Two Isozymes of Dihydroxyacetone Phosphate Reductase in Dunaliella

Abstract

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.

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

“…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). More recently, we have described an active chloroplastic as well as a minor cytosolic isozyme of DHAP reductase in Dunaliella (Gee et al, 1989). The activity in the Dunaliella chloroplastic fraction was stimulated by thioredoxin, NaC1, and Mg2+.…”

“…It has been reported previously that the activity in the chloroplasts of Dunaliella could use either NADH or NADPH without realizing that there were two isoforms present (Marengo et al, 1985;Gee et al, 1988~). However, as shown in Table I11 and Figure 2, both isoforms in the chloroplast can reduce DHAP with either NAClH or NADPH.…”

“…Substrate Specificity, pH Optimum, and K,,, Values DHAP reductase isoforms from leaves are highly specific for NADH (Gee et al, 1988~). It has been reported previously that the activity in the chloroplasts of Dunaliella could use either NADH or NADPH without realizing that there were two isoforms present (Marengo et al, 1985;Gee et al, 1988~).…”

“…In our previous work (Gee et al, 1989), the elution profile for the chloroplast DHAP reductase from DEAE cellulose chromatographic columns often was not symmetrical, indicating that there might be two chloroplastic forms. We have now been able to separate two isoforms from the Dunaliella chloroplast by modifying the DEAE chromatography and by varying the composition of the growth media to alter the ratio of the activities in the chloroplasts.…”

“…NaCl inhibits the chloroplastic isoform of DHAP reductase in higher plants, but activates the algal chloroplastic isoform (Marengo et al, 1985;Gee et al, 1988~). Because of the different properties of the chloroplastic isoform in Dunaliella and in higher plants, we postulated that there may be different isoforms of DHAP reductase specifically involved in glycerol and glyceride synthesis.…”