PURIFICATION AND CHARACTERIZATION OF BOVINE LIVER ORNITHINE DECARBOXYLASE

Abstract

Ornithine decarboxylase has been purified to apparent homogeneity from thioacetamide-stimulated calf liver. The purification process, which has been developed to circumvent the lability of the enzyme, employs ion exchange chromatography, gel filtration, hydroxylapatite chromatography, non-denaturing gel electrophoresis, and sulfhydryl affinity chromatography. The enzyme is purified 71,500-fold to a final specific activity of 286,000 pmol/min/mg protein. Non-denaturing gel electrophoresis indicates a single protein present in the final preparation. The enzyme has a Stokes radius of 3.14 nm as indicated by gel filtration and a monomeric molecular weight of 52,000 daltons as indicated by denaturing gel electrophoresis. The K(m) values for ornithine and pyridoxal phosphate are 0.16 mM and 2.5 μM, respectively. Putrescine inhibits the enzyme (Kᵢ 10mM). The existence of three ionic forms of ornithine decarboxylase is suggested by fractionation of the preparation by gradient sievorptive chromatography. Mammalian ornithine decarboxylase is apparently a metalloenzyme. A variety of structurally distinct metal chelators inhibit the enzyme. A non-chelating analog of the most potent chelator, 1,10-phenanthroline, is without effect. The order of efficacy of the chelators suggests the involvement of a metal from the transition series. Incubation of the enzyme with charcoal or Cibacron Blue-Agarose results in a loss of catalytic activity suggesting that the ornithine decarboxylase may also contain a bound nucleotide.