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We purified and characterized glutaredoxin (thioltransferase), which catalyzes thiol/disulfide exchange reaction, for the first time in plants. The purification procedure employed an immunoabsorbent, antiglutaredoxin-Sepharose. Glutaredoxin was purified about 2,200-fold from rice bran and it appeared to be homogeneous on SDS-PAGE. MALDI-TOF mass spectrometry revealed that the protein has a molecular mass of 11,097.9 Da. Rice glutaredoxin consists of 105 amino acid residues, containing the tetrapeptide -Cys-Phe-Pro (Tyr)-Cys-, which constitutes the active site of Escherichia coli and mammalian glutaredoxins. Inactivation assay also indicated that cysteine residues are responsible for enzyme activity. Kinetic analyses revealed that the enzyme did not exhibit normal Michaelis-Menten kinetics. The enzyme has an optimum pH of about 8.7 with 2-hydroxyethyl disulfide as a substrate. In addition, rice glutaredoxin has dehydroascorbate reductase activity, like mammalian glutaredoxin.

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Enterobacter aerogenes IFO 12010 contains a carbon-phosphorus (C-P) bond cleavage enzyme catalyzing the liberation of inorganic phosphate from various alkyl-and phenylphosphonic acids. The enzyme in the bacterium was found to be composed of two physically different protein components, E2 and E3. The molecular weights of E2 and E3 were 560,000 and 110,000, respectively, and E3 was resolved into two apparently homogeneous subunits. Neither component alone could catalyze the C-P bond cleavage reaction, but the reaction was efficiently catalyzed when the components were mixed.Since the discovery of a natural phosphonic acid, 2-aminoethylphosphonic acid, in 1959 (8), various kinds of phosphonic acids containing a direct carbon-phosphorus (C-P) bond have been found in many living systems. Insecticides, herbicides, fungicides, nerve gases, flame retardants, and several other important synthetic chemicals also contain the C-P bond and have been used in large quantities. Since the C-P bond is highly resistant to chemical hydrolysis and thermal decomposition, the biological breakdown of phosphonic acids is important not only for biochemical studies but also for prevention of overaccumulation of these acids in nature. To date, however, the metabolic route of these unique biological and synthetic molecules has remained obscure because of a failure to detect the enzyme activity responsible for the C-P bond cleavage reaction in cell-free systems (1-7, 14, 17, 18).Recently, we found a C-P bond cleavage enzyme in cell extracts of Enterobacter aerogenes IFO 12010 (15). The enzyme in the bacterium could cleave the C-P bond in various alkyl-and phenylphosphonic acids (such as methylphosphonic acid, phosphonoacetic acid, and phenylphosphonic acid) and was shown to be distinct from the enzyme phosphonatase, which hydrolyzes the C-P bond in phosphonoacetaldehyde (9-11).In order to characterize the properties of the enzyme and elucidate the C-P bond cleavage mechanism, we attempted to purify the C-P bond cleavage enzyme from E. aerogenes IFO 12010. The cells were aerobically grown on a medium [0.5% glucose, 0.1% (NH4)2SO4, 0.01% MgSO4 7H20, 0.02% yeast extract (Pi-free), and 0.4% phosphonoacetic acid (pH 7.2)] at 30°C for 20 h. The cells (38 g [wet weight]) from a 15-liter culture were suspended in 80 ml of 10 mM Tris hydrochloride buffer (pH 7.5) containing 10 mM MgCl2 and 0.2 mM dithiothreitol (buffer A); cell extract was prepared as described previously (15). Pi-free yeast extract was prepared as described previously (15). Phosphonic acids were used after filtration with a membrane filter (Millipore Corp., Bedford, Mass.), and glasswear was rinsed with nitric acid before use.The dialyzed cell extract (protein, 5,600 mg; 130 ml) was applied to a DEAE-cellulose column (4 by 60 cm) equilibrated with buffer A, and proteins were eluted with a linear gradient of KCl (0 to 0.6 M, 4,000 ml) in buffer A. A 20-ml portion was collected every 9 min. The enzyme eluted as a * Corresponding author.single active peak at about 0.05 M KCI. The activ...