NADP+-specific isocitrate dehydrogenase of Escherichia coli

Reeves, Henry C.; Daumy, Gaston O.; Lin, C. C.; Houston, Martin R.

doi:10.1016/0005-2744(72)90964-3

Cited by 89 publications

(37 citation statements)

References 29 publications

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“…The mol wt of90 kD obtained by gel filtration chromatography is similar to that reported for the NADP-IDH from E. coli (10). Also, both enzymes consist of two subunits of identical mol wt (2).…”

Section: Discussionsupporting

confidence: 82%

“…We also observed that the chloroplast fraction contained an NADP-IDH activity much lower than that of the cytosol (results not shown), which was in agreement with the results of Randall and Givan (9). The active forms of NADP-IDH present in the chloroplast and cytosolic fractions were analyzed by isoelectric focusing and a gel stain specific for NADP-IDH (10). There were no bands in the chloroplast fraction which corresponded to the bands present in the cytosolic fraction (data not shown).…”

Section: Discussionmentioning

confidence: 99%

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Purification and Characterization of Cytosolic NADP Specific Isocitrate Dehydrogenase from Pisum sativum

Robertson²,

Reeves³

1987

Plant Physiol.

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Cytosolic NADP-specific isocitrate dehydrogenase was isolated from leaves ofPisum sativum. The purified enzyme was obtained by ammonium sulfate fractionation, ion exchange, affinity, and gel filtration chromatography. The purification procedure yields greater than 50% of the total enzyme activity originally present in the crude extract. The enzyme has a native molecular weight of 90 kilodaltons and is resolved into two catalytically active bands by isoelectric focusing. Purified NADP-isocitrate dehydrogenase exhibited K. values of 23 micromolar for DL-iSocitrate and 10 micromolar for NADP, and displayed optimum activity at pH 8.5 with both Mg2+ and Mn2 .NADP-isocitrate dehydrogenase (threo-Ds-isocitrate: NADP oxidoreductase [decarboxylating]; EC 1.1.1.42) has been studied in several different higher plants (3-5, 8, 9). Randall and Givan (9) used protoplasts as a source of organelles and concluded that in pea leaves NADP-IDH2 was localized primarily in the cytosol (90%), to a lesser degree in the chloroplast, but not at significant levels in peroxisomes or mitochondria. The subcellular localization of NADP-IDH suggests that the two forms of the enzyme may have different functions in the metabolism ofthe plant. The chloroplast form of NADP-IDH is generally believed to supply 2-oxoglutarate for glutamate synthesis (3) while the cytosolic enzyme is thought to function in the movement of reducing power between the mitochondria and cytosol (7). However, little is known about the metabolic role of cystosolic NADP-IDH and its relationship with the NADP-IDH present in the chloroplast. In this study, we have purified the cytosolic NADP-IDH to homogeneity and characterized the enzyme. MATERUILS AND METHODSPurification of NADP-Isocitrate Dehydrogenase. Pisum sativum L. cv Little Marvel seedlings were grown on vermiculite in a controlled environment growth chamber (12 h illumination with an intensity of 100 MtE/m2.s, at 25°C) for 2 weeks. Leaves were cut from the seedlings and blended for 5 s with (1:3 w/v) ice-cold isolation medium (25 mm Hepes-NaOH [pH 7.6], 0.35 M sucrose, 2 mm EDTA, and 2 mm sodium isoascorbate). The homogenate was strained through eight layers of cheesecloth and centrifuged at 27,000g for 15 min at 4°C to remove nuclei, ' This work was supported by Grant-841454 from the National Science Foundation.2 Abbreviations: IDH, isocitrate dehydrogenase; ACB, affinity column buffer.chloroplasts, and other organelles. Crystalline (NH4)2SO4 was added to the supernatant with stirring, until a final concentration of 45% was achieved and was left overnight at 4°C. The precipitate was removed by centrifugation at 27,000g for 15 min.Additional crystalline (NH4)2SO4 was added to the supernatant, to achieve a final concentration of 80% and was mixed overnight at 4°C. The precipitate was collected by centrifugation at 27,000g at 4°C and resuspended in affinity column buffer (ACB), pH 7.0 (10 mm K-phosphate, 5 mm citrate, 2 mm MgC12, 10% glycerol, 1 mm benzamidine, and 0.01% NaN3).The protein suspension was desalte...

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Section: Discussionsupporting

confidence: 82%

Section: Discussionmentioning

confidence: 99%

Purification and Characterization of Cytosolic NADP Specific Isocitrate Dehydrogenase from Pisum sativum

Robertson²,

Reeves³

1987

Plant Physiol.

Self Cite

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“…Activity of the bacterial enzyme, an NADP(H)-specific dimeric enzyme (4,36), is decreased 80% by phosphorylation during acetate growth, allowing effective competition for isocitrate by isocitrate lyase, an enzyme with a substantially higher Km for that substrate (20). Not only is the yeast tricarboxylic acid cycle enzyme apparently not subject to any significant modification by phosphorylation (unpublished observation), the specific activity of NAD(H)-specific isocitrate dehydrogenase increases substantially in acetategrown cells.…”

Section: Discussionmentioning

confidence: 97%

Subunit structure, expression, and function of NAD(H)-specific isocitrate dehydrogenase in Saccharomyces cerevisiae

1990

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“…DTT, which is a protective reagent for SH groups, was effective in preventing loss of activity. Glycerol protects enzyme activity in the purification ofNADP+ -linked isocitrate dehydrogenases from Anacystis nidulans, 19) Escherichia COli,23,24) pig heart,25) and human heart. 26 ) Also in our case, the addition of glycerol to the buffer system increased the recovery of activity, particularly at the desalting steps.…”

Section: Discussionmentioning

confidence: 99%

Purification and Characterization of NADP⁺-Linked Isocitrate Dehydrogenase fromPaecilomyces varioti

Sugiyama

Iida

Tanida

1986

Agricultural and Biological Chemistry

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NADP+-specific isocitrate dehydrogenase of Escherichia coli

Cited by 89 publications

References 29 publications

Purification and Characterization of Cytosolic NADP Specific Isocitrate Dehydrogenase from Pisum sativum

Purification and Characterization of Cytosolic NADP Specific Isocitrate Dehydrogenase from Pisum sativum

Subunit structure, expression, and function of NAD(H)-specific isocitrate dehydrogenase in Saccharomyces cerevisiae

Purification and Characterization of NADP⁺-Linked Isocitrate Dehydrogenase fromPaecilomyces varioti

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NADP+-specific isocitrate dehydrogenase of Escherichia coli

Cited by 89 publications

References 29 publications

Purification and Characterization of Cytosolic NADP Specific Isocitrate Dehydrogenase from Pisum sativum

Purification and Characterization of Cytosolic NADP Specific Isocitrate Dehydrogenase from Pisum sativum

Subunit structure, expression, and function of NAD(H)-specific isocitrate dehydrogenase in Saccharomyces cerevisiae

Purification and Characterization of NADP+-Linked Isocitrate Dehydrogenase fromPaecilomyces varioti

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Purification and Characterization of NADP⁺-Linked Isocitrate Dehydrogenase fromPaecilomyces varioti