Isolation of Dihydroxyacetone Phosphate Reductase from <i>Dunaliella</i> Chloroplasts and Comparison with Isozymes from Spinach Leaves

Gee, Robert; Goyal, Arun; Gerber, Dennis W.; Byerrum, Richard U.; Tolbert, N. E.

doi:10.1104/pp.88.3.896

Cited by 19 publications

(11 citation statements)

References 9 publications

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“…Our previous investigation of DHAP reductase isoforms in leaves had also indicated the presence of a chloroplastic and a cytosolic DHAP reductase (8). However, the purified isoforms described in the present study had quite different Fraction Number…”

Section: Discussionsupporting

confidence: 41%

“…In plants and algae, there are cytosolic and chloroplast stromal forms of DHAP 1 This work was supported by two grants from the U.S. Depart reductases (7,10) that use DHAP from photosynthesis or glycolysis to form glycerol phosphate. So far, DHAP reductases have only been partially purified from spinach (Spinacia oleracea L.) leaves (24), castor bean endosperm (5), Dunaliella tertiolecta (8,15), and Chlamydomonas reinhardtii (18). To study and compare different DHAP reductases from both higher plants and algae at the protein and gene levels, we have purified two forms of DHAP reductase from spinach leaves, one of them to apparent electrophoretic homogeneity, and have generated antibodies against this isoform.…”

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Plant Dihydroxyacetone Phosphate Reductases

Kirsch

Dw²,

Ru³

et al. 1992

Plant Physiol.

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A cytosolic form of dihydroxyacetone phosphate (DHAP) reductase was purified 200,000-fold from spinach (Spinacia oleracea L.) leaves to apparent electrophoretic homogeneity. The purification procedure included anion-exchange chromatography, gel filtration, hydrophobic chromatography, and dye-ligand chromatography on Green-A and Red-A agaroses. The enzyme, prepared in an overall yield of 14%, had a final specific activity of about 500 gmol of DHAP reduced min-1 mg-' protein, a subunit molecular mass of 38 kD, and a native molecular mass of 75 kD. A chloroplastic isoform of DHAP reductase was separated from the cytosolic form by anion-exchange chromatography and partially purified 56,000-fold to a specific activity of 135 Amol min-1 mg-1 protein. Antibodies generated in rabbits against the cytosolic form did not crossreact with the chloroplastic isoform. The two reductases were specific for NADH and DHAP. Although they exhibited some dissimilarities, both isoforms were severely inhibited by higher molecular weight fatty acyl coenzyme A esters and phosphohydroxypyruvate and moderately inhibited by nucleotides. In contrast to previous reports, the partially purified chloroplastic enzyme was not stimulated by dithiothreitol or thioredoxin, nor was the purified cytosolic enzyme stimulated by fructose 2,6-bisphosphate. A third DHAP reductase isoform was isolated from spinach leaf peroxisomes that had been prepared by isopycnic sucrose density gradient centrifugation. The peroxisomal DHAP reductase was sensitive to antibodies raised against the cytosolic enzyme and had a slightly smaller subunit molecular weight than the cytosolic isoform.All organisms need glycerol phosphate for the synthesis of glycerolipids, and some algae use glycerol as an osmoticum (26). NAD+-dependent glycerol-3-phosphate dehydrogenase from muscle and liver has been purified and partially characterized (21 higher plants and algae at the protein and gene levels, we have purified two forms of DHAP reductase from spinach leaves, one of them to apparent electrophoretic homogeneity, and have generated antibodies against this isoform. MATERIALS AND METHODS Plant Material and ChemicalsSpinach (Spinacia oleracea L.) was bought from local markets or grown in growth chambers in soil with 8 h of light (200 EEm-2 s-1) and 16 h of dark at 220C. DEAE-cellulose (DE52) was from Whatman; Sephacryl S-200 and phenyl Sepharose CL-4B were from Pharmacia; the dye-ligand agaroses (Matrex gel Red-A and Matrex gel Green-A) were from the Amicon Corporation. Unless otherwise indicated, chemicals were purchased from Sigma Chemical Company. DHAP, glyceraldehyde phosphate, and hydroxypyruvate phosphate were prepared by hydrolyzing the cyclohexylamine salts as directed by Sigma. Molecular standards for SDS-PAGE and hydroxylapatite (Bio-Gel HTP) were purchased from Bio-Rad. Rabbit muscle DHAP reductase and Percoll were from Sigma.Enzyme Purification pH adjustments of buffers were made at room temperature. Unless otherwise indicated, all purification steps were carried ...

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Section: Discussionsupporting

confidence: 41%

mentioning

confidence: 99%

Plant Dihydroxyacetone Phosphate Reductases

Kirsch

Dw²,

Ru³

et al. 1992

Plant Physiol.

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“….-Isolated chloroplasts from Dunaliella contained the major DHAP reductase isoform for glycerol production. This form was stimulated by salts (4,14) and somewhat stimulated by detergents. The Dunaliella cytosolic form and both forms of DHAP reductase in the spinach leaves were inhibited by salts and by traces of detergents.…”

Section: Effect Of Co2 Concentration During Growth On Dhap Reductase mentioning

confidence: 99%

“…The chloroplast form from Dunaliella has different properties than the two leaf forms, but it was stimulated by E. coli thioredoxin (4). This major Dunaliella enzyme was stimulated by 25 mM Mg2' or 250 mm salts, such as KCl, NaCl, or buffer, whereas both of the higher plant isoforms were inhibited by these salts.…”

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Two Isozymes of Dihydroxyacetone Phosphate Reductase in Dunaliella

Gee¹,

Goyal²,

Byerrum³

et al. 1989

Plant Physiol.

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Two isoforms of dihydroxyacetone phosphate reductase were present in Dunaliella tertiolecta. The major form was located in the chloroplast and the minor form in the cytosol. The chloroplastic reductase eluted first from a DEAE cellulose column followed immediately by the cytosolic form. Both forms were unstable and cold labile. Addition of 5 millimolar dithiothreitol helped to stabilize the enzymes. The cytosolic isoform of DHAP reductase was detected only if the cells were in an active log phase of growth. Then its activity was 20 to 30% of the total reductase activity. When cell cultures entered late log phase of growth the activity of the cytosolic form of the enzyme disappeared, but the chloroplastic form remained. The cytosolic DHAP reductase from Dunalieila has some properties similar to the cytosolic isoform from spinach leaves. Detergents inhibited both enzymes. However, neither form of the algal dihydroxyacetone phosphate reductase was stimulated by fructose 2,6-bisphosphate. In DunalielIa the properties of the chloroplastic form were those expected for glycerol production for osmoregulation, whereas the cytosolic form, like the reductases in leaves, is more likely involved in glycerol phosphate formation for lipid synthesis.We (4) and others (1,7,11,14) have also investigated an extremely active DHAP reductase in Dunaliella chloroplasts, whose major function has been considered to be to produce glycerol for osmoregulation. The chloroplast form from Dunaliella has different properties than the two leaf forms, but it was stimulated by E. coli thioredoxin (4). This major Dunaliella enzyme was stimulated by 25 mM Mg2' or 250 mm salts, such as KCl, NaCl, or buffer, whereas both of the higher plant isoforms were inhibited by these salts. In many investigations with Dunaliella only one form of DHAP reductase had been detected. The presence of two forms of DHAP reductase in leaves in different subcellular locations suggested that Dunaliella also might have other forms of DHAP reductase, particularly a cytosolic form, for lipid synthesis.This report describes a second more labile form of DHAP reductase from Dunaliella which is located in the cytosol. The cytosolic form is less active than the major chloroplast form and measureable activity was not detectable under physiological growth conditions that did not support rapid growth. Partial isolation of the cytosolic isoform of DHAP reductase in Dunaliella and a description of its properties are presented.

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