Purification of urease from Ureaplasma urealyticum

1987 ◽  
Vol 33 (10) ◽  
pp. 857-862 ◽  
Author(s):  
G. W. Stemke ◽  
J. A. Robertson ◽  
M. Nhan
Keyword(s):  
High Performance ◽  
Sodium Phosphate ◽  
Specific Activity ◽  
Ureaplasma Urealyticum ◽  
Major Band ◽  
Jack Bean ◽  
Sodium Phosphate Buffer ◽  
Sds Page ◽  
Lower Molecular Mass ◽  
A Minor

We have purified urease from the Mollicutes, Ureaplasma urealyticum, using high performance liquid chromatography methods and DEAE-Sephadex chromatography. While only small amounts of material could be utilized in these methods, urease was purified at least 180-fold, to yield a major band on SDS–PAGE of 660 00 daltons, a minor band of 64 000 daltons, and several faint bands of lower molecular mass. These results suggest that the 380 000 dalton intact urease is a pentamer or hexamer of these two larger subunits. The highly purified urease from DEAE-Sephadex retained full activity for at least 20 days at 4 °C in sodium phosphate buffer (pH 7.2) with 1% bovine serum albumin. The estimated specific activity of the DEAE peak fractions, 180 IU/μg, is at least 90-fold greater than that of jack bean urease.

scholarly journals Purification and Characterization of Xanthine Oxidase from Liver of the Sheep (Ovis Aries)

2019 ◽  
Vol 1 (4) ◽  
pp. 8-18
Author(s):  
Samir A.M. Zaahkouk ◽  
Doaa A. Darwish ◽  
Hassan M.M. Masoud ◽  
Mohamed M. Abdel-Monsef ◽  
Mohamed S. Helmy ◽  
...  
Keyword(s):  
Xanthine Oxidase ◽  
Specific Activity ◽  
Deae Cellulose ◽  
Ovis Aries ◽  
Major Band ◽  
Sheep Liver ◽  
Sds Page ◽  
Commercially Important ◽  
Important Enzyme

Xanthine oxidase is a commercially important enzyme with wide area of medical applications to develop diagnostic kits. Xanthine oxidase was extracted, purified and characterized from sheep liver (SLXO). The purification procedure involved acetone precipitation and chromatography on DEAE-cellulose and Sephacryl S-300 columns. The sheep liver xanthine oxidase was homogeneously purified 31.8 folds with 3.5 U/mg specific activity and 24.1% recovery. SLXO native molecular weight was 150 kDa and on SDS-PAGE appeared as single major band of 75 kDa representing a homodimer protein. Isoelectric focusing of the purified SLXO resolved into two closely related isoforms with pI values of 5.6 and 5.8. The apparent Km for xanthine oxidase at optimum pH 7.6 was found to be 0.9 mM xanthine. FeCl2 and NiCl2 increased the activity of SLXO, while CuCl2 and ZnCl2 were found to be potent inhibitors of the purified enzyme. Allopurinol inhibits SLXO competitively with one binding site on the purified molecule and Ki value of 0.06 mM.

scholarly journals Comparative Evaluation of Protease Extraction from Leaves of Syzygium polyanthum

2019 ◽  
Vol 19 (2) ◽  
pp. 503 ◽  
Author(s):  
Noorhafiza Yahya ◽  
Zatul Iffah Mohd Arshad ◽  
Siti Kholijah Abdul Mudalip ◽  
Siti Zubaidah Sulaiman ◽  
Rohaida Che Man ◽  
...  
Keyword(s):  
Hydrogen Bond ◽  
Phosphate Buffer ◽  
Comparative Evaluation ◽  
Extraction Method ◽  
Sodium Phosphate ◽  
Specific Activity ◽  
Extraction Methods ◽  
Hydrogen Bond Acceptor ◽  
Sodium Phosphate Buffer

In this study, the comparison of four extraction methods for protease from the leaves of Syzygium polyanthum was evaluated. The methods include sodium phosphate buffer, 1-methyl-8-oxyquinolinium betaine (QB) solvent based extraction, hydrogen-bond acceptor (HBA) solvent based extraction and Trichloroacetic-acetone (TCA) precipitation. The effectiveness of these extraction methods was measured based on the specific activity demonstrated by the crude protease extracted in this experiment. The results indicated that QB solvent-based extraction method could extract the crude protease from the leaves of S. polyanthum with the highest specific activity of 10.204 ± 0.329 U/mg compared to other extraction methods.

scholarly journals Purification and properties of neutral maltase from human granulocytes

1989 ◽  
Vol 263 (3) ◽  
pp. 647-652 ◽  
Author(s):  
P Delqué Bayer ◽  
C Vittori ◽  
P Sudaka ◽  
J Giudicelli
Keyword(s):  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Human Kidney ◽  
Ph Optimum ◽  
Major Band ◽  
Purification And Properties ◽  
A Minor ◽  
Triton X ◽  
Human Polymorphonuclear Leukocytes

A procedure for the purification of neutral maltase from human polymorphonuclear leukocytes is described, involving solubilization with Triton X-100, proteolytic attack and three chromatographic steps: DEAE ion exchange, AcA 22 gel filtration and a second DEAE chromatography. The enzyme was obtained with a final specific activity of 30 units/mg of protein, comparable with that of other neutral maltases previously purified. The Mr of the enzyme was 550,000 as determined by gel filtration. SDS/polyacrylamide-gel electrophoresis, under non-denaturing conditions, led to a major band of 500,000 and a minor one of 260,000, both active, suggesting a polymeric or aggregated form of the protein. The catalytic properties of the human granulocytic neutral maltase were investigated. The pH optimum was around 6. The enzyme exhibited a broad range of substrate specificity, hydrolysing di- and oligosaccharides with alpha (1→2), alpha (1→3) and alpha (1→4) glucosidic linkages. The highest activities were observed for alpha (1→4) glucose oligomers of three to five residues. It was also found to hydrolyse polysaccharides such as starch and glycogen. The results of the inhibition studies are interpreted in terms of the existence of a large site including several subsites. The enzyme properties are broadly similar to those observed for other purified neutral alpha-glucosidases, in particular that of human kidney origin.

scholarly journals Purification and Characterization of protease from Zahdi dates plam seeds (Phoenix dactylifera L.)

2009 ◽  
Vol 6 (4) ◽  
pp. 633-639
Author(s):  
Baghdad Science Journal
Keyword(s):  
Phosphate Buffer ◽  
Sodium Phosphate ◽  
Specific Activity ◽  
Enzyme Stability ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Phoenix Dactylifera ◽  
Sodium Phosphate Buffer ◽  
Phoenix Dactylifera L ◽  
Enzyme Specific Activity

Proteinases (E.C.3.4.21) family are widely distributed in the nature; it was present in animals tissues , plants and microbial cell . Protease was purified from Zahdi seed (Phoenix dactylifera L.) by several steps included ammonium sulphite ppt (75%) saturation and dialyzed against the 80mM sodium phosphate buffer at pH 7.5 . The enzyme specific activity was 407.62 unit/mg protein. The obtained extract was purified by DEAE-Cellulose column followed by gel filtration through Sephacyl S-200 column .The enzyme specific activity ,yield and purification fold were 1873.49 unit/mg protein, 22.99 and 58.42% respectively. The results of protease characterization showed that the molecular weight was 25118 daltons as determined by gel filtration. The optimum temperature of the enzyme activity 35 C for 15 minutes and that for stability was 45 C for 15 minutes, using sodium phosphate buffer at pH 7.5, The optimum pH for the enzyme stability and activity were 8.5 for 15 minute and 7.5 respectively.

scholarly journals Preparation of the lactate oxidase apoenzyme and studies on the binding of flavin mononucleotide to the apoenzyme

1975 ◽  
Vol 145 (1) ◽  
pp. 37-45 ◽  
Author(s):  
Y S Choong ◽  
M G Shepherd ◽  
P A Sullivan
Keyword(s):  
Mycobacterium Smegmatis ◽  
Gel Electrophoresis ◽  
Sodium Phosphate ◽  
Specific Activity ◽  
Native Enzyme ◽  
Flavin Mononucleotide ◽  
Lactate Oxidase ◽  
Extinction Coefficients ◽  
Sodium Phosphate Buffer ◽  
Rapid Binding

1. Lactate oxidase from Mycobacterium smegmatis is completely resolved into free flavin and apoenzyme by treatment with acid (NH4)2SO4. 2. Reconstitution involves rapid binding of FMN, but the recovery of enzyme activity was slower and appeared to be biphasic. 3. The preparation of the holoenzyme obtained differs from the native enzyme in specific activity, extinction coefficients and mobility on disc-gel electrophoresis. 4. Dialysis of this reconstituted enzyme in 0.1 M-sodium phosphate buffer, pH 7.0, at 0 degrees C for 1 week yields a preparation which closely resembles the native enzyme.

scholarly journals Purification and Characterization of Tannin Acyl Hydrolase Produced by Mixed Solid State Fermentation of Wheat Bran and Marigold Flower byPenicillium notatumNCIM 923

2013 ◽  
Vol 2013 ◽  
pp. 1-6 ◽  
Author(s):  
Saswati Gayen ◽  
Uma Ghosh
Keyword(s):  
Solid State ◽  
Molecular Mass ◽  
Wheat Bran ◽  
Solid State Fermentation ◽  
Specific Activity ◽  
Major Band ◽  
Sds Page ◽  
Diethylaminoethyl Cellulose ◽  
Wide Range ◽  
Ammonium Sulphate Fractionation

Tannin acyl hydrolase produced extracellularly by the fungal strainPenicillium notatumNCIM 923 in mixed solid state fermentation of wheat bran and marigold flower in the ratio 4 : 1 was purified from the cell-free extract broth by ammonium sulphate fractionation followed by diethylaminoethyl-cellulose column chromatography. Tannase was purified by 19.89-fold with yield of 11.77%. The specific activity of crude tannase was found to be 1.31 U/mg protein while that of purified tannase was 22.48 U/mg protein. SDS-PAGE analysis indicated that the enzyme is dimeric with one major band of molecular mass 97 kDa and a very light band of molecular mass 43 kDa. Temperature of 35 to 40°C and pH 5 were optimum for tannase activity. The enzyme retained more than 60% of its stability at 60°C and 40% stability at pH 3 and 8, respectively.Kmwas found to be0.33×10-2 M andVmax=40 U/mg. Since the enzyme is active over a wide range of pH and temperature, it could find potential use in the food processing industry.

Purification and some properties of proteinase fromPseudomonas fluorescensNo. 33

1993 ◽  
Vol 60 (2) ◽  
pp. 229-237 ◽  
Author(s):  
Haruto Kumura ◽  
Katsuhiko Mikawa ◽  
Zenichi Saito
Keyword(s):  
Molecular Mass ◽  
Phosphate Buffer ◽  
Sodium Phosphate ◽  
Extracellular Proteinase ◽  
Original Activity ◽  
Sodium Phosphate Buffer ◽  
Sds Page ◽  
Optimum Ph ◽  
Hydrolysis Of ◽  
Optimum Ph And Temperature

SummaryThe extracellular proteinase fromPseudomonas fluorescensNo. 33 was purified to electrophoretic homogeneity by a procedure including precipitation with HC1 and (NH4)2SO4, and column chromatography. The enzyme was purified 170-fold giving a yield of 7 % of the original activity. The molecular mass of the purified enzyme was 48000 by SDS-PAGE. The optimum pH and temperature for the hydrolysis of casein were 8·0–9·8 and 30–35 °C respectively. The enzyme was more thermostable in synthetic milk salts solution than in 0·1 M-sodium phosphate buffer, but was heat-labile at 50 °C in both buffer Systems. The activity was inhibited byo−phenanthroline, Hg2+, Cu2+, Fe2+and, to a lesser extent, Ni2+. Caseins were susceptible to the proteinase, but degradation patterns were dependent on the form of the casein.

Fluorocitrate Neural Dystrophy of the Guinea Pig Cochlea

1975 ◽  
Vol 33 ◽  
pp. 368-369
Author(s):  
G.J. Spector ◽  
C.D. Carr ◽  
I. Kaufman Arenberg ◽  
R.H. Maisel
Keyword(s):  
Hearing Loss ◽  
Hair Cells ◽  
Sodium Phosphate ◽  
Sensory Cells ◽  
Barium Salt ◽  
Perfusion Medium ◽  
Cochlear Hair Cells ◽  
Neural Degeneration ◽  
Sodium Phosphate Buffer ◽  
Cochlear Neurons

All studies on primary neural degeneration in the cochlea have evaluated the end stages of degeneration or the indiscriminate destruction of both sensory cells and cochlear neurons. We have developed a model which selectively simulates the dystrophic changes denoting cochlear neural degeneration while sparing the cochlear hair cells. Such a model can be used to define more precisely the mechanism of presbycusis or the hearing loss in aging man.Twenty-two pigmented guinea pigs (200-250 gm) were perfused by the perilymphatic route as live preparations using fluorocitrate in various concentrations (15-250 ug/cc) and at different incubation times (5-150 minutes). The barium salt of DL fluorocitrate, (C6H4O7F)2Ba3, was reacted with 1.0N sulfuric acid to precipitate the barium as a sulfate. The perfusion medium was prepared, just prior to use, as follows: sodium phosphate buffer 0.2M, pH 7.4 = 9cc; fluorocitrate = 15-200 mg/cc; and sucrose = 0.2M.

Parallel Reduction of Plasma Levels of High and Low Molecular Weight Kininogen in Patients with Cirrhosis

1999 ◽  
Vol 82 (11) ◽  
pp. 1428-1432 ◽  
Author(s):  
Cheryl Scott ◽  
Francesco Salerno ◽  
Elettra Lorenzano ◽  
Werner Müller-Esterl ◽  
Angelo Agostoni ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Liver Disease ◽  
Liver Function ◽  
Plasma Levels ◽  
Plasma Concentrations ◽  
Normal Subjects ◽  
Low Molecular Weight ◽  
Major Band ◽  
Sds Page ◽  
Normal Controls

SummaryLittle is known about the regulation of high-molecular-weight-kininogen (HK) and low-molecular-weight-kininogen (LK) or the relationship of each to the degree of liver function impairment in patients with cirrhosis. In this study, we evaluated HK and LK quantitatively by a recently described particle concentration fluorescence immunoassay (PCFIA) and qualitatively by SDS PAGE and immunoblotting analyses in plasma from 33 patients with cirrhosis presenting various degrees of impairment of liver function. Thirty-three healthy subjects served as normal controls. Patients with cirrhosis had significantly lower plasma levels of HK (median 49 μg/ml [range 22-99 μg/ml]) and LK (58 μg/ml [15-100 μg/ml]) than normal subjects (HK 83 μg/ml [65-115 μg/ml]; LK 80 μg/ml [45-120 μg/ml]) (p < 0.0001). The plasma concentrations of HK and LK were directly related to plasma levels of cholinesterase (P < 0.0001) and albumin (P < 0.0001 and P < 0.001) and inversely to the Child-Pugh score (P < 0.0001) and to prothrombin time ratio (P < 0.0001) (reflecting the clinical and laboratory abnormalities in liver disease). Similar to normal individuals, in patients with cirrhosis, plasma HK and LK levels paralleled one another, suggesting that a coordinate regulation of those proteins persists in liver disease. SDS PAGE and immunoblotting analyses of kininogens in cirrhotic plasma showed a pattern similar to that observed in normal controls for LK (a single band at 66 kDa) with some lower molecular weight forms noted in cirrhotic plasma. A slight increase of cleavage of HK (a major band at 130 kDa and a faint but increased band at 107 kDa) was evident. The increased cleavage of HK was confirmed by the lower cleaved kininogen index (CKI), as compared to normal controls. These data suggest a defect in hepatic synthesis as well as increased destructive cleavage of both kininogens in plasma from patients with cirrhosis. The decrease of important regulatory proteins like kininogens may contribute to the imbalance in coagulation and fibrinolytic systems, which frequently occurs in cirrhotic patients.

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