuro-protective effects of purified Sparassis crispa polysaccharides against l-glutamic acid (l-Glu)-induced differentiated PC12 (DPC12) cell damages and its underlying mechanisms. The Sparassis crispa water extract was purified by a DEAE-52 cellulose anion exchange column and a Sepharose G-100 column. A fraction with a molecular weight of 75 kDa and a diameter of 88.9 nm, entitled SCWEA, was obtained. SCWEA was identified with a triple helix with (1→3)-linked Rha in the backbone, and (1→2) linkages and (1→6) linkages in the side bone. Our results indicated that the pre-treatment of DPC12 cells with SCWEA prior to l-Glu exposure effectively reversed the reduction on cell viability (by 3-(4,5-cimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay) and reduced l-Glu-induced apoptosis (by Hoechst staining). SCWEA decreased the accumulation of intracellular reactive oxygen species, blocked Ca2+ influx and prevented depolarization of the mitochondrial membrane potential in DPC12 cells. Furthermore, SCWEA normalized expression of anti-apoptotic proteins in l-Glu-explored DPC12 cells. These results suggested that SCWEA protects against l-Glu-induced neuronal apoptosis in DPC12 cells and may be a promising candidate for treatment against neurodegenerative disease.

Structural Characterization and Immunostimulatory Activity of a Homogeneous Polysaccharide from Sinonovacula constricta.

Sinonovacula constricta has been widely used as a health food and medicine in China, Japan, and Korea. In the present study, a water-soluble polysaccharide fraction (SCP-1) was prepared from S. constricta by enzyme-assisted extraction and purification of chromatography with DEAE-52 cellulose anion-exchange column and Sephadex G-100 size exclusion column. On the basis of the analytical results of high-performance liquid chromatography, Fourier transform-infrared spectroscopy, methylation analysis, and NMR spectroscopy, SCP-1 was found to have an average molecular weight of 15.63 kDa and a linear backbone of (1→4)-linked α-D-Glcp residue with one branch, α-D-Glcp, attached to the main chain by a (1→6) glycosidic bond at every five α-D-Glcp units. Furthermore, it was found that SCP-1 could significantly increase the viability of macrophages, enhance the capability of macrophage phagocytosis, increase the activity of acid phosphatase, and promote the production of nitric oxide, mouse tumor necrosis factor (TNF)-α, mouse interferon (IFN)-γ, and mouse interleukin (IL)-1β. The results suggest that SCP-1 possesses potent immunomodulating effect and may be explored as a potential biological response modifier.

Purification, characterization and anti-aging capacity of mycelia zinc polysaccharide by Lentinus edodes SD-08.

BackgroundIn the modern society, aging had been a major problem. People may rely on many medicines to delay it. However, lots of medicines were chemosynthetic, and they would do a bad side-effect on human body. Microbial sources could be used as a potential means of producing natural antioxidants. Lentinus edodes, commercial obtained in daily life, had recently become more attractive in physiological research. Zinc was now considered as a major element in assuring the correct functioning of an organism and essential for maintaining coordination of the major homeostatic networks. To investigate the bioconversion of zinc and the physiological effects of their complex (MZPS), the present studies were processed.MethodsMycelia polysaccharides (MPS) and mycelia zinc polysaccharides (MZPS) of Lentinus edodes SD-08 were extracted by hot water leaching and purified by DEAE-52 cellulose anion-exchange column chromatography separately. The zinc content was determined by flame atomic absorption spectrometry. The evaluation of monosaccharide compositions and proportions used gas chromatogram. The analysis of molecular weight used HPGPC chromatogram. The typical structure of polysaccharide was evaluated by IR spectrum. The antioxidant activities in vitro measured through reducing power, the scavenging effects on hydroxyl radical and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals. The anti-aging activities in vivo measured through the total antioxidant capacity (T-AOC), GSH peroxide (GSH-Px), superoxide dismutase (SOD) and the contents of malondialdehyde (MDA).ResultsMPS and MZPS of Lentinus edodes SD-08 were extracted and purified by DEAE-52 cellulose anion-exchange column chromatography separately, and four fractions (MPS-1, MPS-2, MZPS-1 and MZPS-2) were obtained. In addition, MPS composing of rhamnose, arabinose and mannose (molar proportion = 1.75:1.00:3.02) and MZPS containing rhamnose, arabinose, mannose and glucose (molar proportion = 7.19:2.26:1.00:8.39) were investigated by gas chromatography. Infrared spectrum analysis indicated that there were C-H, C=O and -CH2 bonds in MPS and MZPS. MPS also had the typical absorption of -NH3+, -NH2 and -COOH. Compared with MPS, MZPS showed in vitro positive rising of reducing power and certain scavenging effects on hydroxyl radical and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals. MZPS were found to upregulate in vivo the anti-aging activities of total antioxidant capacity (T-AOC), GSH peroxide (GSH-Px), superoxide dismutase (SOD), and decrease the contents of malondialdehyde (MDA).ConclusionsMZPS effectively showed potential anti-aging activities in vivo and antioxidant activities in vitro, and the molecular constituents, chemical bonds and functional groups of MZPS were superior to MPS, suggesting that the MZPS of L. edodes SD-08 could be used as a potential natural antioxidant.

Purification and antioxidant activities of intracellular zinc polysaccharides from Pleurotus cornucopiae SS-03

Intracellular zinc polysaccharides (IZPS) from Pleurotus cornucopiae SS-03 were extracted and purified, and three subfractions (IZPS-1, IZPS-2, and IZPS-3) were separated by DEAE-52 cellulose anion-exchange column chromatography. They showed certain scavenging effects on superoxide anion (O2−) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals, and positive rising of reducing power in vitro. All the subfractions were found upregulated the enzyme activities of superoxide dismutase (SOD), GSH peroxide (GSH-Px) and catalase (CAT) significantly, decreased the contents of malondialdehyde (MDA) and lipid peroxidation (LPO) remarkably in vivo. In addition, five monosaccharides components, including rhamnose, xylose, mannose, glucose, and galactose with the molar ratio of 1.65:0.05:1:1.50:1.07 and contents of 29.25%, 0.78%, 19.45%, 29.84%, and 20.67%, were investigated by gas chromatography.

Production of pectin lyase from Geobacillus pallidus p26, purification, characterization and fruit juice application

Abstract A bacterial strain was isolated from Pasinler hot spring, Erzurum, Turkey. The purified thermophilic isolate was identified as Geobacillus pallidus P26 and used to produce extracellular pectin lyase (EC 4.2.2.10). Pectin lyase enzyme was purified 34 fold by using DEAE-cellulose anion exchange column chromatography and characterized. Molecular weight of the enzyme was determined as 56 kDa by using Sephadex G-100 gel filtration chromatography. Purification of enzyme was verified by SDS-PAGE. The pH- and temperature optima of enzyme were determined (pH 9.0 and 60°C, respectively). Pectin lyase was mostly stable at 50 oC for 24 hours. Its’ activity decreased to 50 % for 24 h at 60°C. KM and Vmax were calculated as 24.8 mg/mL and 2.28 μmol/L min, respectively. Purified pectin lyase was inhibited by Fe3+, Zn2+, Cu2+, Ca2+, Co2+ and Hg2+ but not by Mg2+. The purified pectin lyase enzyme was used for getting fruits juices. It was found that yields of fruits juices increased when they were compared with control.

Characterization of a Novel Organic Solvent Tolerant Protease from a Moderately Halophilic Bacterium and Its Behavior in Ionic Liquids

An extracellular protease was purified from a novel moderately halophilic bacterium Salinivibrio sp. strain MS-7 by the combination of an acetone precipitation (40-80 %) step and a DEAE-cellulose anion exchange column chromatography. Kinetic parameters of the enzyme exhibited V(max) and K(m) of 130 U/mg and 1.14 mg/ml, respectively, using casein as a substrate. The biochemical properties of the enzyme revealed that the 21-kDa protease had a temperature and pH optimum of 50 °C and 8.0, respectively. The enzyme was strongly inhibited by phenylmethylsulfonyl fluoride, Pefabloc SC, chymostatin, and also EDTA, indicating that it belongs to the class of serine metalloproteases. Interestingly, Ba(2+) and Ca(2+) (2 mM) strongly enhanced the enzyme activity, while Fe(2+) and Mg(2+) activated moderately and Zn(2+), Ni(2+), and Hg(2+) decreased the enzyme activity. The effect of organic solvents with different logP on the purified protease revealed complete stability in toluene, ethyl acetate, chloroform, and n-hexane at 10 and 50 % (v/v) and moderate stability even in 50 % of DMSO and ethanol. The behavior of the MS-7 protease in three imidazolium-based ionic liquids exhibited suitable activity in these green solvent systems, especially in 1-hexyl-3-methylimidazolium hexafluorophosphate ([C(6)MIM][PF(6)]). Comparison of the purified protease with other previously reported proteases suggests that strain MS-7 secrets a novel organic solvent-tolerant protease with outstanding activity in organic solvents and imidazolium-based ionic liquids, which could be applied in low water synthetic section of industrial biotechnology.

Purification and characterization of a pectin lyase produced by Geobacillus stearothermophilus Ah22 and its application in fruit juice production

Extracellular pectin lyase (PL) (EC 4.2.2.10) was produced by Geobacillus stearothermophilus Ah22 in solid state fermentation. The PL enzyme was purified 40.8-fold by DEAE-cellulose anion exchange column chromatography and characterized. The molecular weight of the enzyme was determined as 36 kDa by Sephadex G-100 gel filtration chromatography. Purification of the enzyme was verified by SDS-PAGE. The optimum pH and temperature of the enzyme were determined as pH 6.0 and 60°C, respectively. The PL was mostly stable at 40°C. Its activity deceased by 50% after 2 h at 60°C and by 60% after 6 h at 50°C. The Vmax and Km were calculated as 0.47 mg/mL and 355.3 μmol/L·min, respectively. The presence of 10 mM Ca2+, Cu2+, Mn2+, Mg2+, Zn2+, Hg2+, Fe2+ and EDTA, l-cysteine and ascorbic acid significantly enhanced enzyme activity. The purified PL enzyme was used in the production of fruit juices; yields of fruits juice improved significantly compared with controls.

Salicylic acid increased aldose reductase activity and sorbitol accumulation in tomato plants under salt stress

Increased aldose reductase (ALR) activities were detected in the leaf tissues of tomato plants grown for 3 weeks in culture medium containing 10−7 or 10−4 M salicylic acid (SA), and in the roots after the 10−4 M SA pretreatment. The ALR activity changed in parallel with the sorbitol content in the leaves of the SA-treated plants. Salt stress elicited by 100 mM NaCl enhanced the accumulation of sorbitol in the leaves of control plants and as compared with the untreated control the sorbitol content in the SA-pretreated leaves remained elevated under salt stress. DEAE cellulose anionexchange column purification of the protein precipitated with 80 % (NH4)2SO4 revealed two enzyme fractions with ALR activity in both the leaf and the root tissues. The fraction of the leaf extract that was not bound to the column reacted with glucose and glucose-6-P as substrates, whereas glucose was not a substrate for the bound fraction or for root isoenzymes. The root enzyme was less sensitive to salt treatment: 50 mM NaCl caused 30 % inhibition in the leaf extract, whereas the enzyme activity of the root extract was not affected. It is suggested that increased ALR activity and sorbitol synthesis in the leaves of SA-treated tomato plants may result in an improved salt stress tolerance.

Antioxidant activities of different fractions of polysaccharide purified from pulp tissue of litchi (Litchi chinensis Sonn.)

Abstract Three fractions of water-soluble polysaccharide fractions, coded as LFP1, LFP2 and LFP3, were isolated and purified from litchi pulp by DEAE-52 cellulose anion-exchange column and Sephadex G-100 gel-permeation column. Their chemical and physical characteristics were determined by chemical methods, gas chromatography, IR spectrophotometer and gel-permeation chromatography (GPC) with a model 410 refractive index detector. Antioxidant activities of these fractions were investigated using various in vitro assay systems. Results indicated that LFP1 was composed of arabinose, ribose, galactose and glucose, while LFP2 comprised arabinose, rhamnose and glucose. LFP3 was a heteropolysaccharide bounded with protein (2.6%) and composed of arabinose, rhamnose, galactose and glucose. The molecular weights of LFP1, LFP2 and LFP3 were 11.6 × 104, 3.1 × 104 and 2.9 × 104 Da, respectively. LFP3 showed the highest antioxidant activities among the three fractions of polysaccharides, and could be explored as a novel potential antioxidant.

Antioxidant activities of water-soluble polysaccharide extracted from mung bean (Vigna radiata L.) hull with ultrasonic assisted treatment

Ultrasonic assisted extraction and antioxidant activity of water-soluble polysaccharide (MSP) from mung bean ( Vigna radiata L.) hull were investigated. After deproteinization, MSP was isolated and purified by a DEAE-cellulose anion-exchange column and a Sephadex G-100 gel-permeation column. Two acid polysaccharide fractions, MP1 and MP2, were characterized by FT-IR, GC, and gel-permeation chromatography (GPC). Results indicated that MP1 and MP2 were acid heteropolysaccharide with 9.9% and 36.4% of uronic acid contents respectively. The main composition of MP1 was mannose, whereas MP2 consisted of rhamnose and galactose. The molecule weights of MP1 and MP2 were 83 kDa and 45 kDa, respectively. Although, MP2 showed higher hydroxyl radical-scavenging activity, MP1 exhibited higher reducing power and stronger scavenging capacity for superoxide radical and DPPH radicals as well as higher inhibition on self-oxidation of 1,2,3-phentriol than that of MP2. Thus, MP1 should be explored as a potential antioxidant.

Genetic diversity of Trypanosoma evansi in beef cattle based on internal transcribed spacer region

This study was focused on genetic diversity of Trypanosoma evansi which is a widely distributed haemoflagellate of veterinary importance that infects a variety of larger mammals including horses, mules, camels, buffalo, cattle and deer. The genetic diversity of T. evansi of beef cattle LAM19 was accomplished by using phylogenetic analysis based on internal transcribed spacer region (ITS). Blood sample was collected from a naturally infected beef cattle LAM 19 and parasitemia was raised by mouse inoculation. The parasites were collected and isolated by using DE 52 DEAE cellulose anion exchange column prior to DNA extraction. Upon PCR amplification of ITS region, the product of 1300 bp in size was obtained. The ITS nucleotide sequences were analyzed and revealed that it could demonstrate the genetic diversity of T. evansi of beef cattle LAM19. Based on the ITS tree, beef cattle LAM 19 T. evansi were categorized into two main groups where the genetic diversity occurred within Group 1. The data could be applicable for the survey of parasite dynamics, epidemiological studies as well as prevention and control of the disease.

Purification and Properties of NAD-Dependent Malate Dehydrogenase from Mesembryanthemum crystallinum L. Exhibiting Crassulacean Acid Metabolism.

By DEAE-cellulose anion exchange column chromatography, three different forms of NAD-dependent malate dehydrogenase (EC 1.1.1.37) (NAD-MDH) were isolated from leaves of Mesembryanthemum crystallinum L. in the crassulacean acid metabolism mode. NAD-MDH that eluted last from an anion exchange column was purified to a specific activity of 1, 096 units·(mg protein)-1 using Blue Sepharose CL-6B chromatography. Citrate and adenosine 5'-triphosphate effectively inhibited the activity of NAD-MDH. The inhibition of the enzymatic activity by citrate was reversed by inorganic phosphate and the degree of reversal increased with increasing the concentration of oxaloacetate, the substrate of the reaction. The optimal pH for NAD-MDH activity was around 7.5. Citrate inhibited the enzymatic activity over a wide range of pH and caused a shift in the optimal pH. The enzymatic activity in the presence of citrate was increased by adding inorganic phosphate below pH 7.5. On the basic side, higher than pH 7.5, however, the inhibition by citrate was enhanced by adding inorganic phosphate.

Characterization of Serratia marcescens nuclease isoforms by plasma desorption mass spectrometry

Isoforms of Serratia marcescens nuclease found in the natural nuclease produced by S. marcescens and in recombinant nuclease produced by Escherichia coli were structurally characterized by peptide mapping using plasma desorption mass spectrometry. The nuclease isoforms produced and secreted from S. marcescens B10M1, which are present in much greater amounts than in S. marcescens W225 nuclease produced by E. coli, were characterized completely and the information used to facilitate characterization of the recombinant nuclease isoforms. After purification of the nuclease the isoforms were separated on a DEAE-cellulose anion-exchange column and then digested with endoproteinase Lys-C. The peptides generated were isolated by reverse-phase HPLC and their molecular masses determined by plasma desorption mass spectrometry. Comparison of the peptides from the native nuclease, Sm2, and the two isoforms, Sm1 and Sm3, revealed that they differed only in the N-terminus, the latter being found to lack three amino acids in Sm1 and one amino acid in Sm3. No interior post-translational changes were found in either of the three isoforms. Using this information we were able to confirm that Sm1, the isoform lacking three amino acids, was also present in very small amounts in recombinant S. marcescens W225 nuclease produced and excreted by E. coli.