6‐phosphogluconate dehydrogenase from <i>Bacillus stearothermophilus</i>

Pearse, B. M. F.; Harris, J. Ieuan

doi:10.1016/0014-5793(73)80510-1

Cited by 16 publications

(5 citation statements)

References 13 publications

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“…The specific activity of the purified protein was 52units/mg (pH7.5, 300C), which compares with reported values of 20 units/mg (pH 9.0, 250 C) (Pearse & Harris, 1973) and 87units/mg (pH8.0, 430C) (Veronese et al, 1974). Under the latter conditions of pH and temperature our purified protein had a specific activity of 91.6 units/mg.…”

Section: Discussionsupporting

confidence: 81%

The use of various immobilized-triazine affinity dyes for the purification of 6-phosphogluconate dehydrogenase from Bacillus stearothermophilus

Qadri¹,

Dean²

1980

Biochemical Journal

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1. 6-Phosphogluconate dehydrogenase from Bacillus stearothermophilus was purified approximately 260-fold on triazine-immobilized dye columns to a final specific activity of 54 mumol of NADP+ reduced/min per mg of protein and an overall yield of 62%. 2. An investigation of the capacities of different triazine dyes that inhibit 6-phosphogluconate dehydrogenase was carried out. Cibacron Blue F3G-A and Procion Red HE-3B strongly inhibited the enzyme in free solution and were therefore chosen as the ligands in the purification scheme. 3. KCl was found to be the most suitable agent for eluting 6-phosphogluconate dehydrogenase from Procion Red HE-3B-Sepharose 6B. NADP+ could specifically elute 6-phosphogluconate dehydrogenase from Cibacron Blue F3G-A-Sepharose 6B. 4. A study of the effect of temperature on the binding of pure 6-phosphogluconate dehydrogenase to both Cibacron Blue-Sepharose and Procion Red-Sepharose showed that the binding increased with an increase in temperature.

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Section: Discussionsupporting

confidence: 81%

The use of various immobilized-triazine affinity dyes for the purification of 6-phosphogluconate dehydrogenase from Bacillus stearothermophilus

Qadri¹,

Dean²

1980

Biochemical Journal

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“…Tryptophan has been determined spectrophotometrically by the method of Edelhoch (1967). and composed of two apparently identical subunits (Veronese et al, 1973(Veronese et al, , 1974Pearse and Harris, 1973). The enzyme from E. coli and B. stearothermophilus was subjected to sucrose density gradient centrifugation in the same run but in different tubes.…”

Section: Resultsmentioning

confidence: 99%

Isolation and properties of 6-phosphogluconate dehydrogenase from Escherichia coli. Some comparisons with the thermophilic enzyme from Bacillus stearothermophilus

Veronese¹,

Boccù²,

Fontana³

1976

Biochemistry

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6-Phosphogluconate dehydrogenase (6-phospho-D-gluconate:NADP oxidoreductase (decarboxylating), EC 1.1.1.44) of Escherichia coli MREp 600 has been isolated with the purpose of carrying out comparative studies with the thermostable enzyme previously isolated from Bacillus stearothermophilus (Veronese, F.M., Boccù, E.,Fontana, A., Benassi,C.A., and Scoffone, E. (1974), Biochim, Biophys. Acta 334, 31). The purified enzyme appeared homogeneous by the criteria of disc gel electrophoresis with and without sodium dodecyl sulfate, ultracentrifugation, and gel filtration. The enzyme has enzymological and physiochemical properties similar to the enzyme isolated from other sources, including B. stearothermophilus. The E. coli enzyme has a mol wt of 100,000 +/- 3000 and is composed of two apparently identical subunits. The amino acid composition of both the mesophilic and thermophilic enzyme has been determined and found to present large similarities. The E. coli enzyme shows a high degree of specificity for nicotinamide adenine dinucleotide (NADP) and it is inhibited by reduced NADP (NADPH). Cysteine residues are involved in the catalytic activity, since on incubation of the enzyme with p-chloromercuribenzoate or 5.5'-dithiobis(2-nitrobenzoic acid) strong inhibition occurs, activity being restored by treatment with excess of beta-mercaptoethanol. The substrate 6-phosphogluconate protects partially the enzyme from inactivation. Both the mesophilic and thermophilic 6-phosphogluconate dehydrogenases are inactivated by Rose Bengal in the presence of light by similar kinetics and protected against photoinactivation by the enzyme substrate. The E. coli enzyme, on the other hand, showed distinct differences in stability against heat and unfolding agents in respect to the B. stearothermophilus enzyme. Heating at 50 degrees C or incubation in 8 M urea results in rapid inactivation. The gross structure of the mesophilic and thermophilic enzyme was very similar as judged by circular dichroic measurements. The far-ultraviolet circular dichroic spectrum had a negative band centered at about 220 nm. In both cases, the fluorescence emission spectrum indicates that the environment of the tryptophan residues is similar, since both enzymes show an emission maximum at 334 nm upon excitation at 295 nm. Circular dichroism measured at various temperatures between 25 and 80 degrees C showed the mesophilic enzyme to be conformationally stable below about 45 degrees C and the thermophilic enzyme below 60 degrees C. The secondary structure of the E. coli enzyme was very sensitive to the denaturing action of urea, since in 8 M urea it rapidly unfolded. Partial renaturation after urea treatment occurred on dilution with buffer or dialysis, as evidenced by spectral properties of the renatured enzyme. The results show that the mesophilic and thermophilic enzymes are very similar and that differences in thermal stability depend on subtle differences in the architectures of the proteins.

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“…The T. maritima 6PGDH is the most thermostable 6PGDH characterized so far, with half-life times of 48 h and ~140 h at 90°C and 80°C, respectively. This enzyme was far more thermostable than the Bacillus stearothermophilus 6PGDH with a half-life time of about 15 min at 70°C [ 49 ]. The hyper-thermostability of T. maritima 6PGDH makes it possible to simplify its purification by heat precipitation, different from most protein purification technologies, such as chromatography or adsorption/desorption [ 19 , 44 ].…”

Section: Discussionmentioning

confidence: 99%

Overexpression and simple purification of the Thermotoga maritima 6-phosphogluconate dehydrogenase in Escherichia coli and its application for NADPH regeneration

Wang

Zhang

2009

Microb Cell Fact

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6‐phosphogluconate dehydrogenase from Bacillus stearothermophilus

Cited by 16 publications

References 13 publications

The use of various immobilized-triazine affinity dyes for the purification of 6-phosphogluconate dehydrogenase from Bacillus stearothermophilus

The use of various immobilized-triazine affinity dyes for the purification of 6-phosphogluconate dehydrogenase from Bacillus stearothermophilus

Isolation and properties of 6-phosphogluconate dehydrogenase from Escherichia coli. Some comparisons with the thermophilic enzyme from Bacillus stearothermophilus

Overexpression and simple purification of the Thermotoga maritima 6-phosphogluconate dehydrogenase in Escherichia coli and its application for NADPH regeneration

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