The rapid separation of orotate, orotidylate, and uridylate by thin-layer chromatography: Utility in the assay of orotate phosphoribosyltransferase and orotidylate decarboxylase

✍ Scribed by Philip Reyes

• Publisher: Elsevier Science
• Year: 1977
• Tongue: English
• Weight: 498 KB
• Volume: 77
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(77)90249-4

✦ Synopsis

A thin-layer chromatographic method is described that achieves the complete separation of orotidine 5'-monophosphate (OMP), uridine .5'-monophosphate (UMP), and orotate. This is accomplished by developing polyethyleneimine (PEI)-cellulose with 0.2 M LiCI. OMP is retained at or near the origin whereas UMP and orotate migrate with R, values of 0.33 and 0.59, respectively. This method is well suited for studying the conversion of orotate into OMP by orotate phosphoribosyltransferase (PRTase) and the subsequent conversion of OMP into UMP by OMP decarboxylase. The synthesis of OMP and UMP by enzyme preparations from murine leukemia P1534J, mouse liver, and baker's yeast has been quantitated with this method. The results of this study showed that OMP does not normally accumulate in reaction mixtures containing either the mouse liver or P1534J enzyme preparations. This finding is compatible with the presence of a bifunctional enzyme complex comprised of orotate PRTase and OMP decarboxylase. Such a complex is known to exist in P1534J cells and is currently thought to exist in most if not all mammalian tissues. The stoichiometric conversion of orotate into UMP by this complex could easily account for the observed lack of OMP.

Orotate phosphoribosyltransferase (EC 2.4.2.10) and orotidylate decarboxylase (EC 4.1.1.23) participate in the de HOWI synthesis of pyrimidine nucleotides (1). These two enzymes catalyze Reactions [l] and [2], respectively.

Orotate + PP-ribose-P s OMP + PPi, ill OMP --, UMP + CO,.

PI

Assays for orotate PRTase* often involve the overall conversion of orotate into UMP. This process can be followed either spectrophoto-