Purification And Characterization Of Ceql Restriction Endonuclease

1992 ◽  
Vol 47 (11-12) ◽  
pp. 830-834 ◽  
Author(s):  
Zsuzsa Izsvák ◽  
Zsolt Jobbágy ◽  
Ernö Duda
Keyword(s):  
Molecular Mass ◽  
Restriction Endonuclease ◽  
High Speed ◽  
Apparent Size ◽  
Ph Optimum ◽  
Ionic Detergents ◽  
Apparent Homogeneity ◽  
High Concentrations ◽  
Reversible Loss

Ceql, a type II restriction endonuclease, an isoschizomer of EcoRV was purified to apparent homogeneity by a combination of salt precipitation, ion exchange, dye affinity and hydrophobic interaction chromatographies. The crude enzyme was present in the form of large aggregates that could be pelleted by high speed centrifugation. The enzyme was not associated with cellular membranes, though non-ionic detergents lowered the apparent size of the aggregates. The purified enzyme also showed a tendency to form large molecular mass (66-600 kDa) complexes under physiological conditions, in the absence of cleavable DNA. The enzyme formed smaller complexes in the presence of DNA and non-ionic detergents and dissociated into subunits (and undergoes reversible loss of activity) in the presence of high concentrations of salts. According to SDS gel electrophoresis and sedimentation analysis the molecular mass of the monomer 32 ± 2 kDa. The enzyme had a rather broad pH optimum, extending into the alkaline range and lost specificity and activity in buffers below pH 6

Characterization of nuclear structures containing superhelical DNA

1976 ◽  
Vol 22 (2) ◽  
pp. 303-324 ◽  
Author(s):  
P.R. Cook ◽  
I.A. Brazell ◽  
E. Jost
Keyword(s):  
Protein Content ◽  
Ionic Detergents ◽  
High Concentrations ◽  
Nuclear Structures ◽  
Intercalating Agents ◽  
Made In

Structures resembling nuclei but depleted of protein may be released by gently lysing cells in solutions containing non-ionic detergents and high concentrations of salt. These nucleoids sediment in gradients containing intercalating agents in a manner characteristic of DNA that is intact, supercoiled and circular. The concentration of salt present during isolation of human nucleoids affects their protein content. When made in I-95 M NaCl they lack histones and most of the proteins characteristic of chromatin; in 1-0 M NaCl they contain variable amounts of histones. The effects of various treatments on nucleoid integrity were investigated.

scholarly journals Proteases of germinating winged-bean (Psophocarpus tetragonolobus) seeds: purification and characterization of an acidic protease

1996 ◽  
Vol 313 (2) ◽  
pp. 423-429 ◽  
Author(s):  
Rajamma USHA ◽  
Manoranjan SINGH
Keyword(s):  
Physiological Role ◽  
Immunoblot Analysis ◽  
Protein Band ◽  
Winged Bean ◽  
Psophocarpus Tetragonolobus ◽  
Ph Optimum ◽  
Apparent Homogeneity ◽  
Sds Page ◽  
Storage Protein Mobilization

Two major classes of protease are shown to occur in germinating winged-bean (Psophocarpus tetragonolobus) seeds, by assaying extracts at pH 8.0 and pH 5.1 with [14C]gelatin as substrate. At pH 8.0, the activity profile of the enzyme shows a steady rise throughout the period of germination, whereas the activity at the acidic pH is very low up to day 5 and then increases sharply reaching a peak on day 11, followed by an equally sharp decline. The winged-bean acidic protease (WbAP) has been purified to apparent homogeneity, as attested by a single protein band on both PAGE and SDS/PAGE. WbAP is a monomeric enzyme with a molecular mass of 35 kDa and a pH optimum of 6.0. It is a thiol protease that does not belong to the papain family and it has tightly bound Ca2+ as shown by 45Ca2+-exchange studies. Besides gelatin and casein, it hydrolyses a 29 kDa winged-bean protein, indicating a prospective physiological role for it in storage-protein mobilization. Immunoblot analysis shows that it occurs only in the seeds and sprouting tubers of this plant and also that it is synthesized in developing seeds just before desiccation. It appears that the newly synthesized enzyme is inactive, and activation takes place around day 6 of germination. However, neither the mechanism of activation nor the signal that triggers it is clearly understood.

scholarly journals Purification and characterization of human platelet glutathione-S- transferase

Blood ◽  
1986 ◽  
Vol 67 (6) ◽  
pp. 1595-1599
Author(s):  
J Loscalzo ◽  
J Freedman
Keyword(s):  
Specific Activity ◽  
Human Platelet ◽  
Reduced Glutathione ◽  
Human Platelets ◽  
Glutathione S Transferase ◽  
Purification And Characterization ◽  
Ph Optimum ◽  
Complete Purification ◽  
High Concentrations

A glutathione-S-transferase was isolated and purified to homogeneity from human platelets. With a combination of ammonium sulfate fractionation and chromatographic methods, 0.2 mg of pure enzyme was obtained from 9 X 10(11) platelets with a 12% recovery. The purified enzyme had a specific activity of 7.5 U per milligram, representing an approximately 1,100-fold purification. The enzyme was found to be anionic, with an isoelectric point of 4.6. With reduced glutathione as a co-substrate, platelet glutathione-S-transferase was most active with the synthetic substrate, 1-chloro-2,4-dinitrobenzene, less active with 1,2-dichloro-4-nitrobenzene, and essentially inactive with nitroglycerin and 1,2-epoxy-3-(p-nitrophenoxy)-propane. The pH optimum for activity with glutathione and 1-chloro-2,4-dinitrobenzene was 7.0. Indomethacin (1-(p-chlorobenzoyl)-5-methoxy-2-methyindole-3-acetic acid), a chlorobenzene derivative, noncompetitively inhibited human platelet glutathione-S-transferase with an apparent KI of 0.23 mmol/L. This study represents the first complete purification and characterization of a glutathione-S-transferase from platelets. The presence of this enzyme in the platelet, within which high concentrations of reduced glutathione coexist, suggests the potential importance of the platelet in detoxification reactions and in the synthesis of the glutathione adducts of leukotriene metabolism.

Purification and characterization of purine nucleoside phosphorylase from developing embryos of Hyalomma dromedarii

1991 ◽  
Vol 69 (4) ◽  
pp. 223-231 ◽  
Author(s):  
Mamdouh Y. Kamel ◽  
Afaf S. Fahmy ◽  
Abdel H. Ghazy ◽  
Magda A. Mohamed
Keyword(s):  
Purine Nucleoside Phosphorylase ◽  
Catalytic Mechanism ◽  
Kinetic Studies ◽  
Purine Nucleoside ◽  
Hyalomma Dromedarii ◽  
Ph Optimum ◽  
Nucleoside Phosphorylase ◽  
Apparent Homogeneity ◽  
Camel Tick

Purine nucleoside phosphorylase from Hyalomma dromedarii, the camel tick, was purified to apparent homogeneity. A molecular weight of 56 000 – 58 000 was estimated for both the native and denatured enzyme, suggesting that the enzyme is monomeric. Unlike purine nucleoside phosphorylase preparations from other tissues, the H. dromedarii enzyme was unstable in the presence of β-mercaptoethanol. The enzyme had a sharp pH optimum at pH 6.5. It catalyzed the phosphorolysis and arsenolysis of ribo- and deoxyribo-nucleosides of hypoxanthine and guanine, but not of adenine or pyrimidine nucleosides. The Km values of the enzyme at the optimal pH for inosine, deoxyinosine, guanosine, and deoxyguanosine were 0.31, 0.67, 0.55, and 0.33 mM, respectively. Inactivation and kinetic studies suggested that histidine and cysteine residues were essential for activity. The pKa values determined for catalytic ionizable groups were 6–7 and 8–9. The enzyme was completely inactivated by thiol reagents and reactivated by excess β-mercaptoethanol. The enzyme was also susceptible to pH-dependent photooxidation in the presence of methylene blue, implicating histidine. Initial velocity studies showed an intersecting pattern of double-reciprocal plots of the data, consistent with a sequential mechanism.Key words: Acarina, Hyalomma dromedarii, purine nucleoside phosphorylase, kinetics, active site, catalytic mechanism.

scholarly journals Purification and characterization of an extracellular (1 → 6)-β-glucanase from the filamentous fungus Acremonium persicinum

1996 ◽  
Vol 316 (3) ◽  
pp. 841-846 ◽  
Author(s):  
Stuart M. PITSON ◽  
Robert J. SEVIOUR ◽  
Barbara M. McDOUGALL ◽  
Bruce A. STONE ◽  
Maruse SADEK
Keyword(s):  
Molecular Mass ◽  
Filamentous Fungus ◽  
Gel Filtration ◽  
Purification And Characterization ◽  
Eisenia Bicyclis ◽  
Ph Optimum ◽  
Hydrolysis Products ◽  
Sds Page ◽  
Hydrolysis Of

An endo-(1 → 6)-β-glucanase has been isolated from the culture filtrates of the filamentous fungus Acremonium persicinum and purified by (NH4)2SO4 precipitation followed by anion-exchange and gel-filtration chromatography. SDS/PAGE of the purified enzyme gave a single band with an apparent molecular mass of 42.7 kDa. The enzyme is a non-glycosylated, monomeric protein with a pI of 4.9 and pH optimum of 5.0. It hydrolysed (1 → 6)-β-glucans (pustulan and lutean), initially yielding a series of (1 → 6)-β-linked oligoglucosides, consistent with endo-hydrolytic action. Final hydrolysis products from these substrates were gentiobiose and gentiotriose, with all products released as β-anomers, indicating that the enzyme acts with retention of configuration. The purified enzyme also hydrolysed Eisenia bicyclis laminarin, liberating glucose, gentiobiose, and a range of larger oligoglucosides, through the apparent hydrolysis of (1 → 6)-β- and some (1 → 3)-β-linkages in this substrate. Km values for pustulan, lutean and laminarin were 1.28, 1.38, and 1.67 mg/ml respectively. The enzyme was inhibited by N-acetylimidazole, N-bromosuccinimide, dicyclohexylcarbodi-imide, Woodward's Regent K, 2-hydroxy-5-nitrobenzyl bromide, KMnO4 and some metal ions, whereas D-glucono-1,5-lactone and EDTA had no effect.

scholarly journals Characterization of the hydroxymethylglutaryl-CoA lyase precursor, a protein targeted to peroxisomes and mitochondria

1996 ◽  
Vol 315 (1) ◽  
pp. 71-75 ◽  
Author(s):  
Lyudmila I. ASHMARINA ◽  
Marie-France ROBERT ◽  
Marc-André ELSLIGER ◽  
Grant A. MITCHELL
Keyword(s):  
Molecular Mass ◽  
Gel Filtration ◽  
Leader Peptide ◽  
Mitochondrial Targeting ◽  
Ph Optimum ◽  
Sds Page ◽  
Peroxisomal Targeting ◽  
Quaternary Structures ◽  
Hydroxymethylglutaryl Coa Lyase

We previously showed that human liver hydroxymethylglutaryl-CoA (HMG-CoA) lyase (HL; EC 4.1.3.4) is found in both mitochondria and peroxisomes. HL contains a 27-residue N-terminal mitochondrial targeting sequence which is cleaved on mitochondrial entry, as well as a C-terminal Cys-Lys-Leu peroxisomal targeting motif. Because peroxisomal HL has a greater molecular mass and more basic pI value than mitochondrial HL, we predicted that peroxisomal HL retains the mitochondrial leader. To test this hypothesis, we expressed both the precursor (pHL) and mature (mHL) peptides in Escherichia coli and studied their properties. pHL purified by ion-exchange and hydrophobic chromatography had a pI of 7.6 on FPLC chromatofocusing and a molecular mass of 34.5 kDa on SDS/PAGE, similar to our findings for peroxisomal HL. For purified mHL, pI (6.2) and molecular mass (32 kDa) values resemble those of mitochondrial HL. Purified pHL is similar to mHL in Km for HMG-CoA (44.8 μM), kcat (6.3 min-1) and pH optimum (9.0–9.5). However, the quaternary structures of pHL and mHL differ. On Superose 12 FPLC gel filtration and also on ultrafiltration, both in the presence and in the absence of HMG-CoA, pHL behaves as a monomer whereas mHL migrates as a dimer. We conclude that the HL precursor is probably identical to peroxisomal HL, that its catalytic properties resemble those of mature mitochondrial HL, and that the mitochondrial leader peptide prevents dimerization of pHL.

scholarly journals Purification and characterization of an Ins(1,3,4,5)P4 binding protein from pig platelets: possible identification of a novel non-neuronal Ins(1,3,4,5)P4 receptor

1995 ◽  
Vol 305 (1) ◽  
pp. 139-143 ◽  
Author(s):  
P J Cullen ◽  
A P Dawson ◽  
R F Irvine
Keyword(s):  
Molecular Mass ◽  
Binding Protein ◽  
High Specificity ◽  
Purification And Characterization ◽  
Tissue Protein ◽  
High Affinity ◽  
Apparent Homogeneity ◽  
Kd Values ◽  
Neuronal Tissues

A novel Ins(1,3,4,5)P4-binding protein has been purified to apparent homogeneity from solubilized membranes derived from pig platelets. It has a high affinity for Ins(1,3,4,5)P4 (Kd 6.3 +/- 0.4 nM), a Bmax of 2.5-6.0 nmol/mg of protein, and a high specificity for Ins(1,3,4,5)P4 [Kd values for Ins(1,3,4,5,6)P5, InsP6, GroPtdIns(3,4,5)P3, Ins(1,4,5)P3, Ins(3,4,5,6)P4 and L-Ins(1,3,4,5)P4 of 85.0 +/- 4.1 nM, 800.0 +/- 20.2 nM, 65.6 +/- 2.6 nM, > 10 microM, 793.3 +/- 55.6 nM and 81.0 +/- 5.9 nM respectively]. The protein has an apparent molecular mass of 104 kDa, suggesting that this peripheral tissue protein may be different from Ins(1,3,4,5)P4 binding proteins previously isolated from neuronal tissues.

scholarly journals (335) Purification and Characterization of ADP-glucose Pyrophosphorylase from Apple Leaves

HortScience ◽  
2005 ◽  
Vol 40 (4) ◽  
pp. 1083C-1083
Author(s):  
Rui Zhou ◽  
Lailiang Cheng
Keyword(s):  
Enzyme Activity ◽  
Thermal Inactivation ◽  
Specific Activity ◽  
Purification And Characterization ◽  
Apple Leaves ◽  
Apparent Homogeneity ◽  
Adp Glucose Pyrophosphorylase ◽  
High Concentrations ◽  
Dual Functions

Apple leaf ADP-glucose pyrophosphorylase was purified over 1400-fold to apparent homogeneity with a specific activity of 58.9 units per mg of protein. The enzyme was activated by 3-phosphoglycerate (PGA) and inhibited by inorganic phosphate (Pi) in the ADPG synthesis direction. In the pyrophosphorolysis direction, however, high concentrations of PGA (>2.5 mm) inhibited the enzyme activity. The enzyme was resistant to thermal inactivation with a T0.5 (temperature at which 50% of the enzyme activity is lost after 5 min of incubation) of 52 °C. Incubation with 2 mm PGA or 2 mm Pi increased T0.5 to 68 °C. Incubation with 2 mm dithiothreitol (DTT) decreased T0.5 to 42 °C, whereas inclusion of 2 mm PGA in the DTT incubation maintained T0.5 at 52 °C. DTT-induced decrease in thermal stability was accompanied by monomerization of the small subunits. Presence of PGA in the DTT incubation did not alter the monomerization of the small subunits of the enzyme induced by DTT. These findings indicate that the binding of PGA may have dual functions in regulating apple leaf AGPase activity—activating the enzyme and rendering the enzyme with a conformation more stable to thermal inactivation.

scholarly journals Purification and characterization of a nutritionally controlled endodeoxyribonuclease from Streptomyces glaucescens

1992 ◽  
Vol 281 (1) ◽  
pp. 231-237 ◽  
Author(s):  
J F Aparicio ◽  
C Hardisson ◽  
J Sánchez
Keyword(s):  
Gel Chromatography ◽  
Purification And Characterization ◽  
Ph Optimum ◽  
Double Stranded Dna ◽  
Bivalent Cations ◽  
Apparent Homogeneity ◽  
Absolute Requirement ◽  
Hydrolysis Of ◽  
Streptomyces Glaucescens

Streptomyces glaucescens has a DNAase whose synthesis is under nutritional control. We have purified this enzyme to apparent homogeneity by phosphocellulose chromatography followed by heparin-agarose, Cibacron Blue F3-GA-Sepharose and Sephadex G-75 chromatography and MonoQ f.p.l.c. The enzyme had an apparent Mr of 39,600 and a pI of approx. 8.15. The Mr of the native enzyme estimated by gel chromatography was 49,000. The DNAase had a pH optimum of 7.5 and an absolute requirement for bivalent cations in the reaction buffer. It was inhibited by high salt concentrations, chelating agents or phosphate-containing compounds and was stimulated by dimethyl sulphoxide. The activity was greatly diminished unless dithiothreitol or 2-mercaptoethanol was included in the reaction mixture. Reagents such as Hg2+ or iodoacetate strongly inhibited the enzyme. The nuclease hydrolysed both double-stranded and single-stranded DNA, showing greater affinity for double-stranded DNA, and no detectable hydrolysis of RNA. The enzyme produced nicks in double-stranded DNA, generating 3′-hydroxy and 5′-phosphate termini, and degraded circular DNA.

scholarly journals Positive co-operative interaction between the subunits of CeqI restriction endonuclease

1992 ◽  
Vol 286 (1) ◽  
pp. 85-88 ◽  
Author(s):  
Z Jobbágy ◽  
Z Izsvák ◽  
E Duda
Keyword(s):  
Restriction Endonuclease ◽  
Dna Sequences ◽  
Hill Coefficient ◽  
Computer Assisted ◽  
Physiological Conditions ◽  
Ionic Detergents ◽  
Catalytically Active ◽  
High Concentrations ◽  
Assisted Analysis ◽  
Tetrameric Form

CeqI restriction endonuclease, an isoschizomer of EcoRV, forms complexes of 12-20 subunits under physiological conditions, in the absence of DNA. These molecules partially dissociate in the presence of DNA sequences recognized by CeqI or in the presence of non-ionic detergents. In solutions containing high concentrations of salts (e.g. 1 M-NaCl), the enzyme dissociates into subunits, concomitantly losing its activity. According to our experiments, it is the tetrameric form of the enzyme that binds the DNA and represents the catalytically active molecule. Analysis of the enzyme kinetics revealed a positive co-operative interaction between the subunits of the enzyme. Computer-assisted analysis of these data yielded a Hill coefficient of approx. 1.35, suggesting two binding sites per tetrameric enzyme molecule, two subunits per palindromic recognition site.

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