Chloroplastic glyceride isoform of dihydroxyacetone phosphate reductase from Dunaliella tertiolecta : purification and characterization

摘要

The Chloroplastic glyceride isoform of dihydroxyacetone phosphate reductase (Gly-DHAPR) in the photosynthetic unicellular green algae, Dunaliella, plays key role in the synthesis of glycerol-P and glycerides. A four-step procedure has been developedto purify the Gly-DHAPR from the chloroplasts of Dunaliella tertiolecta. The enzyme was purified 462-fold to apparent electrophoretic homogeneity by precipitation of Rubisco by polyethylene glycol-4000, and successive chromatography on DEAE cellulose, Sephacryl S-200, and Red Agarose. The overall yield of the purified enzyme was 5.1 percent with a specific activity of 425 #mu#mol . min~(-1) . mg~(-1) protein, and a subunit molecular mass of 37 kD. The Gly-DHAPR had little preference for NADH or NADPH,but was highly specific for DHAP. The purified enzyme was slightly stimulated by 50 mM NaCl, KCl or by 25 mM MgCl_2. Detergents, lipids, fatty acids, or long-chain acyl-CoA derivatives inhibited the Gly-DHAPR. The Gly-DHAPR differs in properties from theother chloroplastic osmoregulatory isoform of DHAP reductase from Dunaliella, but has significant similarities with the glyceride isoforms from higher plants for glycerol-P and triglyceride synthesis.