scholarly journals A novel alkaline protease with antiproliferative activity from fresh fruiting bodies of the toxic wild mushroom Amanita farinosa.

2011 ◽  
Vol 58 (4) ◽  
Author(s):  
Jian Sun ◽  
Yongchang Zhao ◽  
Hongmei Chai ◽  
Hexiang Wang ◽  
Tzi Bun Ng
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Fruiting Bodies ◽  
Wild Mushroom ◽  
Sds Page ◽  
Human Hepatoma Hepg2 Cells

A novel protease with a molecular mass of 15 kDa was purified from fresh fruiting bodies of the wild mushroom Amanita farinosa. The purification protocol entailed anion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, cation exchange chromatography on SP-Sepharose, and gel filtration by fast protein liquid chromatography on Superdex 75. The protease was unadsorbed on DEAE-cellulose but adsorbed on Affi-gel blue gel and SP-Sepharose. It demonstrated a single 15-kDa band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS/PAGE) and a 15-kDa peak in gel filtration. The optimal pH and optimal temperature of the protease were pH 8.0 and 65 °C, respectively. Proliferation of human hepatoma HepG2 cells was inhibited by the protease with an IC(50) of 25 µM. The protease did not have antifungal or ribonuclease activity.

scholarly journals Purification and characterization of an antibacterial protein from dried fruiting bodies of the wild mushroom Clitocybe sinopica.

2010 ◽  
Vol 57 (1) ◽  
Author(s):  
Suyue Zheng ◽  
Qinghong Liu ◽  
Guoqing Zhang ◽  
Hexiang Wang ◽  
Tzi Bun Ng
Keyword(s):  
Antibacterial Activity ◽  
Molecular Mass ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Exchange Chromatography ◽  
Fruiting Bodies ◽  
Wild Mushroom ◽  
Antibacterial Protein

A novel antibacterial protein with a molecular mass of 44 kDa has been isolated from dried fruiting bodies of the wild mushroom Clitocybe sinopica. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis showed that the protein was composed of two subunits each with a molecular mass of 22 kDa. Its N-terminal amino-acid sequence, SVQATVNGDKML, has not been reported for other antimicrobial proteins. The purification protocol included ion exchange chromatography on DEAE-cellulose, CM-cellulose and Q-Sepharose, and gel filtration by fast protein liquid chromatography on Superdex 75. The antibacterial protein was adsorbed on all three ion exchangers. The antimicrobial activity profile of the protein against tested bacterial and fungal strains disclosed that it possessed potent antibacterial activity against Agrobacterium rhizogenes, A. tumefaciens, A. vitis, Xanthomonas oryzae and X. malvacearum with a minimum inhibitory concentration mostly below 0.6 microM. However, it had no antibacterial activity against Pseudomonas batatae, Erwinia herbicola, Escherichia coli, and Staphylococcus aureus, and no antifungal activity against Setosphaeria turcica, Fusarium oxysporum, Verticillium dahliae, Bipolaris maydis, and B. sativum. The antibacterial antivity against A. tumefaciens was stable after exposure to 20-60 degrees C for 30 min and to pH 4-9 for 1 h.

Purification and characterization of the extracellular α-amylase activity of the yeast Schwanniomyces alluvius

1987 ◽  
Vol 65 (10) ◽  
pp. 899-908 ◽  
Author(s):  
F. Moranelli ◽  
M. Yaguchi ◽  
G. B. Calleja ◽  
A. Nasim
Keyword(s):  
Amino Acid ◽  
Amylase Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Exchange Chromatography ◽  
Proteinase K ◽  
Ph Optimum ◽  
Sds Page

The extracellular α-amylase activity of the yeast Schwanniomyces alluvius has been purified by anion-exchange chromatography on DEAE-cellulose and gel-filtration chromatography on Sephadex G-100. Sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS–PAGE) and N-terminal amino acid analysis of the purified sample indicated that the enzyme preparation was homogeneous. The enzyme is a glycoprotein having a molecular mass of 52 kilodaltons (kDa) estimated by SDS–PAGE and 39 kDa by gel filtration on Sephadex G-100. Chromatofocusing shows that it is an acidic protein. It is resistant to trypsin but sensitive to proteinase K. Its activity is inhibited by the divalent cation chelators EDTA and EGTA and it is insensitive to sulfhydryl-blocking agents. Exogenous divalent cations are inhibitory as are high concentrations of monovalent salts. The enzyme has a pH optimum between 3.75 and 5.5 and displays maximum stability in the pH range of 4.0–7.0. Under the conditions tested, the activity is maximal between 45 and 50 °C and is very thermolabile. Analysis of its amino acid composition supports its acidic nature.

scholarly journals Mouse Uterine Alkaline Phosphatase: Improved Purification by Affinity Chromatography and Further Characterization of the Enzyme

1980 ◽  
Vol 33 (3) ◽  
pp. 279 ◽  
Author(s):  
RN Murdoch ◽  
Louise E Buxton ◽  
DJ Kay
Keyword(s):  
Alkaline Phosphatase ◽  
Affinity Chromatography ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Apparent Molecular Weight ◽  
Anion Exchange Chromatography ◽  
Pregnant Mouse ◽  
Exchange Chromatography ◽  
Enzyme Preparations

An improved procedure for the purification of alkaline phosphatase from about 10 g of day 7 pregnant mouse uterine tissue is described. Following homogenization, the procedure involved solubilization and extraction with 0�8% (v/v) Triton X-lOO and 20% (v/v) n-butanol, ammonium sulfate precipitation, concanavalin A-Sepharose 4B affinity chromatography, DEAE-cellulose anion-exchange chromatography and Sephacryl S200 gel filtration. On subjecting 2162-fold purified enzyme preparations to polyacrylamide-gel electrophoresis, a single band of protein coincident with the zone of enzyme activity and having an apparent molecular weight of 205 OOO� lOOOO was identified. Affinity chromatography yielded the largest increase in purity of any step in the procedure and established the glycoprotein nature of the uterine enzyme.

scholarly journals Cadmium-binding proteins of rat testes. Characterization of a low-molecular-mass protein that lacks identity with metallothionein

1984 ◽  
Vol 220 (3) ◽  
pp. 811-818 ◽  
Author(s):  
M P Waalkes ◽  
S B Chernoff ◽  
C D Klaassen
Keyword(s):  
Metal Binding ◽  
Gel Electrophoresis ◽  
Binding Proteins ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Polyacrylamide Gel ◽  
Or Groups

Cadmium-binding proteins in the cytosol of testes from untreated rats were separated by Sephadex G-75 gel filtration. Three major testicular metal-binding proteins (TMBP), or groups of proteins, with relative elution volumes of approx. 1.0 (TMBP-1), 1.7 (TMBP-2) and 2.4 (TMBP-3) were separated. Elution of Zn-binding proteins exhibited a similar pattern. TMBP-3 has previously been thought to be metallothionein (MT), and hence this protein was further characterized and compared with hepatic MT isolated from Cd-treated rats. Estimation of Mr by gel filtration indicated a slight difference between MT (Mr 10000) and TMBP-3 (Mr 8000). Two major forms of MT (MT-I and MT-II) and TMBP-3 (TMBP-3 form I and TMBP-3 form II) were obtained after DEAE-Sephadex A-25 anion-exchange chromatography, with the corresponding subfractions being eluted at similar conductances. Non-denaturing polyacrylamide-gel electrophoresis on 7% acrylamide gels indicated that the subfractions of TMBP-3 had similar mobilities to those of the corresponding subfractions of MT. However, SDS (sodium dodecyl sulphate)/12% (w/v)-polyacrylamide-gel electrophoresis resulted in marked differences in migration of the two corresponding forms of MT and TMBP-3. Co-electrophoresis of MT-II and TMBP-3 form II by SDS/polyacrylamide-gel electrophoresis revealed two distinct proteins. Amino acid analysis indicated much lower content of cysteine in the testicular than in the hepatic proteins. TMBP-3 also contained significant amounts of tyrosine, phenylalanine and histidine, whereas MT did not. U.v.-spectral analysis of TMBP-3 showed a much lower A250/A280 ratio than for MT. Thus this major metal-binding protein in testes, which has been assumed to be MT is, in fact, a quite different protein.

Purification and some properties of the extracellular α-amylase from Aspergillus awamori

1985 ◽  
Vol 31 (2) ◽  
pp. 149-153 ◽  
Author(s):  
Resham S. Bhella ◽  
Illimar Altosaar
Keyword(s):  
Enzyme Activity ◽  
Gel Electrophoresis ◽  
Culture Medium ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Aspergillus Awamori ◽  
Original Activity

Alpha-amylase was purified from the extracellular culture medium of Aspergillus awamori by means of ethanol precipitation. Sephacryl-200 gel filtration and anion-exchange chromatography on Dowex (AG1-X4) resin. The enzyme preparation was found to be homogeneous by means of sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The purified enzyme had a molecular weight of 54 000 ± 2 500 and its isoelectric point was pH 4.2. The enzyme was found to be most active between pH 4.8 and 5.0 and was stable between pH 3.5 and 6.5. The optimal temperature for the enzyme activity was around 50 °C and the enzyme was stable for at least 1 h up to 45 °C retaining more than 80% of its original activity. The Km (37 °C, pH 5.3) for starch hydrolysis was 1.0 g∙L−1 and maltose inhibited the enzyme activity uncompetitively with a K1 value of 20.05 g∙L−1

ISOLATION OF LH, FSH AND TSH FROM HUMAN PITUITARIES AFTER THE REMOVAL OF HGH

1974 ◽  
Vol 77 (3) ◽  
pp. 485-497 ◽  
Author(s):  
P. A. Torjesen ◽  
T. Sand ◽  
N. Norman ◽  
O. Trygstad ◽  
I. Foss
Keyword(s):  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Disc Electrophoresis ◽  
Exchange Chromatography ◽  
Polyacrylamide Gel ◽  
Molecular Weights ◽  
Pituitary Glands

ABSTRACT Highly purified human LH, FSH and TSH were isolated from batches of 300 frozen pituitary glands (200 g) by pH, acetone and ethanol fractionation, Sephadex gel filtration, ion-exchange chromatography on DEAE-cellulose and CM-Sephadex, and preparative polyacrylamide-gel electrophoresis. Sodium dodecyl-sulphate (SDS) polyacrylamide gel electrophoresis was used in order to check the purity, the identity and the molecular weight of the purified LH, FSH and TSH. This procedure showed that the hormone preparations consisted of two subunits with molecular weights of: LH: 21 300 and 17 900, FSH: 22 100 and 18 300 and TSH: 20 800 and 16 400. The purity of the hormone preparations was also evaluated by analytical disc electrophoresis at pH 8.9. The purified hormone preparations had radioimmunological activity as follows: LH: 20 000 IU/mg, FSH: 16 500 IU/mg and TSH: 5 IU/mg. All preparations had high biological potency.

Purification, properties, and partial amino acid sequence of chitinase from a marine Alteromonas sp. strain O-7

1992 ◽  
Vol 38 (9) ◽  
pp. 891-897 ◽  
Author(s):  
Hiroshi Tsujibo ◽  
Yukio Yoshida ◽  
Katsushiro Miyamoto ◽  
Chiaki Imada ◽  
Yoshiro Okami ◽  
...  
Keyword(s):  
Amino Acid ◽  
Marine Bacterium ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Molecular Size ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Amino Acid Residues ◽  
Amino Terminal

Chitinase (EC 3.2.1.14) was isolated from the culture supernatant of a marine bacterium, Alteromonas sp. strain O-7. The enzyme (Chi-A) was purified by anion-exchange chromatography (DEAE-Toyopearl 650 M) and gel filtration (Sephadex G-100). The purified enzyme showed a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The molecular size and pI of Chi-A were 70 kDa and 3.9, respectively. The optimum pH and temperature of Chi-A were 8.0 and 50 °C, respectively. Chi-A was stable in the range of pH 5–10 up to 40 °C. Among the main cations, such as Na+, K+, Mg2+, and Ca2+, contained in seawater, Mg2+ stimulated Chi-A activity. N-Bromosuccinimide and 2-hydroxy-5-nitrobenzyl bromide inhibited Chi-A activity. The amino-terminal 27 amino acid residues of Chi-A were sequenced. This enzyme showed sequence homology with chitinases from terrestrial bacteria such as Serratia marcescens QMB1466 and Bacillus circulons WL-12. Key words: marine bacterium, Alteromonas sp., chitinase.

scholarly journals PURIFICATION AND BIOCHEMICAL CHARACTERIZATION OF A T/TN SPECIFIC LECTIN FROM LEPECHINIA BULLATA SEEDS (LAMIACEAE)

2017 ◽  
Vol 9 (10) ◽  
pp. 165
Author(s):  
Wilches Torres A. ◽  
Rojas Caraballo J. ◽  
Sanabria E. ◽  
Reyes MontaÑo E ◽  
FernÁndez Alonso Jl ◽  
...  
Keyword(s):  
Isoelectric Focusing ◽  
Biochemical Characterization ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Reducing Conditions ◽  
Sds Page ◽  
Unique Band

Objective: This study focused on purifying and characterizing a lectin from Lepechinia bullata (L. bullata) seeds, and determining its specificity towards tumour-associated carbohydrate-antigens.Methods: Pigments were removed by washing the seeds with NH4OH 0.1 M pH 9.4 and treating the crude extracts with Pectinex®. The purification procedure consisted of anion exchange chromatography on diethylaminoethyl (DEAE)-Sephadex followed by affinity chromatography. For the characterization, the phase was used polyacrylamide gel electrophoresis-sodium dodecyl sulphate (SDS-PAGE), isoelectric focusing, hemagglutination assays, enzyme-linked lectinosorbent assay (ELLA) and thermal shift assay (TSA).Results: 6.2 mg of lectin were obtained from 100 g of seeds. It was able to agglutinate enzymatically treated erythrocytes with a minimal required lectin concentration of 7 μg. ml-1. Strong binding to asialo bovine submaxillary mucine (aBSM) was determined, corroborating Tn recognition.The isoelectric focusing showed a unique band at pH 8.5. Lectin pure shown bands at 28, 48 and 93 kDa by SDS-PAGE, with an incomplete dissociation of the last species despite trying several reduction conditions. By preparative electrophoresis under different conditions, three species were observed too, in all fractions one band at 28 kDa on Tricine-PAGE in reducing and no reducing conditions were found.Amino acid composition, carbohydrate content, thermal stability and Ca2+and Mn2+requirements were determined. N-acetylgalactosamine (GalNAc) and desialylated mucins inhibited the agglutinant activity on human cells. Fetuin inhibited hemagglutination of rabbit erythrocytes.Conclusion: A new lectin was isolated and characterized from L. bullata seeds, it recognizes T/Tn antigen and shows some similarities with other Lamiaceae lectins.

Purification and characterization of a β-glucosidase from solid-state cultures ofHumicola griseavar.thermoidea

1996 ◽  
Vol 42 (1) ◽  
pp. 1-5 ◽  
Author(s):  
Edivaldo Ximenes Ferreira Filho
Keyword(s):  
Solid State ◽  
High Performance ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
High Performance Liquid Chromatographic ◽  
Exchange Chromatography ◽  
Glucosidase Activity ◽  
Apparent Homogeneity ◽  
Sds Page

The thermophilic fungus Humicola grisea var. thermoidea produced β-glucosidase activity when grown in a solid-state culture on wheat bran as carbon source. A β-glucosidase was purified to apparent homogeneity by ultrafiltration, gel filtration chromatography on Sephacryl S-100, and ion-exchange chromatography on S-Sepharose, as judged by sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS–PAGE) on a 12.5% (w/v) slab gel. The enzyme had a molecular mass of 82 and 156 kDa, as estimated by SDS–PAGE and gel filtration on a high performance liquid chromatographic column, respectively, suggesting that the native enzyme may consist of two identical subunits. The purified enzyme was thermostable at 60 °C for 1 h with a half-life of 15 min at 65 °C, and displayed optimum activity at 60 °C and a pH range of 4.0–4.5. The Kmand Vmaxvalues for p-nitrophenyl β-D-glucopyranoside were determined to be 0.316 mM and 0.459 IU∙mL−1, respectively. D-Glucose, D-gluconic acid lactone, Hg2+, Cu2+, and Mn2+inhibited β-glucosidase activity. The enzyme activity was competitively inhibited by D-glucose (Ki = 0.6 mM). The purified enzyme was very active against cellobiose and p-nitrophenyl β-D-glucopyranoside.Key words: Humicola, β-glucosidase, purification, characterization.

scholarly journals Hydrophobic-interaction chromatography and anion-exchange chromatography in the presence of acetonitrile. A two-step purification method for human prolactin

1981 ◽  
Vol 199 (3) ◽  
pp. 619-627 ◽  
Author(s):  
Steven C. Hodgkinson ◽  
Philip J. Lowry
Keyword(s):  
Anion Exchange ◽  
Hydrophobic Interaction ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Deae Cellulose ◽  
Hydrophobic Interaction Chromatography ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Preparative Scale ◽  
Human Prolactin

Described is a two-chromatographic-step preparative-scale technique for the purification of human prolactin from a frozen pituitary homogenate. The method utilizes hydrophobic interaction chromatography on the mildly hydrophobic adsorbent phenyl-Sepharose CL-4B and anion-exchange chromatography on DEAE-cellulose in the presence of acetonitrile. Human prolactin was solubilized at pH10.0 after a prior extraction of pituitaries at pH4.0, the acid pH being ineffective at solubilizing human prolactin but capable of solubilizing large amounts of interfering protein. An 11-fold increase in the potency of the solubilized human prolactin was achieved in this manner. Prolactin could be adsorbed to phenyl-Sepharose at low ionic strengths (I<0.01); few other proteins were adsorbed under these conditions. This is a demonstration of the hydrophobic nature of human prolactin. The amount of phenyl-Sepharose was limited to the minimum (35mg of protein/g of phenyl-Sepharose) necessary to adsorb human prolactin, further reducing the uptake of other pituitary protein. Desorption was achieved by using an acetonitrile gradient (0–30%, v/v), resulting in a purification of human prolactin of 85-fold and recovery of 78%. Acetonitrile (20%, v/v) was also included in all buffers for DEAE-cellulose chromatography, increasing the resolution and recovery of human prolactin, apparently by minimizing non-ionic interactions with the matrix. Prolactin (10mg) was recovered from 63g if pituitaries, an overall recovery of 58%. It was homogeneous by gel filtration and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, contained less than 0.1% somatotropin (growth hormone), on iodination demonstrated more than 95% binding to excess anti-(human prolactin) serum and could be displaced from anti-(human prolactin) serum in a manner indistinguishable from the serum of a patient with a human prolactin-secreting adenoma.

Sign in / Sign up

Export Citation Format

Share Document