Abstract
Arginine decarboxylase (ADC, EC 4.1.1.9) is a key enzyme in the biosynthesis of polyamines in higher plants, whereas ornithine decarboxylase represents the sole pathway of polyamine biosynthesis in animals. Previously, we characterized a genomic clone from Dianthus caryophyllus, in which the deduced polypeptide of ADC was 725 amino acids with a molecular mass of 78 kDa. In the present study, the ADC gene was subcloned into the pGEX4T1 expression vector in combination with glutathione S-transferase (GST). The fusion protein GST–ADC was water-soluble and thus was purified by sequential GSTrap–arginine affinity chromatography. A thrombin-mediated on-column cleavage reaction was employed to release free ADC from GST. Hiload superdex gel filtration FPLC was then used to obtain a highly purified ADC. The identity of the ADC was confirmed by immunoblot analysis, and its specific activity with respect to 14C-arginine decarboxylation reaction was determined to be 0.9 CO 2 pkat mg –1 protein. K m and V max of the reaction between ADC and the substrate were 0.077 ± 0.001 mM and 6.0 ± 0.6 pkat mg –1 protein, respectively. ADC activity was reduced by 70% in the presence of 0.1 mM Cu 2+ or CO 2+, but was only marginally affected by Mg 2+, or Ca 2+ at the same concentration. Moreover, spermine at 1 mM significantly reduced its activity by 30%.
Published Version
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