Purification of ornithine decarboxylase from Physarum polycephalum with characterization and quantitation of the catalytic unit utilizing 14 C-αdifuoromethylornithine
Wilson, J. Michael
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Ornithine decarboxylase (L-ornithine carboxy-lyase, E.C. 220.127.116.11) is the rate limiting enzyme of the polyamine biosynthetic pathway in eukaryotic cells. The enzyme's lability and low intracellular concentration (<0.02% of total protein) has, in the past, hampered attempts to obtain sufficient quantities of highly purified enzyme fractions for biochemical analyses. Consequently the molecular properties of ornithine decarboxylase are not well understood. In this project, large-scale culturing of Physarum polycephalum was achieved, making available log-phase microplasmodia in large quantities. This allowed subsequent extensive purification of ornithine decarboxylase in quantities needed for biochemical analyses. From fermenter-grown microplasmodia (60 liters) active form (A-form) ornithine decarboxylase«has been purified greater than 4-900 fold by acetone precipitation and column chromatography with DEAE-Cellulose, Sephacryl S-300, DEAE-Sepha- cel, Ultrogel AcA-34- and hydroxylapatite. Quantitative measurement of the catalytic unit has been established with the use of the radioactive, enzyme activated, irreversible inhibitor ¹⁴C-αDFMO. The molecular weight of the catalytic unit has been conclusively demonstrated to be approximately 52,000 in SDS polyacrylamide gel analysis. The specific activity of the catalytic unit has been calculated to be 1.62 x 10⁶ units/mg. The specific activity of the most highly purified enzyme fractions (1.18 x 10⁶ units/mg) reflects the level of purification achieved (4-990 fold) in attempts to obtain active enzyme homogeneity (6807 fold). These results have directly characterized a purification system by which high concentrations of active ornithine decarboxylase are obtainable along with the demonstration of the specific activity and molecular weight of the catalytic unit. We have introduced a novel technique for specific measurement of the catalytic unit of the enzyme and a compound which will serve as a powerful tool in further studies involving ornithine decarboxylase.