Radioassay of orotic acid phosphoribosyltransferase and orotidylate decarboxylase utilizing a high-voltage paper electrophoresis technique or an improved 14CO 2-release method

Abstract

Orotic acid phosphoribosyltransferase (EC 2.4.2.10) and orotidylate decarboxylase (EC 4.1.1.23) can be assayed independently of one another by the high voltage paper electrophoresis method described here, which separates orotic acid, OMP, and UMP, the substrates and/or products of these enzymes, from each other. The relative migration of other compounds, mainly other nucleotides, their bases, or other intermediates of the UMP biosynthetic pathway, has also been recorded. The method has allowed us to observe that OMP is not released to any significant degree from the enzyme complex of these two enzymes that occurs in Ehrlich ascites cells; rather orotic acid is converted stoichiometrically by the enzyme complex to UMP. For purification of the enzyme complex, we have found the release of 14CO 2 from [ 14C]carboxyl-labeled orotic acid (when phosphoribosyl pyrophosphate and Mg 2+ are present) preferable to the HVPE method as a routine assay procedure. The most economical CO 2-absorbant for the assay of the enzyme complex or for orotidylate decarboxylase (and possibly for other enzymes which release CO 2) is an NaOH-soaked paper strip. As detailed here, its use allows one to repeatedly reuse the scintillation vials and fluid.