scholarly journals Purification and properties of DNA polymerase from Bacillus caldotenax

1992 ◽  
Vol 287 (3) ◽  
pp. 971-977 ◽  
Author(s):  
J A Burrows ◽  
C R Goward
Keyword(s):  
Enzyme Activity ◽  
Dna Polymerase ◽  
Maximum Activity ◽  
Equimolar Mixture ◽  
High Concentration ◽  
Bivalent Cation ◽  
Absolute Requirement ◽  
Purification And Properties ◽  
Calf Thymus ◽  
Km Value

A thermostable DNA polymerase was prepared from Bacillus caldotenax by using a four-step chromatography procedure. The protein exists as a monomer of M(r) 94,000, has a pI of 4.9 and has no associated 3′-5′ or 5′-3′-exonuclease activities or endonuclease activity. The temperature optimum of the enzyme was about 70 degrees C and the pH for maximum activity was about 7.5. The enzyme has an absolute requirement for a bivalent cation, and maximum activity was obtained at the unusually high concentration of 70 mM-MgCl2. Mg2+ could be replaced by MnCl2 or CoCl2, with decreased activity, at the lower optimal concentrations of 1 mM and 2.5 mM respectively. Enzyme activity was inhibited in the presence of 2′,3′-dideoxy-TTP, arabinosyl-CTP and aphidicolin. Enzyme activity was stimulated with KCl concentrations of about 100 mM, and concentrations of univalent salts above about 150 mM inhibited activity. The enzyme could use activated calf thymus DNA, poly(dA).p(dT)10 or primed single-stranded phage M13 DNA as a template and maximum activity was obtained with poly(dA).p(dT)10. The enzyme was inactive on unprimed single-stranded DNA, double-stranded DNA and polyribonucleotide template/primer. The apparent Km values for individual dNTPs, determined with the other dNTPs at saturating concentrations, were 5.7 microM (dCTP), 6.3 microM (dATP, dGTP) and 6.4 microM (dTTP). The Km value for the overall incorporation of each dNTP from an equimolar mixture of all four dNTPs was 24.7 microM. The kcat. value was about 1.05 s-1. The kcat./Km value was 0.16-0.18 M-1.s-1 for individual dNTPs and 0.04 for the incorporation of an equimolar mixture of all four dNTPs. Some of the properties of the enzyme show it may be classified as an alpha-Type DNA polymerase.

scholarly journals DNA polymerases from Chlamydomonas reinhardii. Purification and properties

1978 ◽  
Vol 171 (1) ◽  
pp. 231-240 ◽  
Author(s):  
C A Ross ◽  
W J Harris
Keyword(s):  
Dna Polymerase ◽  
Nuclear Dna ◽  
Dna Polymerases ◽  
Deae Cellulose ◽  
Single Species ◽  
Bivalent Cation ◽  
Chlamydomonas Reinhardii ◽  
Synchronous Cultures ◽  
Purification And Properties ◽  
Calf Thymus

Three DNA polymerase activities, A, B and C, were identified in extracts of exponentially growing synchronous cultures of Chlamydomonas reinhardii, and DNA polymerases A and B were characterized in detail. Both enzymes have the same binding affinity for DEAE-cellulose at pH 7.8, but can be distinguished from each other by their behaviour on phosphocellulose and DNA-agarose. ‘Activated’ calf thymus DNA was used as template, and the pH, K+ and bivalent-cation optima were measured. DNA polymerase A sediments at 5.3 S in glycerol gradients, with an apparent mol.wt. of 90000-100000. Polymerase B sediments between 8S and 10S in 100mM-KCl, the predominant species having an apparent mol.wt. of 200000. In 200mM-KCl, polymerase B dissociates to a single species, which sediments at 5.8S. A 3S species was found in aged preparations of both enzymes. The activity of polymerase B from cells harvested during nuclear DNA synthesis is twice that found in Chlamydomonas at other times during the cell cycle.

scholarly journals Increase in alkaline phosphatase activity in calvaria cells cultured with diphosphonates

1979 ◽  
Vol 183 (1) ◽  
pp. 73-81 ◽  
Author(s):  
R Felix ◽  
H Fleisch
Keyword(s):  
Alkaline Phosphatase ◽  
Protein Synthesis ◽  
Enzyme Activity ◽  
Phosphatase Activity ◽  
Alkaline Phosphatase Activity ◽  
Heat Inactivation ◽  
High Concentration ◽  
Control Value ◽  
Km Value ◽  
Inhibitor Of Protein Synthesis

1. Dichloromethanediphosphonate and to a lesser degree 1-hydroxyethane-1,1-diphosphonate, two compounds characterized by a P-C-P bond, increased the alkaline phosphatase activity of cultured rat calvaria cells up to 30 times in a dose-dependent fashion. 2. Both diphosphonates also slightly inhibited the protein synthesis in these cells. 3. Thymidine, an inhibitor of cell division, did not inhibit the induction of the enzyme, indicating that the increase in enzyme activity was not due to the formation of a specific population of cells with high alkaline phosphatase activity. 4. The effect on alkaline phosphatase was suppressed by the addition of cycloheximide, an inhibitor of protein synthesis. 5. After subculturing the stimulated cells in medium without diphosphonates, the enzyme activity fell almost to the control value. 6. Bovine parathyrin diminished the enzyme activity of the control cells and the cells treated with dichloromethanediphosphonate; however, at high concentration the effect of parathyrin was greater on the diphosphonate-treated cells than on the control cells. 7. The electrophoretic behaviour, heat inactivation, inhibition by bromotetramisole or by phenylalanine, and the Km value of the induced enzyme were identical with that of the control enzyme.

scholarly journals Free and Immobilised β-Glucosidases in Oenology: Biotechnological Characterisation and Its Effect on Enhancement of Wine Aroma

2021 ◽  
Vol 12 ◽  
Author(s):  
Pilar Fernández-Pacheco ◽  
Beatriz García-Béjar ◽  
Ana Briones Pérez ◽  
María Arévalo-Villena
Keyword(s):  
Volatile Compounds ◽  
Hydrolytic Activity ◽  
Maximum Activity ◽  
Maximum Temperature ◽  
Wine Aroma ◽  
High Concentration ◽  
Aroma Precursors ◽  
Km Value ◽  
Immobilised Enzymes ◽  
Optimal Ph

In grapes, monoterpenes and norisoprenoids are in the form of non-volatile compounds, flavourless glycosides which could enhance the aroma of wines after its hydrolysis using β- glucosidases enzymes. It is known that the use of immobilised enzymes offers advantages such as reusability and easy recuperation. In this study, a commercial β-glucosidase was immobilised by absorption in sodium alginate. Biotechnological characteristics and terpen hydrolysis (hydrolysis aroma precursors) in muscat wines were studied after treatment with both free and immobilised commercial β- glucosidase with two different concentrations. It was revealed that both forms shared an optimal pH (4.5) and a maximum temperature (64°C), even an increment on the activity between 40and 60°C. A similar Km value has been determined while Vmax from the immobilised enzyme was higher than the free (3.35 and 2.52 μmol min–1 mg–1, respectively). Additionally, the immobilised enzyme showed a better hydrolytic activity during 24 h, and its reusability has been proven. Regarding enzymatic hydrolysis in grape must, the best results were observed for the highest concentration of free β-glucosidase although glucose release was also determined for the immobilised enzyme along the days. In contrast, maximum activity was reached by the immobilised β-glucosidase in less time but in no case equalled the free ones. Finally, volatile compound liberation in wines treated with free or immobilised enzymes was analysed using HRGC-MS. Liberation for both enzymes and the greatest concentrations of some volatiles were detected when a double dose of the free β-glucosidase was used. Nevertheless, the wines treated with the immobilised β-glucosidase showed a high concentration of some volatile compounds such as nerol or geraniol.

Effect of nitric oxide on Sertoli cell microtubule of piglets

2009 ◽  
Vol 6 (3) ◽  
pp. 257-263 ◽  
Author(s):  
Yang Li ◽  
Wang Xian-zhong ◽  
Yang Meng-bo ◽  
Zhang Jia-hua
Keyword(s):  
Nitric Oxide ◽  
Enzyme Activity ◽  
Protein Kinase ◽  
Cell Viability ◽  
Sodium Nitroprusside ◽  
Sertoli Cell ◽  
Sertoli Cells ◽  
High Concentration ◽  
Treatment Results ◽  
Anti Oxidant

AbstractTo illustrate the effect of nitric oxide (NO) on the microtubules of Sertoli cells (SC), SCs of piglets were treated with sodium nitroprusside (SNP). Changes in cell viability, anti-oxidant activity, enzyme activity and p38 mutagen-activated protein kinase (p38MAPK) activation were detected. The results were as follows. A low concentration of NO can keep SC microtubule and cell viability normal, and a high concentration of NO could increase p38MAPK activation, decrease anti-oxidant activity and transferrin secretion, and destroy the structure and distribution of the microtubules. The results suggest that SNP treatment results in an increase in NO in SCs and decreased cell anti-oxidant activity. The high concentration of NO destroys cell microtubules by activating p38MAPK.

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