Purification and characterization of monodehydroascorbate reductase from soybean root nodules.
Soybean (Glycine max (L.) Merr.) root nodules contain the enzymes of the ascorbate-glutathione cycle as an important defense against activated forms of oxygen. A key enzyme in this cycle--monodehydroascorbate reductase (MR)--was purified 646-fold and appeared as a single band on SDS-PAGE with silver or Coomassie blue staining. Purified MR contained 0.7 mol FAD/mol enzyme and had a specific activity of 288 mumol NADH oxidized.min-1.mg protein-1. The enzyme was a single subunit occurring as two isozymes (MR I and MR II) with Mr values of 39,000 and 40,000. Isoelectric focusing revealed that each isozyme consisted of two forms with pl values of 4.6 to 4.7. Ferricyanide and 2,6-dichlorophenol-indophenol were effective as electron acceptors. The purified enzyme did not possess leghemoglobin reductase activity. Inhibition by p-chloromercuribenzoate indicated the involvement of a thiol group in MR activity. The Km values were 5.6, 150, and 7 microM for NADH, NADPH, and monodehydroascorbate, respectively. The pH optimum was 8 to 9. The N-terminal sequence of 10 amino acids of MR II had little homology to known protein sequences.
Dalton
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DA
Department of Biology, Reed College, Portland, Oregon 97202.
Langeberg
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Robbins
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M
D000595:Amino Acid Sequence; D002384:Catalysis; D008969:Molecular Sequence Data; D009247:NADH, NADPH Oxidoreductases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification*; D010940:Plant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification*; D013025:Glycine max / Q000737:chemistry / Q000201:enzymology*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D010940:Plant Proteins; D009247:NADH, NADPH Oxidoreductases; C023927:monodehydroascorbate reductase (NADH)