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.

HETEROGENEITY OF INSULIN: I. ISOLATION OF A CHROMATOGRAPHICALLY PURIFIED, HIGH-POTENCY INSULIN AND SOME OF ITS PROPERTIES

1966 ◽  
Vol 44 (8) ◽  
pp. 1171-1181 ◽  
Author(s):  
W. W. Dillon ◽  
R. G. Romans
Keyword(s):  
Biological Activity ◽  
Gel Electrophoresis ◽  
Sodium Phosphate ◽  
Chemical Degradation ◽  
Minor Components ◽  
Slight Improvement ◽  
High Potency ◽  
Sodium Phosphate Buffer ◽  
A Value ◽  
Main Component

A slight improvement in the chromatography of insulin on Amberlite IRC-50 resin in 7 M urea −0.13 M sodium phosphate buffer, pH 6.05, at 3 °C revealed the presence of at least four components.The major component was found to have a potency greater than that of any other purified insulin hitherto reported and substantiated. The biological activity of the minor components varied from a value close to that of the starting insulin down to very low or negligible values, indicating a loss of biological activity with increasing chemical degradation. The potency studies were quantitative.Starch-gel electrophoresis showed that the main component was not completely homogeneous, traces of other components being present.A highly fluorescent, colored material was separated from among the minor components and may be of interest in relation to allergy produced by some insulins.The separation reported here is discussed in relation to other fractionation procedures.

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 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 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.

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.

Low Molecular Weight Trypsin-Plasmin Inhibitors Isolated from Papain Treated Urinary Trypsin Inhibitor

1982 ◽  
Vol 47 (01) ◽  
pp. 014-018 ◽  
Author(s):  
H Sumi ◽  
N Toki ◽  
S Takasugi ◽  
S Maehara ◽  
M Maruyama ◽  
...  
Keyword(s):  
Trypsin Inhibitor ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Chromatography ◽  
Urinary Trypsin Inhibitor ◽  
Plasmin Activity ◽  
Molecular Weights ◽  
Plasmin Inhibitors

SummaryPapain treatment of human urinary trypsin inhibitor (UTI67; mol. wt. 43,000 by SDS-polyacrylamide gel electrophoresis, specific activity 1,897 U/mg protein) produced four new protease inhibitors, which were highly purified by gel chromatography on Sephadex G-100 and isoelectric focusing. The purified inhibitors (UTI26, UTI9-I, UTI9-II, and UTI9-III) were shown to be homogeneous by polyacrylamide disc gel electrophoresis, and had apparent molecular weights of 26,000, 9,000, 9,000, and 9,800, respectively, by sodium dodecyl sulfate gel electrophoresis. During enzymatic degradation of UTI67, the amino acid compositions changed to more basic, and the isoelectric point increased from pH 2.0 (UTI67) to pHs 4.4, 5.2, 6.6, and 8.3 (UTI26, UTI9-I, UTI9-II, and UTI9-III), respectively. Both the parent and degraded inhibitors had anti-plasmin activity as well as antitrypsin and anti-chymotrypsin activities. Much higher anti-plasmin/anti-trypsin and anti-plasmin/anti-chymotrypsin activities were observed in the degraded inhibitors than in the parent UTI67. They competitively inhibited human plasmin with Ki values of 1.13 X 10-7 - 2.12 X 10-6 M (H-D-Val-Leu-Lys-pNA substrate). The reactions were very fast and the active site of the inhibitors to plasmin was thought to be different from that to trypsin or chymotrypsin.

scholarly journals Structural properties of homogeneous protein disulphide-isomerase from bovine liver purified by a rapid high-yielding procedure

1983 ◽  
Vol 213 (1) ◽  
pp. 225-234 ◽  
Author(s):  
N Lambert ◽  
R B Freedman
Keyword(s):  
Amino Acid ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sedimentation Coefficient ◽  
Bovine Liver ◽  
Polyacrylamide Gel ◽  
Protein Disulphide Isomerase

Protein disulphide-isomerase from bovine liver was purified to homogeneity as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, two-dimensional electrophoresis and N-terminal amino acid analysis. The preparative procedure, a modification of that of Carmichael, Morin & Dixon [(1977) J. Biol. Chem. 252, 7163-7167], is much faster and higher-yielding than previous procedures, and the final purified material is of higher specific activity. The enzyme has Mr 57 000 as determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, both in the presence and in the absence of thiol compounds. Gel-filtration studies on Sephadex G-200 indicate an Mr of 107 000, suggesting that the native enzyme is a homodimer with no interchain disulphide bonds. Ultracentrifugation studies give a sedimentation coefficient of 3.5S, implying that the enzyme sediments as the monomer. The isoelectric point, in the presence of 8 M-urea, is 4.2, and some microheterogeneity is detectable. The amino acid composition is comparable with previous analyses of this enzyme from bovine liver and of other preparations of thiol:protein disulphide oxidoreductases whose relation to protein disulphide-isomerase has been controversial. The enzyme contains a very high proportion of Glx + Asx residues (27%). The N-terminal residue is His. The pure enzyme has a very small carbohydrate content, determined as 0.5-1.0% by the phenol/H2SO4 assay. Unless specific steps are taken to remove it, the purified enzyme contains a small amount (5 mol/mol of enzyme) of Triton X-100 carried through the purification.

scholarly journals A Monoacylglycerol Lipase from Mycobacterium smegmatis Involved in Bacterial Cell Interaction

2010 ◽  
Vol 192 (18) ◽  
pp. 4776-4785 ◽  
Author(s):  
Rabeb Dhouib ◽  
Françoise Laval ◽  
Frédéric Carrière ◽  
Mamadou Daffé ◽  
Stéphane Canaan
Keyword(s):  
Mycobacterium Smegmatis ◽  
Wild Type Strain ◽  
Specific Activity ◽  
Cell Interaction ◽  
Monoacylglycerol Lipase ◽  
Homologous Proteins ◽  
Dependent Activity ◽  
Hydrolysis Of

ABSTRACT MSMEG_0220 from Mycobacterium smegmatis, the ortholog of the Rv0183 gene from M. tuberculosis, recently identified and characterized as encoding a monoacylglycerol lipase, was cloned and expressed in Escherichia coli. The recombinant protein (rMSMEG_0220), which exhibits 68% amino acid sequence identity with Rv0183, showed the same substrate specificity and similar patterns of pH-dependent activity and stability as the M. tuberculosis enzyme. rMSMEG_0220 was found to hydrolyze long-chain monoacylglycerol with a specific activity of 143 ± 6 U mg−1. Like Rv0183 in M. tuberculosis, MSMEG_0220 was found to be located in the cell wall. To assess the in vivo role of the homologous proteins, an MSMEG_0220 disrupted mutant of M. smegmatis (MsΔ0220) was produced. An intriguing change in the colony morphology and in the cell interaction, which were partly restored in the complemented mutant containing either an active (ComMsΔ0220) or an inactive (ComMsΔ0220S111A) enzyme, was observed. Growth studies performed in media supplemented with monoolein showed that the ability of both MsΔ0220 and ComMsΔ0220S111A to grow in the presence of this lipid was impaired. Moreover, studies of the antimicrobial susceptibility of the MsΔ0220 strain showed that this mutant is more sensitive to rifampin and more resistant to isoniazid than the wild-type strain, pointing to a critical structural role of this enzyme in mycobacterial physiology, in addition to its function in the hydrolysis of exogenous lipids.

Effects of sodium phosphate buffer on horseradish peroxidase thermal stability

2008 ◽  
Vol 93 (2) ◽  
pp. 569-574 ◽  
Author(s):  
L. Haifeng ◽  
L. Yuwen ◽  
C. Xiaomin ◽  
W. Zhiyong ◽  
W. Cunxin
Keyword(s):  
Thermal Stability ◽  
Horseradish Peroxidase ◽  
Phosphate Buffer ◽  
Sodium Phosphate ◽  
Sodium Phosphate Buffer
Sign in / Sign up

Export Citation Format

Share Document