The rapid purification of 3-hydroxybutyrate dehydrogenase and malate dehydrogenase on triazine dye affinity matrices

Scawen, Michael D.; Darbyshire, John F.; Harvey, Michael J.; Atkinson, Tony

doi:10.1042/bj2030699

Cited by 46 publications

(9 citation statements)

References 19 publications

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“…Similar purification procedures have been employed previously for the isolation of MDH from a variety of gram-positive and gram-negative bacteria, including two Rhodobacter species (17,21,22). In general, triazine dye affinity columns bind dehydrogenases quite tightly, and enzyme inhibition studies have shown that the dye interacts with the substrate or cofactor binding sites of many dehydrogenases.…”

Section: Discussionmentioning

confidence: 99%

Malate dehydrogenase in phototrophic purple bacteria: purification, molecular weight, and quaternary structure

Tayeh¹,

Madigan²

1987

J Bacteriol

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The citric acid cycle enzyme malate dehydrogenase was purified to homogeneity from the nonsulfur purple bacteria Rhodobacter capsulatus, Rhodospirillum rubrum, Rhodomicrobium vannielii, and Rhodocyclus purpureus. Malate dehydrogenase was purified from each species by either a single-or a two-step protocol; triazine dye affinity chromatography was the key step in purification of malate dehydrogenase in all cases. Purification of malate dehydrogenase resulted in a 130-to 240-fold increase in malate dehydrogenase specific activity, depending on the species, with recoveries ranging from 30 to 70%. Homogeneity of malate dehydrogenase preparations from the four organisms was determined by sodium dodecyl sulfate and nondenaturing polyacrylamide gel electrophoresis; a single protein band was observed in purified preparations by both techniques. The molecular weight of native malate dehydrogenases was determined by four independent methods and estimated to be in the range of 130,000 to 140,000 for the enzyme from R. capsulatus, R. rubrum, and R. vannielii and 57,000 for that from R. purpureus. It is concluded that malate dehydrogenase from R. capsulatus, R. rubrum, and R. vannielii is a tetramer composed of four identical subunits, while the enzyme from R. purpureus is a dimer composed of two identical subunits.

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Section: Discussionmentioning

confidence: 99%

Malate dehydrogenase in phototrophic purple bacteria: purification, molecular weight, and quaternary structure

Tayeh¹,

Madigan²

1987

J Bacteriol

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“…Among these supports, one of the first to be used for preparative use was agarose [9] or silica [10]. However, the use of agarose was limited, because it cannot withstand high pressure and high flow-rates and may suffer microbial degradation of its backbone resulting in ligand leakage.…”

Section: Introductionmentioning

confidence: 99%

Chitosan microspheres as immobilized dye affinity support for catalase adsorption

Shentu

Song

et al. 2005

International Journal of Biological Macromolecules

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“…These same immobilized dyes can be used to purify enzymes (Lowe et al, 1980;Atkinson et al, 1981), e.g. glycerokinase from B. stearothermophilus (Scawen et al, 1983), hexokinase from pig heart (Farmer & Easterby, 1982), malate dehydrogenase (EC 1.1.1.37) and 3-hydroxybutyrate dehydrogenase (EC 1.1.1.30) from Rhodopseudomonas spheroides (Scawen et al, 1982) and alcohol dehydrogenase (EC 1.1.1.1) from horse muscle (Roy & Nishikawa, 1979). In all these instances an immobilized dye was found to have a much higher binding capacity than an equivalent nucleotide ligand, as well as being much less expensive and easier to couple to a chromatography matrix.…”

Section: Introductionmentioning

confidence: 99%

The purification and characterization of glucokinase from the thermophile Bacillus stearothermophilus

Goward¹,

Hartwell²,

Atkinson³

et al. 1986

Biochemical Journal

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Homogeneous glucokinase (EC 2.7.1.2) from the thermophile Bacillus stearothermophilus was isolated on the large scale by using four major steps: precipitation of extraneous material at pH 5.5, ion-exchange chromatography on DEAE-Sepharose, pseudo-affinity chromatography on Procion Brown H-3R-Sepharose 4B and gel filtration on Ultrogel AcA 34. The purified enzyme had a specific activity of about 330 units/mg of protein and was shown to exist as a dimer of subunit Mr 33,000. Kinetic parameters for the enzyme were determined with a variety of substrates. The glucokinase was highly specific for alpha-D-glucose, and the only other sugar substrate utilized was N-acetyl-alpha-D-glucosamine. The enzyme shows Michaelis-Menten kinetics, with a Km value of 150 microM for alpha-D-glucose. The glucokinase was maximally active at pH 9.0.

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The rapid purification of 3-hydroxybutyrate dehydrogenase and malate dehydrogenase on triazine dye affinity matrices

Cited by 46 publications

References 19 publications

Malate dehydrogenase in phototrophic purple bacteria: purification, molecular weight, and quaternary structure

Malate dehydrogenase in phototrophic purple bacteria: purification, molecular weight, and quaternary structure

Chitosan microspheres as immobilized dye affinity support for catalase adsorption

The purification and characterization of glucokinase from the thermophile Bacillus stearothermophilus

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