scholarly journals Component analysis and characterization of a nuclear deoxyribonucleotidase

1994 ◽  
Vol 298 (3) ◽  
pp. 727-732 ◽  
Author(s):  
K Ford ◽  
J Waltho ◽  
D Hornby
Keyword(s):  
Mammalian Cells ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Hydrolytic Activity ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Nuclear Fraction ◽  
Unidentified Species ◽  
Rf Values

We have previously reported the identification of a novel activity residing in the nuclear fraction of mammalian cells that selectively binds and hydrolyses deoxyribonucleoside triphosphates. Incubation of this protein with [alpha-32P]dATP leads to the appearance of a retarded band relative to free dATP when the reaction is analysed on non-denaturing polyacrylamide gels. We now show that the retarded species comprises the product of dATP hydrolysis (dADP or dAMP) bound to an as yet unidentified species. We have termed this complex the ‘product-nucleotide binding particle’ or PNBP*. Through a combination of continuous elution polyacrylamide-gel electrophoresis and gel-filtration chromatography, we demonstrate that the hydrolytic activity (dNTPase) is distinct from the radiolabelled species detected in gel-retardation experiments. T.l.c. confirms that the labelled product does not share RF values associated with a range of mono-, di- and tri-phosphate deoxyribonucleotide standards, and gel-filtration experiments suggest a molecular mass for PNBP* of between 2.5 and 3 kDa. The ability of purified PNBP* to retain its nucleotide ligand after a number of denaturing processes suggests that the ligand is covalently bound. The recovery of dNTPase activity from both gel-filtration and ion-exchange chromatography reveals that the as yet unliganded PNBP* (or a precursor form) is associated with the dNTPase enzyme as part of the active complex, prior to addition of dATP.

Preparation and Characterization of NH2-Terminal Fibrinogen Bβ Fragments from N-DSK of Human Fibrinogen

1984 ◽  
Vol 51 (01) ◽  
pp. 016-021 ◽  
Author(s):  
S Birken ◽  
G Agosto ◽  
B Lahiri ◽  
R Canfield
Keyword(s):  
High Performance ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Reverse Phase ◽  
Human Fibrinogen ◽  
Human Volunteers ◽  
Cnbr Cleavage ◽  
Two Peaks

SummaryIn order to investigate the early release of NH2-terminal plasmic fragments from the Bβ chain of fibrinogen, substantial quantities of Bβ 1-42 and Bβ 1-21 are required as immunogens, as radioimmunoassay standards and for infusion into human volunteers to determine the half-lives of these peptides. Towards this end methods that employ selective proteolytic cleavage of these fragments from fibrinogen have been developed. Both the N-DSK fragment, produced by CNBr cleavage of fibrinogen, and Bβ 1-118 were employed as substrates for plasmin with the finding of higher yields from N-DSK. Bβ 1-42 and Bβ 1-21 were purified by gel filtration and ion-exchange chromatography on SP-Sephadex using volatile buffers. When the purified preparation of Bβ 1-42 was chromatographed on reverse-phase high performance liquid chromatography, two peaks of identical amino acid composition were separated, presumably due either to pyroglutamate or to amide differences.

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.

Enolase from Lobster (Homarus americanus)

1971 ◽  
Vol 28 (6) ◽  
pp. 879-882 ◽  
Author(s):  
M. John Chapman ◽  
Christopher Chin ◽  
Finn Wold
Keyword(s):  
Heat Treatment ◽  
Ion Exchange ◽  
Ammonium Sulfate ◽  
Catalytic Properties ◽  
Gel Filtration ◽  
Homarus Americanus ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Acetone Fractionation

Enolase has been isolated from lobster muscle by acetone fractionation, heat treatment, ammonium sulfate fractionation, gel filtration, and ion-exchange chromatography. Preliminary characterization of the pure enzyme shows that the catalytic properties are very similar to those of the enolases from rabbit and fish.

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 Purification and Characterization of Two Endo-β-1,4-glucanases from Mollusca, Ampullaria crossean

2005 ◽  
Vol 37 (10) ◽  
pp. 702-708 ◽  
Author(s):  
Yan-Hong Li ◽  
Rui Guo ◽  
Qiu-Yu Yin ◽  
Ming Ding ◽  
Si-Liang Zhang ◽  
...  
Keyword(s):  
Anion Exchange ◽  
Carboxymethyl Cellulose ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecylsulfate ◽  
Gel Filtration ◽  
Residual Activity ◽  
Hydrolytic Activity ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
The Stability

Abstract Two novel endo-β-1,4-glucanases, EG45 and EG27, were isolated from the gastric juice of mollusca, Ampullaria crossean, by anion exchange, hydrophobic interaction, gel filtration and a second round of anion exchange chromatography. The purified proteins EG45 and EG27 appeared as a single band on sodium dodecylsulfate polyacrylamide gel electrophoresis with a molecular mass of 45 kDa and 27 kDa, respectively. The optimum pH for CMC activity was 5.5 for EG45 and 4.4-4.8 for EG27. The optimum temperature range for EG27 was broad, between 50 °C and 60 °C; for EG45 it was 50 °C. The analysis on the stability of these two endo-β-1,4-glucanases showed that EG27 was acceptably stable at pH 3.0-11.0 even when the incubation time was prolonged to 24 h at 30 °C, whereas EG45 remained relatively stable at pH 5.0-8.0. About 85% of the activity of EG27 could be retained upon incubation at 60 °C for 24 h. However, less than 10% residual activity of EG45 was detected at 50 °C. Among different kinds of substrates, both enzymes showed a high preference for carboxymethyl cellulose. EG45, in particular, showed a carboxymethyl cellulose hydrolytic activity of 146.5 IU/mg protein. Both enzymes showed low activities to xylan (from oat spelt) and Sigmacell 101, and they were inactive to p-nitrophenyl-β-D-cellobioside, salicin and starch.

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.

Multiplicity of extracellular β-(1,4)-endoglucanases of Bacteroides succinogenes S85

1984 ◽  
Vol 30 (7) ◽  
pp. 930-937 ◽  
Author(s):  
H. E. Schellhorn ◽  
C. W. Forsberg
Keyword(s):  
Ion Exchange ◽  
Microcrystalline Cellulose ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Glucose Production ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Reducing Sugar ◽  
Sugar Production ◽  
Endoglucanase Activity

During growth on 0.2% (w/v) microcrystalline cellulose, Bacteroides succinogenes S85 produces endoglucanase activity which can be separated by centrifugation into sedimentable and nonsedimentable fractions. The sedimentable activity, after solubilization with Triton X-100, was resolved into four components by ion-exchange chromatography and these were further fractionated by nondenaturing polyacrylamide gel electrophoresis (PAGE). The nonsedimentable activity contained three enzymic components as determined by gel filtration. Like the preparations derived from the sedimentable fraction, these components yielded a multiplicity of endoglucanases when electrophoresed under nondenaturing conditions. The fractions obtained by ion-exchange chromatography and by gel filtration were assayed for endoglucanase activity by both viscometric assays and reducing sugar production using carboxymethylcellulose as the substrate. Plots of the fluidity change in the enzyme–substrate preparation in relation to reducing sugar production revealed the presence of two distinct groups of endoglucanases differing in catalytic activity. Two of the components from the nonsedimentable fraction had more exoglucanase-like activity than either the third nonsedimentable fraction or any of the four fractions derived from the sedimentable material. These two enzymes could be further differentiated on the basis of glucose production from microcrystalline cellulose and by their relative activity toward p-nitrophenyl cellobioside, a chromogenic substrate.

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 a metalloproteinase inhibitor from human rheumatoid synovial fluid

1985 ◽  
Vol 231 (3) ◽  
pp. 505-510 ◽  
Author(s):  
E Mercer ◽  
T E Cawston ◽  
M de Silva ◽  
B L Hazleman
Keyword(s):  
Synovial Fluid ◽  
Culture Medium ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Serine Proteinase ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Metalloproteinase Inhibitor ◽  
Bacterial Collagenase ◽  
Rheumatoid Synovial Fluid

A metalloproteinase inhibitor present in human rheumatoid synovial fluid was purified by a combination of heparin-Sepharose chromatography, concanavalin A-Sepharose chromatography, ion-exchange chromatography and gel filtration. The Mr of the purified inhibitor was 28000 by SDS/polyacrylamide-gel electrophoresis and 30000 by gel filtration. The inhibitor blocked the activity of the metalloproteinases collagenase, gelatinase and proteoglycanase, but not thermolysin or bacterial collagenase. The serine proteinase trypsin was not inhibited. The inhibitory activity was lost after treatment with trypsin (0.5 micrograms/ml) at 37 degrees C for 30 min, 4-aminophenylmercuric acetate (1 mM) at 37 degrees C for 3 h, after incubation for 30 min at 90 degrees C and by reduction and alkylation. These properties suggest that the inhibitor closely resembles the tissue inhibitor of metalloproteinases (‘TIMP’) recently purified from connective-tissue culture medium.

scholarly journals The cellulolytic enzymes of Botryodiplodia theobromae Pat. Separation and characterization of cellulases and β-glucosidases

1979 ◽  
Vol 177 (1) ◽  
pp. 9-19 ◽  
Author(s):  
Gabriel M. Umezurike
Keyword(s):  
Molecular Weight ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Cellulolytic Enzymes ◽  
Botryodiplodia Theobromae ◽  
Molecular Weights ◽  
Low Concentrations ◽  
High Concentrations

1. Filtrates from cultures of different ages of Botryodiplodia theobromae Pat. were fractionated by gel filtration, ion-exchange chromatography and polyacrylamide-gel electrophoresis. 2. Five cellulases (C1, C2, C3, C4 and C5) were found, and their molecular weights, estimated by gel filtration, were 46000–48000 (C1), 30000–35000 (C2), 15000–18000 (C3), 10000–11000 (C4) and 4800–5500 (C5). 3. Cellulase C5 was absent from old culture filtrates. 4. Cellulase C1 had little or no activity on CM-cellulose (viscometric assay), but degraded cotton flock and Whatman cellulose powder to give cellobiose only. 5. The other components (C2–C5) produced cellobiose and smaller amounts of glucose and cellotriose from cellulosic substrates and were more active in lowering the viscosity of CM-cellulose. 6. The ratio of activities assayed by viscometry and by the release of reducing sugars from CM-cellulose increased with decrease in the molecular weights of cellulases C2–C5. 7. Cellobiose inhibited the activities of the cellulases, but glucose stimulated at low concentrations although it inhibited at high concentrations. 8. A high-molecular-weight β-glucosidase (component B1, mol.wt. 350000–380000) predominated in filtrates from young cultures, but a low-molecular-weight enzyme (B4, mol.wt. 45000–47000) predominated in older filtrates. 9. Intermediate molecular species of β-glucosidase (B2, mol.wt. 170000–180000; B3, mol.wt. 83000–87000) were also found. 10. Cellulases C2–C5 acted in synergism with C1, particularly in the presence of β-glucosidase.

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