Osmoregulatory isoform of dihydroxyacetone phosphate reductase from Dunaliella tertiolecta: purification and characterization.
The osmoregulatory isoform of dihydroxyacetone phosphate (DHAP) reductase (Osm-DHAPR) is an enzyme unique to Dunaliella, photosynthetic unicellular green algae adapted to extreme environments. This is the first report of purification of an isoform of DHAP reductase from Dunaliella, specifically the osmoregulatory isoform that is involved in the synthesis of free glycerol for osmoregulation in extreme environments, such as high salinity. The Osm-DHAPR is cold labile, inactivated by ammonium sulfate, forms a strong complex with Rubisco, and is unstable in the absence of glycerol. These difficulties have been addressed, and a four-step procedure has been developed to purify the Osm-DHAPR from Dunaliella tertiolecta: precipitation of Rubisco by polyethylene glycol, followed by successive chromatography on DEAE cellulose, Sephacryl S-200, and Red Agarose. Yield of the purified enzyme was 3.6%, with a specific activity of 938 micromol.min-1.mg-1 of protein and a subunit molecular mass of approximately 38 kDa. A maximum specific activity of 2580 micromol.min-1.mg-1 of protein could be achieved by assay with 150 mM NaCl. The Osm-DHAPR had little preference for NADH or NADPH, but it is highly specific for DHAP. Other metabolites of glycolysis, the tricarboxylic acid cycle, and the C3 reductive photosynthetic carbon cycle were not reduced by the enzyme. The purified enzyme was stimulated three-fold by 150 to 250 mM NaCl/KCl and by 25 mM MgCl2. Detergents, lipids, or long-chain acyl CoA derivatives, all of which inhibited the chloroplastic glyceride form of DHAP reductase, did not affect the activity of Osm-DHAPR. The Osm-DHAPR has different properties than the other chloroplastic isoform of DHAP reductase from plants and algae for glycerol phosphate formation and triglyceride synthesis.
Ghoshal
Durba
D
Department of Biology, College of Science and Engineering, Duluth, Minnesota 55812, USA.
Mach
David
D
Agarwal
Mamta
M
Goyal
Archana
A
Goyal
Arun
A
D000460:Chlorophyta / Q000201:enzymology*; D005993:Glycerolphosphate Dehydrogenase / Q000302:isolation & purification* / Q000378:metabolism; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism; D011189:Potassium Chloride / Q000378:metabolism; D012965:Sodium Chloride / Q000378:metabolism; D014882:Water-Electrolyte Balance
D007527:Isoenzymes; D012965:Sodium Chloride; D011189:Potassium Chloride; D005993:Glycerolphosphate Dehydrogenase; C096203:glycerol-3-phosphate dehydrogenase (NAD(P)+)