scholarly journals Purification and some characteristics of an oestrogen sulphotransferase from guinea pig adrenal gland and its non-identity with adrenal pregnenolone sulphotransferase

1990 ◽  
Vol 268 (3) ◽  
pp. 759-764 ◽  
Author(s):  
R Hobkirk ◽  
M A Glasier ◽  
L Y Brown
Keyword(s):  
Adrenal Glands ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
High Specific Activity ◽  
Protein Band ◽  
Thiol Groups ◽  
Reducing Conditions ◽  
Main Protein ◽  
Salt Fractionation

An oestrogen sulphotransferase, active towards both oestrone and oestradiol, and of high specific activity, is present in cytosol prepared from adrenal glands of both sexes of English Shorthair and Hartley guinea pigs. The ovarian and testicular cytosolic activities of this enzyme are markedly low in comparison with the adrenal activity. The adrenal enzyme is distinct from an accompanying pregnenolone sulphotransferase as judged by f.p.l.c. gel filtration, chromatofocusing, and differences in activation brought about by the addition of thiol groups. The oestrogen sulphotransferase behaved as a 67 kDa protein on a Sephadex G100 column and as a 48 kDa protein on f.p.l.c. gel-filtration columns. Two forms of the enzyme with apparent pI values of 6.1 and 5.5 were eluted during f.p.l.c. chromatofocusing. Sequential salt fractionation, f.p.l.c. gel filtration and elution from an agarose-hexane-adenosine-3′,5′-diphosphate affinity gel has resulted in a preparation which, when resubmitted to f.p.l.c. gel filtration, yields a considerably purified oestrogen sulphotransferase. When submitted to SDS/polyacrylamide-gel electrophoresis under reducing conditions, a main protein band of 34-36 kDa is observed. It is suggested that the enzyme may exist as a dimer in the cytosol.

scholarly journals Purification of the enzyme NADPH: protochlorophyllide oxidoreductase

1981 ◽  
Vol 195 (1) ◽  
pp. 83-92 ◽  
Author(s):  
N S Beer ◽  
W T Griffiths
Keyword(s):  
Specific Activity ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
High Specific Activity ◽  
Sucrose Density Gradient ◽  
Sucrose Density Gradient Centrifugation ◽  
Triton X ◽  
Etioplast Membranes

A procedure for the purification of the enzyme NADPH:protochlorophyllide oxidoreductase is described. This involves fractionation of sonicated oat etioplast membranes by discontinuous-sucrose-density-gradient centrifugation, which gives membranes in which the enzyme is present at a high specific activity. The enzyme is solubilized from the membranes with Triton X-100, followed by gel filtration of the extract; enzyme activity is eluted in fractions corresponding to a mol.wt of approx. 35000. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of the enzyme-containing fractions from gel filtration shows two peptides, of mol.wts. approx. 35000 and 37000.

Purification and Properties of a Neutral Protease from Soil Bacterium WM 122

1977 ◽  
Vol 37 (03) ◽  
pp. 556-565 ◽  
Author(s):  
S. E Papaioannou ◽  
W. J Marsheck
Keyword(s):  
Methyl Ester ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
High Specific Activity ◽  
Soil Bacterium ◽  
Ester Hydrochloride ◽  
Apparent Homogeneity ◽  
Purification And Properties ◽  
Hydrolysis Of

SummaryAn extracellular protease SN 687, secreted by the soil bacterium isolate WM 122, has been purified by means of gel filtration, ammonium sulfate precipitation, DEAE-Sephadex and hydroxylapatite chromatography. Apparent homogeneity was ascertained by Polyacrylamide gel electrophoresis. The protease was inactivated by ethylenediamine tetracetic acid (EDTA) but not by diisopropylfluorophosphate (DFP), and it was partially inhibited by serum inhibitors. SN 687 was shown to be of high specific activity against casein and fibrin, but it did not hydrolyze L- lysine -methyl ester dihydrochloride (LME), p-tosyl-L-arginine-methyl ester hydrochloride (TAME) and N-benzoyl-L-tyrosine-ethyl ester hydrochloride (BTEE) synthetic substrates. The optimum pH for hydrolysis of casein was 7.5 and the molecular weight, as determined by gel filtration, was 31,000.

COMPARISON OF DIFFERENT CELL DISRUPTION METHODS AND CELL EXTRACTANT BUFFERS FOR RECOMBINANT BROMELAIN EXPRESSED IN E.COLI BL21-A1

2015 ◽  
Vol 77 (24) ◽  
Author(s):  
Zatul Iffah Mohd Arshad ◽  
Azura Amid ◽  
Muhd. Ezza Faiez Othman
Keyword(s):  
Phosphate Buffer ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
High Specific Activity ◽  
Protein Band ◽  
E Coli ◽  
Sodium Phosphate Buffer ◽  
Pre Treatment

The often-encountered problem such as protein degradation has driven various methods of cell lysis in obtaining recombinant protein post fermentation. In this paper, we compare methods such as homogenization, sonication, sonication with lysozyme and chemical lysis using B-PER reagent with lysozyme to extract the recombinant bromelain from E. coli BL21-AI. The sonication process is found to be the most effective in releasing recombinant bromelain without any pre-treatment. To obtain the high quality of protein from sonication method, the influence of different extractant buffer was investigated including phosphate buffer saline (PBS), PBS containing cysteine and EDTA (PBS-CE), and sodium phosphate buffer containing cysteine and EDTA (EB-CE). The highest specific enzyme activity was obtained when it was extracted with EB-CE buffer. Under sodium dodecyl sulfate polyacrylamide gel electrophoresis, the recombinant bromelain showed protein band at 55kDa. In conclusion, the sonication method with extractant buffer containing 100mM phosphate buffer pH7.0 with 15 mM cysteine and 2 mM EDTA (EB-CE) was shown to give high specific activity of recombinant bromelain.

scholarly journals Purification, characterization and application of novel alkaline protease from new Bacillus cereus UV-15 mutant

2017 ◽  
Vol 7 (4) ◽  
pp. 1 ◽  
Author(s):  
Sreedevi Basavaraju ◽  
Chandrasekhar Kathera ◽  
Pramoda Kumari Jasti
Keyword(s):  
Bacillus Cereus ◽  
Ammonium Sulphate ◽  
Alkaline Protease ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Industrial Applications ◽  
Tween 20 ◽  
Original Activity ◽  
Protease Enzyme

The alkaline protease produced by Bacillus cereus UV-15 mutant was purified by precipitation with ammonium sulphate and gel filtration through sephadex G-100. The enzyme has shown to have a molecular weight of 29kDa by SDS polyacrylamide gel electrophoresis. The extracted protease enzyme was purified by 16.64 fold through ammonium sulphate precipitation and chromatography separation in Sephadex G-100. The purified protease had a specific activity of 2915 (U/mg). The zymogram also revealed a clear hydrolytic zone due to proteolytic activity, which coincided with the band obtained with SDS–PAGE. The enzyme was remained active and stable at pH 8-11, with an optimum at pH 10.0. The protease was stable in the temperature ranging from 40°C to 60°C, but gradually decreased at temperature 70°C. The optimum temperature for protease activity was determined at 60°C. The enzyme showed stability towards non-ionic and anionic surfactants, and oxidizing agents. At 1% concentration of Tween-20 and Tween-80, the enzyme retained 78% and 94% relative activity respectively. Alkaline protease retained 95% activity toward 0.5% concentration of the anionic detergent SDS. The enzyme showed compatibility at 50°C with commercial detergents such as Ariel, Surf excel, Rin, wheel, Tide and Nirma. In the presence of Ariel and Rin the enzyme retained about 72 and 75% of the original activity respectively. The supplementation of the enzyme in detergents could improve the cleansing performance towards the blood stains and suggested to be used as a detergent additive. The enzyme also removed goat hide hairs completely after 15 hr of incubation. These characteristics may make the enzyme suitable for several industrial applications, especially in leather industries.

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 Molecular Characterization of a Recombinant Manganese Superoxide Dismutase fromLactococcus lactisM4

2014 ◽  
Vol 2014 ◽  
pp. 1-9
Author(s):  
Boon Hooi Tan ◽  
Thean Chor Leow ◽  
Hooi Ling Foo ◽  
Raha Abdul Rahim
Keyword(s):  
Superoxide Dismutase ◽  
Active Sites ◽  
Manganese Superoxide Dismutase ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Maximal Activity ◽  
Gel Filtration Chromatography ◽  
T7 Promoter

A superoxide dismutase (SOD) gene ofLactococcus lactisM4 was cloned and expressed in a prokaryotic system. Sequence analysis revealed an open reading frame of 621 bp which codes for 206 amino acid residues. Expression ofsodAunder T7 promoter exhibited a specific activity of 4967 U/mg when induced with 1 mM of isopropyl-β-D-thiogalactopyranoside. The recombinant SOD was purified to homogeneity by immobilised metal affinity chromatography and Superose 12 gel filtration chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blot analyses of the recombinant SOD detected a molecular mass of approximately 27 kDa. However, the SOD was in dimer form as revealed by gel filtration chromatography. The purified recombinant enzyme had a pI of 4.5 and exhibited maximal activity at 25°C and pH 7.2. It was stable up to 45°C. The insensitivity of this lactococcal SOD to cyanide and hydrogen peroxide established that it was a MnSOD. Although it has 98% homology to SOD ofL. lactisIL1403, this is the first elucidated structure of lactococcal SOD revealing active sites containing the catalytic manganese coordinated by four ligands (H-27, H-82, D-168, and H-172).

scholarly journals Purification, characterization and inhibition of human skin collagenase

1978 ◽  
Vol 169 (2) ◽  
pp. 265-276 ◽  
Author(s):  
David E. Woolley ◽  
Robert W. Glanville ◽  
Dennis R. Roberts ◽  
John M. Evanson
Keyword(s):  
Human Skin ◽  
Culture Medium ◽  
Serum Proteins ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Polyacrylamide Gel ◽  
Collagen Fibrils ◽  
Ph Optimum

1. The neutral collagenase released into the culture medium by explants of human skin tissue was purified by ultrafiltration and column chromatography. The final enzyme preparation had a specific activity against thermally reconstituted collagen fibrils of 32μg of collagen degraded/min per mg of enzyme protein, representing a 266-fold increase over that of the culture medium. Electrophoresis in polyacrylamide disc gels showed it to migrate as a single protein band from which enzyme activity could be eluted. Chromatographic and polyacrylamide-gel-elution experiments provided no evidence for the existence of more than one active collagenase. 2. The molecular weight of the enzyme estimated from gel filtration and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis was approx. 60000. The purified collagenase, having a pH optimum of 7.5–8.5, did not hydrolyse the synthetic collagen peptide 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-d-Arg-OH and had no non-specific proteinase activity when examined against non-collagenous proteins. 3. It attacked undenatured collagen in solution at 25°C, producing the two characteristic products TCA(¾) and TCB(¼). Collagen types I, II and III were all cleaved in a similar manner by the enzyme at 25°C, but under similar conditions basement-membrane collagen appeared not to be susceptible to collagenase attack. At 37°C the enzyme attacked gelatin, producing initially three-quarter and one-quarter fragments of the α-chains, which were degraded further at a lower rate. As judged by the release of soluble hydroxyproline peptides and electron microscopy, the purified enzyme degraded insoluble collagen derived from human skin at 37°C, but at a rate much lower than that for reconstituted collagen fibrils. 4. Inhibition of the skin collagenase was obtained with EDTA, 1,10-phenanthroline, cysteine, dithiothreitol and sodium aurothiomaleate. Cartilage proteoglycans did not inhibit the enzyme. The serum proteins α2-macroglobulin and β1-anti-collagenase both inhibited the enzyme, but α1-anti-trypsin did not. 5. The physicochemical and enzymic properties of the skin enzyme are discussed in relation to those of other human collagenases.

Partial characterization of a novel oestrogen-induced protein in the rat adenohypophysis

1993 ◽  
Vol 10 (3) ◽  
pp. 345-357
Author(s):  
X Casabiell ◽  
J L Zugaza ◽  
C M Pombo ◽  
L Bokser ◽  
N Mulet ◽  
...  
Keyword(s):  
Incubation Medium ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Limited Proteolysis ◽  
Electrophoretic Pattern ◽  
High Specific Activity ◽  
Tumour Cell Line ◽  
Pituitary Cells ◽  
Tissue Extracts

ABSTRACT In order to detect putative markers of prolactin-secreting pituitary tumours, adult rats were subjected to long-term oestrogenization with oestradiol benzoate (OE2) on a monthly basis. At 6 months, anterior pituitaries were dissected and incubated either as tissue fragments or as dispersed cells with a S]methionine mix for labelling. Proteins released into the incubation medium and from tissue extracts were further analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and fluorography. Oestrogen induced the appearance in the incubation medium of a protein (OE2 band) with an Mr of 38 000 under reducing conditions, and high specific activity. Surprisingly, such a protein was not detected in tissue extracts. The OE2 band was detectable by 7 days after the first dose of oestrogen, and remained throughout 1 year of treatment. The tumour cell line GH3 showed a similar OE2 band which was further enhanced by oestrogens. The protein was observed similarly in both female and male pituitary donors, either intact or gonadectomized, and also in rats of different strains, suggesting that its appearance was independent of the strain of rat and gonadal status. Furthermore, the OE2 band was specific for pituitary cells and not produced by other oestrogenized tissues. No alteration in the rate of generation or the electrophoretic pattern of the OE2 band was observed when pituitary cells from oestrogenized rats were metabolically labelled while being incubated with tunicamycin. Furthermore, a system for glycan detection, adsorption to Concanavalin A or incubation with endoglycosidase F also failed to show a clear amount of glycosylation of the oestrogen-induced protein. Both immunoprecipitation experiments and time-limited proteolysis with V8 protease ruled out the possibility that the OE2 band could be structurally related to either GH or prolactin. In conclusion, oestrogens induce the generation of a new monocatenary protein with an apparent Mr of 38 000, which has at least one intramolecular disulphide loop and is not glycosylated. The OE2 band was detected only in incubation medium and never in tissue extracts.

Purification and some properties of the hemolytic toxin aerolysin

1981 ◽  
Vol 59 (6) ◽  
pp. 430-435 ◽  
Author(s):  
J. Thomas Buckley ◽  
L. Nicole Halasa ◽  
Karen D. Lund ◽  
Sheila MacIntyre
Keyword(s):  
Aeromonas Hydrophila ◽  
Culture Supernatant ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Active Components ◽  
Original Culture ◽  
Hemolytic Action ◽  
Hemolytic Toxin ◽  
Salt Fractionation

Aerolysin, the hemolytic toxin produced by Aeromonas hydrophila, has been purified by a combination of salt fractionation, gel filtration, and ion-exchange and hydroxyapatite chromatography. The resulting protein has a molecular weight of 51 500 and appears homogeneous by polyacrylamide gel electrophoresis in sodium dodecyl sulphate. It is free of detectable protease and phospholipase activities. The purified protein can be separated into two active components with pIs of 5.39 and 5.46 by isoelectric focusing. Both components are found in the original culture supernatant indicating that the multiplicity is not due to proteolysis during isolation. Purified aeroiysin is unstable even at 25 °C and its hemolytic action is inhibited by certain reducing agents including ferrous iron and cysteine. It appears to be the only toxin hemolytic to human cells that is produced by A. hydrophila under the conditions described.

scholarly journals Studies on the subunits of human myeloperoxidase

1984 ◽  
Vol 222 (3) ◽  
pp. 701-709 ◽  
Author(s):  
R L Olsen ◽  
C Little
Keyword(s):  
Heat Treatment ◽  
Amino Acid ◽  
Molecular Mass ◽  
Amino Acid Composition ◽  
Acid Composition ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Additional Species ◽  
Main Protein

The subunit composition of human myeloperoxidase was studied with the use of sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and gel filtration. The subunit pattern observed depended on the manner in which the enzyme was treated before analysis. Reduction before heat treatment in detergent led to two main protein species (Mr 57 000 and 10 500), whereas reduction during or after heat treatment yielded an additional species of Mr 39 000. Heating without any reductive pretreatment yielded the 39 000-Mr form as the major electrophoretic species. Carbohydrate staining showed large amounts of sugar on the 57 000-Mr species and little on the 10 500-Mr form. Significant amounts of haem were associated with this latter subunit. Haem also seemed to be associated with the 57 000-Mr form but not with the 39 000-Mr one. These three subunit forms were isolated and their amino acid composition analysed. The 57 000-Mr and 39 000-Mr forms had very similar amino acid composition and yielded an apparently identical collection of fragments on incubation with CNBr. Once separated, the subunits could not be interconverted. Generally, minor amounts of other molecular-mass forms were observed. The nature of the various molecular-mass forms originating from myeloperoxidase is discussed.

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