Plasmodium falciparum: purification, properties, and immunochemical study of ornithine decarboxylase, the key enzyme in polyamine biosynthesis.
Ornithine decarboxylase, the rate-limiting enzyme in the polyamine biosynthetic pathway has been purified 7,600 fold from Plasmodium falciparum by affinity chromatography on a pyridoxamine phosphate column. The partially purified enzyme was specifically tagged with radioactive DL-alpha-difluoromethylornithine and subjected to polyacrylamide gel electrophoresis under denaturing conditions. A major protein band of 49 kilodalton was obtained while with the purified mouse enzyme, a typical 53 kilodalton band, was observed. The catalytic activity of parasite enzyme was dependent on pyridoxal 5'-phosphate and was optimal at pH 8.0. The apparent Michaelis constant for L-ornithine was 52 microM. DL-alpha-difluoromethylornithine efficiently and irreversibly inhibited ornithine decarboxylase activity from P. falciparum grown in vitro or Plasmodium berghei grown in vivo. The Ki of the human malarial enzyme for this inhibitor was 16 microM. Ornithine decarboxylase activity in P. falciparum cultures was rapidly lost upon exposure to the direct product, putrescine. Despite the profound inhibition of protein synthesis with cycloheximide in vitro, parasite enzyme activity was only slightly reduced by 75 min of treatment, suggesting a relatively long half-life for the malarial enzyme. Ornithine decarboxylase activity from P. falciparum and P. berghei was not eliminated by antiserum prepared against purified mouse enzyme. Furthermore, RNA or DNA extracted from P. falciparum failed to hybridize to a mouse ornithine decarboxylase cDNA probe. These results suggest that ODC from P. falciparum bears some structural differences as compared to the mammalian enzyme.
D000818:Animals; D002846:Chromatography, Affinity; D004247:DNA; D000518:Eflornithine / Q000378:metabolism; D005057:Eukaryotic Cells / Q000201:enzymology; D006801:Humans; D007106:Immune Sera; D008322:Mammals; D009693:Nucleic Acid Hybridization; D009955:Ornithine Decarboxylase / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D065108:Ornithine Decarboxylase Inhibitors; D010962:Plasmodium berghei / Q000201:enzymology; D010963:Plasmodium falciparum / Q000201:enzymology*; D011073:Polyamines / Q000096:biosynthesis; D012333:RNA, Messenger
D007106:Immune Sera; D065108:Ornithine Decarboxylase Inhibitors; D011073:Polyamines; D012333:RNA, Messenger; D004247:DNA; D009955:Ornithine Decarboxylase; D000518:Eflornithine