Differential Inhibition and Activation of Two Leaf Dihydroxyacetone Phosphate Reductases

Abstract

The chloroplastic and cytosolic forms of spinach (Spinacia oleracea cv Long Standing Bloomsdale) leaf NADH:dihydroxyacetone phosphate (DHAP) reductase were separated and partially purified. The chloroplastic form was stimulated by dithiothreitol, reduced thioredoxin, dihydrolipoic acid, 6-phosphogluconate, and phosphate; the cytosolic isozyme was stimulated by fructose 2,6-bisphosphate but not by reduced thioredoxin. End product components that severely inhibited both forms of the reductase included lipids and free fatty acids, membranes, and glycerol phosphate. In addition, two groups of inhibitory peptides were obtained from the fraction precipitated by 70 to 90% saturation with (NH4)2SO4. Chromatography of this fraction on Sephadex G-50 revealed a peptide peak of about 5 kilodaltons which inhibited the chloroplastic DHAP reductase and a second peak containing peptides of about 2 kilodaltons which inhibited the cytosolic form of the enzyme. Regulation of the reduction of dihydroxyacetone phosphate from the C3 photosynthetic carbon cycle or from glycolysis is a complex process involving activators such as thioredoxin or fructose 2,6-bisphosphate, peptide and lipid inhibitors, and intermediary metabolites. It is possible that fructose 2,6-bisphosphate increases lipid production by stimulating DHAP reductase for glycerol phosphate production as well as inhibiting fructose 1,6-bisphosphatase to stimulate glycolysis. tors are so effective, no activity had earlier been detectable in crude leaf homogenates or chloroplast preparations (3, 6). Enzyme activity could only be measured by first precipitating a protein fraction with 35 to 70% saturated (NH4)2SO4 followed by dialysis. However, it has now been possible to detect the DHAP reductase in a crude homogenate after centrifugation at 110,000g for 1 hr and addition of BSA to remove most lipids and membranes and addition of DTI to stimulate the chloroplastic form.In addition to NADH:DHAP reductase, thioredoxin regulates the activity of a number of chloroplast enzymes, and, therefore, stimulation of the DHAP reductase isozyme in the chloroplast by thioredoxin is consistent with previous findings with other chloroplast enzymes. An increase in the activity of DHAP reductase would lead to increased production of glycerol phosphate, which in turn would be used for lipid synthesis or might yield glycerol by dephosphorylation.The present study indicates that Fru 2,6-P2 stimulates the cytoplasmic DHAP reductase, which could also result in increased lipid synthesis or amounts of glycerol in the cytoplasm. Recent investigations (7-11) have indicated that Fru 2,6-P2 also inhibits cytosolic Fru 1,6-P2 phosphatase to block the formation of sucrose and promote glycolysis. Thus, regulation of the cytosolic DHAP reductase isozyme appears to be another cytoplasmic systems that is regulated by Fru 2,6-P2. MATERIALS AND METHODSThere are two forms of sn-3-phosphoglycerol dehydrogenase (EC 1.1.1.8) or NADH:DHAP2 reductase in leaves (3,6). In the leaf, the isozyme in the chloroplast...

Two Isozymes of Dihydroxyacetone Phosphate Reductase in Dunaliella

“…The chloroplast form was stimulated fivefold by spinach thioredoxin but only threefold by Escherichia coli thioredoxin (2). The cytoplasmic form was stimulated twofold by Fru 2,6-P2 and activity was further enhanced by the presence of Mg2' at a 1:1 ratio of Mg2+ to Fru 2,6-P2 (3,6). The levels of activity of the two DHAP reductases in leaves depended on the physiological state of the plant (5 …”

“…Chloroplast and cytoplasmic forms ofthe DHAP2 reductase activity of sn glycerol-3-phosphate:NAD oxidoreductase (EC 1.1.1.8) have been isolated from higher plants (2)(3)(4)(5). The chloroplast form was stimulated fivefold by spinach thioredoxin but only threefold by Escherichia coli thioredoxin (2).…”

Two Isoforms of Dihydroxyacetone Phosphate Reductase from the Chloroplasts of Dunaliella tertiolecta

“…Because the enzyme was from the late log phase, the glyceride form had less activity. the two forms were differentially regulated by inhibitors and activators of the glyceride form as previously described for leaf chloroplasts (Gee et al, 1988b). The glyceride form was inhibited by detergents, lipids, or long-chain acyl-COA derivatives, regardless of whether it was isolated from chloroplasts, from leaves, or from Dunaliella.…”

“…Leaves of higher plants contain both a chloroplastic and a cytoplasmic DHAP reductase form (Gee et al, 1988a(Gee et al, , 1988b. The presence of DHAP reductase has also been reported in Dunaliella tertiolecta (Haus and Wegman, 1984;Marengo et al, 1985; and Dunaliella parva (Gimmler and Lotter, 1982), and the enzyme was considered to be localized in the chloroplast (Brown et al, 1982;Gimmler and Lotter, 1982).…”

“…The activity as the glyceride form i n leaf homogenates could not be measured until after DEAE cellulose chromatography, due to the remova1 of low mo1 wt inhibitors, such as fatty acids and other membrane components (Gee et al, 1988a(Gee et al, , 1988b. However, the Dunaliella osmoregulator form was not inhibited by fatty acids and membrane components (next sections), so the nature of the inhibitors in the Dunaliella extract remains unknown.…”