Assay of Mammalian Ornithine Decarboxylase Activity Using [¹⁴C]Ornithine

Abstract

L-Ornithine decarboxylase (EC 4.1.1.17) (ODC) catalyzes the conversion of L-ornithine to putrescine and CO₂. ODC is dependent on pyridoxal 5′-phosphate (PLP) and thiol-reducing agents for activity (1). At least two key active site residues of ODC are known. Lysine 69 was recently identified as the residue in mouse ODC that forms a Schiff base with PLP and α-difluoromethyl-ornithine, an enzyme-activated irreversible inhibitor of ODC, forms adducts at both lysine 69 and cysteine 360 (2). It has been shown that mutation of cysteine 360 to alanine (C360A) results in an enzyme not only having greatly reduced activity, but the activity of this mutant is much more stable than wild-type ODC in the absence of dithiothreitol. The high dependence of ODC on thiol-reducing agents for activity is, therefore, likely to be a result of a need to maintain cysteine 360 in a reduced state (3).