In plant metabolism glycerol phosphate must be produced for the synthesis of various phospholipids, sulfolipids, galactolipids, and triglycerides. The metabolic reaction in leaf tissue of higher plants for glycerol phosphate synthesis is catalyzed by a DHAP2 reductase which has been partially purified from spinach leaves and castor bean endosperm, and which catalyzes the reduction of DHAP at pH 7.0 using NADH as the reductant (6,17 No. 12290. 2Abbreviations: DHAP, dihydroxyacetone phosphate; ME, mercaptoethanol; PVPP, polyvinylpolypyrrolidone. metabolism must be highly controlled, as is expected of compounds and enzymes at metabolic branch points.A number of free living algae produce glycerol as a major product of photosynthesis rather than accumulate starch or sucrose. Zooxanthellae, symbiotic, unicellular algae in the polyps of reef building corals and other marine invertebrates, excrete to their host up to 40% of their photosynthate as glycerol (14, 15).The halotolerant alga, Dunaliella tertiolecta (5,20) and Chiamydomonas (1 1)

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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...

A dihydroxyacetone phosphate (DHAP) reductase has been isolated in 50% yield from Dunaliella tertiokecta by rapid chromatography on diethylaminoethyl cellulose. The activity was located in the chloroplasts. The enzyme was cold labile, but if stored with 2 molar glycerol, most of the activity was restored at 30°C after 20 minutes. The spinach (Spinacia oleracea L.) reductase isoforms were not activated by heat treatment. Whereas the spinach chloroplast DHAP reductase isoform was stimulated by leaf thioredoxin, the enzyme from Dunaliella was stimulated by reduced Escherichia coli thioredoxin. The reductase from Dunaliella was insensitive to surfactants, whereas the higher plant reductases were completely inhibited by traces of detergents. The partially purified, coldinactivated reductase from Dunaliella was reactivated and stimulated by 25 millimolar Mg2+ or by 250 millimolar salts, such as NaCI or KCI, which inhibited the spinach chloroplast enzyme. Phosphate at 3 to 10 millimolar severely inhibited the algal enzyme, whereas phosphate stimulated the isoform in spinach chloroplasts. Phosphate inhibition of the algal reductase was partially reversed by the addition of NaCI or MgCl2 and totally by both. In the presence of 10 millimolar phosphate, 25 millimolar MgC92, and 100 millimolar NaCl reduced thioredoxin causes a further twofold stimulation of the algal enzyme. The Dunaliella reductase utilized either NADH or NADPH with the same pH maximum at about 7.0. The apparent K. (NADH) was 74 micromolar and K.(NADPH) was 81 micromolar. Apparent V.,,,, was 1100 umoles DHAP reduced per hour per milligram chlorophyll for NADH, but due to NADH inhibition highest measured values were 350 to 400. The DHAP reductase from spinach chloroplasts exhibited little activity with NADPH above pH 7.0. Thus, the spinach chloroplast enzyme appears to use NADH in vivo, whereas the chloroplast enzyme from Dunaliella or the cytosolic isozyme from spinach may utilize either nucleotide.

Young or mature rosette leaves from spinach (Spinacia oleracea L.) plants growing in the field, in the greenhouse, or in a growth chamber under a regimen of 8 hours light and 16 hours dark contained 15 to 50 nanomoles per minute per gram wet weight of NADH:dihydroxyacetone phosphate reductase activity. Of this activity, 75 to 87% was the chloroplastic isoform and 25 to 13% was the cytosolic form. When plants were induced to senesce, as measured by stem elongation and flowering, the percentage of the two reductase isoforms in rosette or stem leaves changed to about 12% as the chloroplastic and 88% as the cytosolic isoform. The change in enzyme activity of the rosette leaves occurred within 3 days, before phenotypic changes were observed. Likewise, when plants senesced in continuous darkness, the percentage of chloroplastic to cytosolic reductase changed from 80:20% to 25:75% after 62 hours before changes in total protein or chlorophyll occurred. The ratio of activities did not change in the first 16 hours of darkness or ovemight. In each case the change in ratio resulted from about a 75% decrease in activity of the chloroplastic isoform and up to 14-fold increase in cytosolic isoform. In spinach leaves purchased at a local market primarily only the cytosolic isoform remained. When plants were retumed to normal day-nights, after 62 hours in continuous darkness, the activity of the chloroplastic isoform increased, but not to control levels after 3 days, while the cytosolic enzyme decreased within 1 day to normal day-night values. Changes in activity were not due to changes during In vitro assays in activation by thioredoxin for the chloroplastic isoform or fructose 2,6-phosphate for the cytosolic isoform.