Polyamine synthesis in plants: isolation and characterization of spermidine synthase from soybean (Glycine max) axes |
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Authors: | Sang Oh Yoon Yong Sun Lee Sung Ho Lee Young Dong Cho |
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Affiliation: | Department of Biochemistry, College of Science, Yonsei University, Seoul 120-749, South Korea |
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Abstract: | Spermidine synthase (EC 2.5.1.16) was purified to homogeneity for the cytosol of soybean (Glycine max) axes using ammonium sulfate fractionation and chromatography on DEAE-Sephacel, Sephacryl S-300, ω-aminooctyl-Sepharose and ATPA-Sepharose. The molecular mass of the enzyme estimated by gel filtration and SDS–PAGE is 74 kDa. Cadaverin and 1,6-diaminohexane could not replace putrescine as the aminopropyl acceptor. Kinetic behaviors of the substrate are consistent with a ping pong mechanism. The kinetic mechanism is further supported by direct evidence confirming the presence of an aminopropylated enzyme and identification of product, 5′-deoxy-5′-methylthioadenosine, prior to adding putrescine. The Km values for decarboxylated S-adenosylmethionine and putrescine are 0.43 μM and 32.45 μM, respectively. Optimum pH and temperature for the enzyme reaction are 8.5 and 37°C, respectively. The enzyme activity is inhibited by N-ethylmaleimide and DTNB, but stimulated by Co2+, Cu2+ and Ca2+ significantly, suggesting that these metal ions could be the cellular regulators in polyamine biosynthesis. |
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Keywords: | Spermidine synthase Purification Ping pong mechanism MTA, 5′-deoxy-5′-methylthioadenosine RIA, radioimmunoassay DTT, dithiothreitol MTA–BSA, 5′-deoxy-5′-methylthioadenosine-conjugated bovine serum albumin TLC, thin layer chromatography PBS, phosphate-buffered saline ACC, 1-aminocyclopropane-1-carboxylic acid |
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