Purification, characterization, and antifungal activity of chitinase from Fusarium chlamydosporum, a mycoparasiteto groundnut rust, Puccinia arachidis

1998 ◽  
Vol 44 (7) ◽  
pp. 646-651 ◽  
Author(s):  
N Mathivanan ◽  
V Kabilan ◽  
K Murugesan
Keyword(s):  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Puccinia Arachidis ◽  
Fusarium Chlamydosporum ◽  
Groundnut Rust ◽  
Germ Tubes

Chitinase (EC 3.2.1.14) was isolated from the culture filtrate of Fusarium chlamydosporum and purified by ion-exchange chromatography and gel filtration. The molecular mass of purified chitinase was 40 kDa as estimated by sodium dodecyl sulfate – polyacrylamide gel electrophoresis. Chitinase was optimally active at a pH of 5 and stable from pH 4 to 6 and up to 40°C. Among the metals and inhibitors tested, mercuric chloride completely inhibited the enzyme activity. The activity of chitinase was high on colloidal and pure chitin. The purified chitinase inhibited the germination of uredospores of Puccinia arachidis and also lysed the walls of uredospores and germ tubes. The results from these experiments indicated that chitinase of F. chlamydosporum plays an important role in the biocontrol of groundnut rust. Key words: Fusarium chlamydosporum, chitinase, purification, Puccinia arachidis, uredospores.

scholarly journals Purification of rabbit bone inhibitor of collagenase

1981 ◽  
Vol 195 (1) ◽  
pp. 159-165 ◽  
Author(s):  
T E Cawston ◽  
W A Galloway ◽  
E Mercer ◽  
G Murphy ◽  
J J Reynolds
Keyword(s):  
Tissue Culture ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Serine Proteinases ◽  
Bacterial Collagenase ◽  
Rabbit Bone

1. Rabbit bones in tissue culture synthesize an inhibitor of collagenase during the first 4 days of culture. 2. The inhibitor was purified by a combination of gel filtration, concanavalin A--Sepharose chromatography, ion-exchange chromatography and zinc-chelate affinity chromatography. 3. The purified inhibitor migrated as a single band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and had a mol.wt. of 28000. 4. The inhibitor blocked the activity of the metalloproteinases collagenase, gelatinase, neutral proteinase III (proteoglycanase), human leucocyte collagenase and gelatinase, but not thermolysin or bacterial collagenase. The serine proteinases plasmin and trypsin were not inhibited. 5. The inhibitor interacted with purified rabbit bone collagenase with 1:1 stoichiometry. 6. The inhibitory activity was lost after incubation for 1 h at 90 degrees C, after treatment with trypsin (250 micrograms/ml) at 37 degrees C for 30 min and after reduction and alkylation.

scholarly journals α-Lactalbumin and lactose concentrations in rat milk during lactation

1981 ◽  
Vol 194 (1) ◽  
pp. 149-154 ◽  
Author(s):  
K R Nicholas ◽  
P E Hartmann ◽  
B L McDonald
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Significant Negative Correlation ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Biologically Active ◽  
Alpha Lactalbumin

Homogeneous rat alpha-lactalbumin was prepared from whey by chromatography on DEAE-Sephadex A-50 and Ultrogel AcA 44. Two biologically active forms of alpha-lactalbumin were apparent after ion-exchange chromatography, but on gel filtration the combined forms were eluted as a single peak with a molecular weight of approx. 33000. The molecular weight when determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis was 15100. Antiserum to alpha-lactalbumin was prepared from rabbits, and single radial immunodiffusion was used to measure the concentration of alpha-lactalbumin in milk expressed from rats during lactation and for 2 days after the cessation of lactation. A significant positive correlation (r = + 0.89) between the concentrations of alpha-lactalbumin and lactose was obtained for the first 20 days of lactation. This is consistent with the suggestion that alpha-lactalbumin may control the concentration of lactose in milk. However, a significant negative correlation (r = -0.91) between the concentration of alpha-lactalbumin and lactose was obtained for 2 days after the cessation of lactation on day 20.

scholarly journals Isolation, partial purification and characterization of phospholipase A2 from Naja Katiensis venom

2021 ◽  
Vol 13 (2) ◽  
pp. 107-112
Author(s):  
C.F. Okechukwu ◽  
P.L. Shamsudeen ◽  
R.K. Bala ◽  
B.G. Kurfi ◽  
A.M. Abdulazeez
Keyword(s):  
Ion Exchange ◽  
Phospholipase A2 ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Partial Purification ◽  
Crude Venom

The most effective and acceptable therapy for snakebite victims is the immediate administration of antivenin which is limited by problems of hypersensitivity reactions in some individuals and its inability to resolve the local effects of the venom. The aim of this study was to isolate, partially purify and characterize phospholipase A2 from Naja Katiensis venom. Phospholipase A2 was partially purified via a two-step process: gel filtration on Sephadex G-75 and ion exchange chromatography using CM Sephadex, and subjected to SDS-PAGE analysis. From the results, the specific activity of the partially purified PLA2 decreased from 0.67μmol/min/mg in crude venom to 0.29μmol/min/mg after ion exchange chromatography with a yield of 5% and purification fold of 0.43. The optimum temperature of the purified PLA2 was found to be 35ºC and optimum p.H of 7. velocity studies for the determination of kinetic constants using L-a-lecithin as substrate revealed a Km  of 1.47mg/ml and Vmax  of 3.32μ moles/min/mg. The sodium dodecyl sulphate polyacrylamide gel electrophoresis of the purified PLA2 showed a distinct band with molecular weight estimated to be 14KDa. In conclusion, the present study shows that phospholipase A2 was isolated, purified and characterized. This may serve as a promising candidate for future development of a novel anti-venin drug.

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

Charge Properties of Mitochondrial Matrix Proteins

1975 ◽  
Vol 53 (11) ◽  
pp. 1150-1157 ◽  
Author(s):  
P. M. Strasberg ◽  
K. B. Freeman
Keyword(s):  
Gel Electrophoresis ◽  
Matrix Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Matrix Proteins ◽  
Mitochondrial Matrix ◽  
Total Fraction ◽  
Acidic Nature

Proteins of the rat liver mitochondrial matrix have been separated into anionic (acidic), cationic (basic), and neutral groups by electrophoresis. These groups represent 69, 8, and 23% of the total matrix protein, respectively, compared to 69, 21, and 10% for the cytosol protein. The acidic nature of the mitochondrial matrix proteins has been confirmed by cellulose ion-exchange chromatography, isoelectric focusing in sucrose gradients, and amino acid analysis. The anionic, cationic, and neutral matrix proteins were separated by sodium dodecyl sulfate – polyacrylamide gel electrophoresis into 18, 6, and 5 bands, respectively, compared to 22 bands for the total fraction. The significance of the charge properties of these proteins in terms of mitochondrial biogenesis is discussed.

scholarly journals Dephosphorylation of Phytate by Using theAspergillus niger Phytase with a High Affinity for Phytate

1999 ◽  
Vol 65 (10) ◽  
pp. 4682-4684 ◽  
Author(s):  
Tadashi Nagashima ◽  
Tatsuya Tange ◽  
Hideharu Anazawa
Keyword(s):  
Phytic Acid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Aspergillus Ficuum ◽  
Michaelis Constant ◽  
High Affinity ◽  
Significant Difference

ABSTRACT A phytase (EC 3.1.3.8 ) with a high affinity for phytic acid was found in Aspergillus niger SK-57 and purified to homogeneity in four steps by using ion-exchange chromatography (two types), gel filtration, and chromatofocusing. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme gave a single stained band at a molecular mass of approximately 60 kDa. The Michaelis constant of the enzyme for phytic acid (18.7 ± 4.6 μM) was statistically analyzed. In regard to the orthophosphate released from phytic acid, a significant difference between a lowKm phytase from A. niger SK-57 and a high Km phytase from Aspergillus ficuum was recognized.

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.

scholarly journals Purification of plasminogen activators from human seminal plasma

1978 ◽  
Vol 171 (2) ◽  
pp. 435-444 ◽  
Author(s):  
D Propping ◽  
L J D Zaneveld ◽  
P F Tauber ◽  
G F B Schumacher
Keyword(s):  
Seminal Plasma ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Plasminogen Activators ◽  
Exchange Chromatography ◽  
Human Seminal Plasma ◽  
Molecular Weights ◽  
Wide Ph Range

Two plasminogen activators (1 and 2) were isolated from human seminal plasma by hiigh-speed centrifugation, Sephadex-gel filtration and ion-exchange chromatography. The activators were shown to be homogeneous by polyacrylamide-disc -gel electrophoresis at pH 8.3 and 4.5, and by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The molecular weights of activators 1 and 2 were estimated as 69 000 and 74 000. Their amino acid compositions are very similar, both being high in aspartic acid, glutamic acid, serine, glycine and leucine, and low in methionine, tryptophan, tyrosine, isoleucine and histidine. Activators 1 and 2 each possess 16 cysteine residues. Both activators have isoelectric points of approx. 7.0, are stable over a wide pH range at temperatures up to 60 degrees C, but lose activity at higher temperatures, particularly under very basic or acidic conditions. They are not inhibited by EDTA, Mg2+ and Ca2+ at 10 mM concentrations, but their activity decreases on addition of 10 mM-cysteine or Fe2+ and 6-aminohexanoate or sera from pregnant women. The precipitin band formed between urokinase and its antiserum is continuous with the precipitin bands formed between the seminal plasminogen activators and the urokinase antiserum. Antisera to urokinase inhibit both the activity of urokinase and the seminal plasminogen activators.

Biosynthèse "In Vitro" de l'Hormone Béta-Lipotropique de Boeuf

1974 ◽  
Vol 52 (5) ◽  
pp. 349-358 ◽  
Author(s):  
X. Bertagna ◽  
M. Lis ◽  
C. Gilardeau ◽  
M. Chrétien
Keyword(s):  
Amino Acids ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Usual Method ◽  
Pituitary Hormone ◽  
Melanocyte Stimulating Hormone

Sheep beta-lipotropic hormone (β-LPH) is a pituitary hormone made of 90 amino acids and having a portion of its sequence (41–58) identical with the structure of beta-melanocyte-stimulating hormone (β-MSH). We hypothetized that β-LPH could be the biological precursor of β-MSH. We studied the biosynthesis of these two molecules by monitoring the incorporation of radioactive amino acids in beef pituitary slices. We separated β-LPH from the other radioactive proteins with the usual method of purification described previously and we characterized the proteins by ion-exchange chromatography, gel filtration, and polyacrylamide gel electrophoresis. Our results show that the pituitary slices synthesized a radioactive β-LPH which has all the characteristics of non-radioactive β-LPH. However, in the conditions used, we could not demonstrate any biosynthesis of β-MSH after 4 h incubation. These results suggest that the conversion of β-LPH into β-MSH, if it exists, is a slow process and should be studied in more prolonged incubations.

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