Characterization of arginine decarboxylase from Dianthus caryophyllus.
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 (14)C-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%.
Ha
Byung Hak
BH
Department of Biotechnology, Graduate School of Biotechnology, Korea University, Seoul 136-701, Republic of Korea.
Cho
Ki Joon
KJ
Choi
Yu Jin
YJ
Park
Ky Young
KY
Kim
Kyung Hyun
KH
D002118:Calcium / Q000737:chemistry / Q000494:pharmacology; D002262:Carboxy-Lyases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D029749:Dianthus / Q000201:enzymology* / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry / Q000494:pharmacology; D019216:Metals, Heavy / Q000737:chemistry / Q000494:pharmacology; D010940:Plant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011700:Putrescine / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013095:Spermidine / Q000494:pharmacology; D013096:Spermine / Q000494:pharmacology
D004791:Enzyme Inhibitors; D019216:Metals, Heavy; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D013096:Spermine; D002262:Carboxy-Lyases; C019722:arginine decarboxylase; D008274:Magnesium; D002118:Calcium; D013095:Spermidine; D011700:Putrescine