Polyamine synthesis in plants: isolation and characterization of spermidine synthase from soybean (Glycine max) axes

Spermidine synthase (EC 2.5.1.16) was purified to homogeneity for the cytos ol of soybean (Glycine max ) axes using ammonium sulfate fractionation and chromatography on DEAE-Sephacel, Sephacryl S-300, omega-aminooctyl-Sepharos e and ATPA-Sepharose. The molecular mass of the enzyme estimated by gel fil tration and SDS-PAGE is 74 kDa. Cadaverin and 1,6-diaminohexane could not r eplace putrescine as the aminopropyl acceptor. Kinetic behaviors of the sub strate are consistent with a ping pong mechanism. The kinetic mechanism is further supported by direct evidence confirming the presence of an aminopro pylated enzyme and identification of product, 5'-deoxy-5'-methylthioadenosi ne, prior to adding putrescine. The K-m values for decarboxylated S-adenosy lmethionine and putrescine are 0.43 mu M and 32.45 mu M, respectively. Opti mum pH and temperature for the enzyme reaction are 8.5 and 37 degrees C, re spectively. 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. (C) 2000 Elsevier Science B.V. All rights reserved.