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 ...