A haemagglutinin from the medicinal fungus Cordyceps militaris

2009 ◽  
Vol 29 (5) ◽  
pp. 321-327 ◽  
Author(s):  
Jack H. Wong ◽  
Hexiang Wang ◽  
Tzi B. Ng
Keyword(s):  
Gel Filtration ◽  
Exchange Chromatography ◽  
Cordyceps Militaris ◽  
Medicinal Mushrooms ◽  
Medicinal Fungus ◽  
Haemagglutinating Activity ◽  
Medicinal Fungi ◽  
Terminal Amino ◽  
Ph Range ◽  
Hepatoma Hepg2

There are only a few reports on agglutinins from ascomycete and medicinal fungi. An HA (haemagglutinin), with an N-terminal amino acid sequence different from those of known lectins, was isolated in the present study from dried fruiting bodies of the medicinal ascomycete fungus Cordyceps militaris. The purification protocol consisted of affinity chromatography, ion-exchange chromatography and gel filtration. The haemagglutinating activity of the HA could not be inhibited by simple sugars or heparin, and was stable over the pH range 2–13 and up to 60°C. Chemical modification of tryptophan and tyrosine residues had no effect. The HA exhibited some antiproliferative activity towards hepatoma (HepG2) cells and inhibited HIV-1 reverse transcriptase (IC50=10 μM). However, it did not exhibit antifungal activity, mitogenic activity towards splenocytes, nitric oxide-inducing activity towards macrophages or RNase activity. The results of the present study add to the meagre information pertaining to agglutinins from ascomycete and medicinal mushrooms. It is revealed in this study that C. militaris HA differs from other ascomycete mushroom HAs in a variety of biochemical characteristics.

Isolation and characterization of a lectin with potentially exploitable activities from caper (Capparis spinosa) seeds

2009 ◽  
Vol 29 (5) ◽  
pp. 293-299 ◽  
Author(s):  
Sze Kwan Lam ◽  
Qi Feng Han ◽  
Tzi Bun Ng
Keyword(s):  
Gel Filtration ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Mouse Splenocytes ◽  
Isolation And Characterization ◽  
Haemagglutinating Activity ◽  
Capparis Spinosa ◽  
Ph Range ◽  
Induced Apoptosis ◽  
Mcf 7

A dimeric 62-kDa lectin exhibiting a novel N-terminal amino acid sequence was purified from caper (Capparis spinosa) seeds. The purification protocol involved anion-exchange chromatography, cation-exchange chromatography and, finally, gel filtration by FPLC on Superdex 75. Approx. 100-fold purification was achieved. The haemagglutinating activity of the lectin, which was stable in the pH range 1–12 and up to 40°C, could be inhibited by D(+) galactose, α-lactose, raffinose and rhamnose at 1 mM concentration, by 25 mM L(+)-arabinose and by 100 mM D(+)GlcN (glucosamine). The lectin potently inhibited HIV-1 reverse transcriptase with an IC50 of 0.28 μM and proliferation of both hepatoma HepG2 and breast cancer MCF-7 cells with an IC50 of approx. 2 μM. It induced apoptosis in HepG2 and MCF-7 cells. It manifested a weaker mitogenic activity on mouse splenocytes than ConA (concanavalin A). It inhibited mycelial growth in Valsa mali with an IC50 of 18 μM.

scholarly journals Purification and properties of three (1→3)-β-d-glucanase isoenzymes from young leaves of barley (Hordeum vulgare)

1993 ◽  
Vol 289 (2) ◽  
pp. 453-461 ◽  
Author(s):  
M Hrmova ◽  
G B Fincher
Keyword(s):  
Hordeum Vulgare ◽  
Elevated Temperatures ◽  
Gel Filtration ◽  
Degree Of Polymerization ◽  
Exchange Chromatography ◽  
Purification And Properties ◽  
Young Leaves ◽  
Monomeric Proteins ◽  
Ph Range ◽  
Hydrolysis Of

Three (1->3)-beta-D-glucan glucanohydrolase (EC 3.2.1.39) isoenzymes GI, GII and GIII were purified from young leaves of barley (Hordeum vulgare) using (NH4)2SO4 fractional precipitation, ion-exchange chromatography, chromatofocusing and gel-filtration chromatography. The three (1->3)-beta-D-glucanases are monomeric proteins of apparent M(r)32,000 with pI values in the range 8.8-10.3. N-terminal amino-acid-sequence analyses confirmed that the three isoenzymes represent the products of separate genes. Isoenzymes GI and GII are less stable at elevated temperatures and are active over a narrower pH range than is isoenzyme GIII, which is a glycoprotein containing 20-30 mol of hexose equivalents/mol of enzyme. The preferred substrate for the enzymes is laminarin from the brown alga Laminaria digitata, an essentially linear (1->3)-beta-D-glucan with a low degree of glucosyl substitution at 0-6 and a degree of polymerization of approx. 25. The three enzymes are classified as endohydrolases, because they yield (1->3)-beta-D-oligoglucosides with degrees of polymerization of 3-8 in the initial stages of hydrolysis of laminarin. Kinetic analyses indicate apparent Km values in the range 172-208 microM, kcat. constants of 36-155 s-1 and pH optima of 4.8. Substrate specificity studies show that the three isoenzymes hydrolyse substituted (1->3)-beta-D-glucans with degrees of polymerization of 25-31 and various high-M(r), substituted and side-branched fungal (1->3;1->6)-beta-D-glucans. However, the isoenzymes differ in their rates of hydrolysis of a (1->3;1->6)-beta-D-glucan from baker's yeast and their specific activities against laminarin vary significantly. The enzymes do not hydrolyse (1->3;1->4)-beta-D-glucans, (1->6)-beta-D-glucan, CM-cellulose, insoluble (1->3)-beta-D-glucans or aryl beta-D-glycosides.

Physicochemical properties of a novel α-L-arabinofuranosidase fromRhizomucor pusillusHHT-1

2001 ◽  
Vol 47 (8) ◽  
pp. 767-772 ◽  
Author(s):  
A KM Shofiqur Rahman ◽  
Shinya Kawamura ◽  
Masahiro Hatsu ◽  
M M Hoq ◽  
Kazuhiro Takamizawa
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Molecular Mass ◽  
Gel Filtration ◽  
Hydrolytic Activity ◽  
Exchange Chromatography ◽  
Ph Optimum ◽  
Rhizomucor Pusillus ◽  
Terminal Amino ◽  
Metal Cofactors

The zygomycete fungus Rhizomucor pusillus HHT-1, cultured on L(+)arabinose as a sole carbon source, produced extracellular α-L-arabinofuranosidase. The enzyme was purified by (NH4)2SO4fractionation, gel filtration, and ion exchange chromatography. The molecular mass of this monomeric enzyme was 88 kDa. The native enzyme had a pI of 4.2 and displayed a pH optimum and stability of 4.0 and 7.0–10.0, respectively. The temperature optimum was 65°C, and it was stable up to 70°C. The Kmand Vmaxfor p-nitrophenyl α-L-arabinofuranoside were 0.59 mM and 387 µmol·min–1·mg–1protein, respectively. Activity was not stimulated by metal cofactors. The N-terminal amino acid sequence did not show any similarity to other arabinofuranosidases. Higher hydrolytic activity was recorded with p-nitrophenyl α-L-arabinofuranoside, arabinotriose, and sugar beet arabinan; lower hydrolytic activity was recorded with oat–spelt xylan and arabinogalactan, indicating specificity for the low molecular mass L(+)-arabinose containing oligosaccharides with furanoside configuration.Key words: α-L-arabinofuranosidase, enzyme purification, amino acid sequence, Rhizomucor pusillus.

Isolation of Fasciola hepatica haemoglobin

Parasitology ◽  
1995 ◽  
Vol 111 (2) ◽  
pp. 209-215 ◽  
Author(s):  
S. McGonigle ◽  
J. P. Dalton
Keyword(s):  
Fasciola Hepatica ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Amino Acid Sequence Analysis ◽  
Terminal Amino Acid ◽  
Terminal Amino ◽  
Haemoglobin Molecule

SUMMARYA haemoprotein released in vitro by adult Fasciola hepatica was purified by gel filtration chromatography on Sephacryl S-200 and ion-exchange chromatography on DEAE-Sepharose. The molecule, with an apparent molecular weight of > 200 kDa, contains a haem group and has absorption spectra characteristics similar to haemoglobins. N-terminal amino acid sequence analysis revealed no similarity between the F. hepatica haemoglobin and other vertebrate or invertebrate haemoglobins. Antibodies to the haemoglobin molecule can be detected in the sera of F. hepatica-infected bovines as early as 1 week after infection.

A novel mucin sulphatase from human faeces: its identification, purification and characterization

1992 ◽  
Vol 82 (4) ◽  
pp. 447-454 ◽  
Author(s):  
H. H. Tsai ◽  
D. Sunderland ◽  
G. R. Gibson ◽  
C. A. Hart ◽  
J. M. Rhodes
Keyword(s):  
High Performance ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Purification And Characterization ◽  
Ph Optimum ◽  
Human Faeces ◽  
Colonic Mucin ◽  
Inorganic Sulphate ◽  
Normal Human ◽  
Ph Range

1. Colonic mucus is heavily sulphated and it is likely that this contributes considerably to its resistance to degradation by bacterial enzymes. The presence of a mucin-desulphating enzyme in faeces could therefore be very important in determining the rate of degradation of secreted mucus and hence the level of protection of the mucosa. 2. A novel assay for mucin sulphatase has been developed using biologically labelled human colonic [35S]sulphomucin as a substrate and a mucin sulphatase has been purified from faeces by sequential high-performance gel filtration and ion-exchange chromatography. 3. The mucin sulphatase has been shown to have a pH optimum of 4.5 and activity over the pH range 3–7. It has a pI of 4.0 and is inhibited by inorganic sulphate and phosphate. The purified enzyme preparation gave a single band on electrophoresis with a molecular mass of 15000 Da. It has a Km of 41.9 mmol/l and a Vmax. of 1.17 katal/ kg for glucose 6-sulphate. The enzyme was also shown to enhance fivefold the deglycosylation of [3H]glucosamine-labelled mucin by a faecal mucin glycosidase preparation. 4. Two bacteroides spp. isolated from normal human faeces, Bacteroides fragilis and B. thetaiotaomicron, were found to be producers of mucin-desulphating enzymes. 5. Mucin sulphatase is likely to be critical in determining the rate of enzymic degradation of secreted colonic mucin.

Human Leucocyte Urokinase Inhibitor - Purification, Characterization and Comparative Studies Against Different Plasminogen Activators

1985 ◽  
Vol 54 (04) ◽  
pp. 750-755 ◽  
Author(s):  
M Kopitar ◽  
B Rozman ◽  
J Babnik ◽  
V Turk ◽  
D E Mullins ◽  
...  
Keyword(s):  
Plasminogen Activator ◽  
Inhibitory Activity ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
Plasminogen Activator Inhibitor ◽  
Exchange Chromatography ◽  
Sds Page ◽  
Peripheral Leukocytes ◽  
Ph Range ◽  
Activator Inhibitor

SummaryA plasminogen activator inhibitor (PA-I) which inhibits primarily plasminogen activator of the urokinase type (u-PA) was isolated from the cytosol of human peripheral leukocytes. The inhibitor was isolated using ion exchange chromatography, gel filtration and FPLC. This inhibitor has an apparent molecular weight of 45 kDa, determined by SDS-PAGE, and a pi of 5.5-5.7. The inhibitor is a fast reacting inhibitor, is thermally unstable and is inactivated outside the pH range 7-9. Treatment of cytosol to pH 9 for 30 min at 37° C resulted in a large increase in inhibitory activity. Antibodies against human placental UK-I completely quenched the inhibitory activity of human leucocyte UK-I.

scholarly journals A Laccase with Antiproliferative and HIV-I Reverse Transcriptase Inhibitory Activities from the Mycorrhizal FungusAgaricus placomyces

2012 ◽  
Vol 2012 ◽  
pp. 1-8 ◽  
Author(s):  
Jian Sun ◽  
Qing-Jun Chen ◽  
Qing-Qin Cao ◽  
Ying-Ying Wu ◽  
Li-Jing Xu ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Mycorrhizal Fungus ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Hep G2 ◽  
Immunodeficiency Virus ◽  
Terminal Amino ◽  
Hiv 1 ◽  
Mcf 7

A novel 68 kDa laccase was purified from the mycorrhizal fungusAgaricus placomycesby utilizing a procedure that comprised three successive steps of ion exchange chromatography and gel filtration as the final step. The monomeric enzyme exhibited the N-terminal amino acid sequence of DVIGPQAQVTLANQD, which showed only a low extent of homology to sequences of other fungal laccases. The optimal temperature forA. placomyceslaccase was 30°C, and optimal pH values for laccase activity towards the substrates 2,7′-azinobis[3-ethylbenzothiazolone-6-sulfonic acid] diammonium salt (ABTS) and hydroquinone were 5.2 and 6.8, respectively. The laccase displayed, at 30°C and pH 5.2,Kmvalues of 0.392 mM towards hydroquinone and 0.775 mM towards ABTS. It potently suppressed proliferation of MCF 7 human breast cancer cells and Hep G2 hepatoma cells and inhibited human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) activity with an IC50of 1.8 μM, 1.7 μM, and 1.25 μM, respectively, signifying that it is an antipathogenic protein.

scholarly journals Purification and Characterization of a Prolyl Aminopeptidase from Debaryomyces hansenii

2003 ◽  
Vol 69 (1) ◽  
pp. 227-232 ◽  
Author(s):  
Tomás Bolumar ◽  
Yolanda Sanz ◽  
M.-Concepción Aristoy ◽  
Fidel Toldrá
Keyword(s):  
Enzyme Activity ◽  
Gel Filtration ◽  
Debaryomyces Hansenii ◽  
Cysteine Proteases ◽  
Anion Exchange Chromatography ◽  
Cell Extract ◽  
Exchange Chromatography ◽  
Prolyl Aminopeptidase ◽  
Ph Range ◽  
Meat Fermentation

ABSTRACT A prolyl aminopeptidase (PAP) (EC 3.4.11.5) was isolated from the cell extract of Debaryomyces hansenii CECT12487. The enzyme was purified by selective fractionation with protamine and ammonium sulfate, followed by two chromatography steps, which included gel filtration and anion-exchange chromatography. The PAP was purified 248-fold, with a recovery yield of 1.4%. The enzyme was active in a broad pH range (from 5 to 9.5), with pH and temperature optima at 7.5 and 45°C. The molecular mass was estimated to be around 370 kDa. The presence of inhibitors of serine and aspartic proteases, bestatin, puromycin, reducing agents, chelating agents, and different cations did not have any effect on the enzyme activity. Only iodoacetate, p-chloromercuribenzoic acid, and Hg2+, which are inhibitors of cysteine proteases, markedly reduced the enzyme activity. The Km for proline-7-amido-4-methylcoumarin was 40 μM. The enzyme exclusively hydrolyzed N-terminal-proline-containing substrates. This is the first report on the identification and purification of this type of aminopeptidase in yeast, which may contribute to the scarce knowledge about D. hansenii proteases and their possible roles in meat fermentation.

Purification of Clostridium perfringens type C theta toxin

1973 ◽  
Vol 19 (8) ◽  
pp. 881-885 ◽  
Author(s):  
A. H. W. Hauschild ◽  
A. Lecroisey ◽  
J. E. Alouf
Keyword(s):  
Clostridium Perfringens ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Ammonium Sulfate Precipitation ◽  
Terminal Amino Acid ◽  
Single Protein ◽  
Dodecyl Sulfate ◽  
Type C ◽  
Terminal Amino ◽  
Anionic Polyacrylamide

Clostridium perfringens type C theta toxin was purified by ammonium sulfate precipitation, gel filtration, and ion-exchange chromatography. The purified toxin was free of alpha, beta, delta, and kappa toxins. Electrophoresis of the toxin in both the anionic polyacrylamide disc gel and in the polyacrylamide dodecyl sulfate gel yielded single protein bands. L-Lysine was determined as the sole N-terminal amino acid. The specific hemolytic activities of two purified preparations were 3.6 × 106 and 4.8 × 106 HU/mg N; the specific toxicities were 8.1 × 103 and 7.7 × 103 mouse MLD/mg N. The molecular weight determined by the polyacrylamide – dodecyl sulfate method was 74 000.

scholarly journals Screening and Purification of a Chymotrypsin Inhibitor from Entrolobium Saman Seeds

2010 ◽  
Vol 15 ◽  
pp. 19
Author(s):  
Maher Ali. Maqtari ◽  
A.B. Mohamed. Saad
Keyword(s):  
Molecular Weight ◽  
Enzyme Inhibitor ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Molar Ratio ◽  
Apparent Dissociation Constant ◽  
Chymotrypsin Inhibitor ◽  
Ammonium Sulphate Precipitation ◽  
Wide Ph Range ◽  
Ph Range

A chymotrypsin inhibitor was isolated and purified from the seeds of Enterolobium saman  (Leguminaceae family) by extraction with 100 mM phosphate buffer, heat treatment, ammonium sulphate precipitation, ion-exchange chromatography on DEAEcellulose and filtration through Sephadex G-75. The final preparation appeared to be homogeneous by both chromatographic and electrophoretic analyses. ESCI had a molecular weight of about 17,890 and an isoelectric point of 5.8. ESCI inhibited bovine chymotrypsin at an inhibitor-enzyme molar ratio of 1:2. The inhibition mode of chymotrypsin inhibitor was competitive on bovine chymotrypsin. Investigation has been carried out on the complex formed between chymotrypsin and chymotrypsin inhibitor by physico-chemical methods. An apparent dissociation constant (Ki) of 9.05 X 10-8 M has been calculated for the complex. This enzyme-  inhibitor complex was isolated by gel filtration on Sephadex G-75 and a molecular weight of 43.000 was estimated for the complex. The inhibitor did not have any effect on other proteinases, such as papain, bromelin, elastase, α -amylase, trypsin and pepsin. The chemical modification of lysine residues indicated that –NH2  groups are not essential for the activity of ESCI toward chymotrypsin. The inhibitor was an acidic protein and was stable over a wide pH range of 2-12 and temperature range of 10o C-97o C. 

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