Purification of orotate phosphoribosyltransferase and orotidylate decarboxylase by affinity chromatography on Sepharose dye derivatives

✍ Scribed by Philip Reyes; Richard B. Sandouist

• Publisher: Elsevier Science
• Year: 1978
• Tongue: English
• Weight: 723 KB
• Volume: 88
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(78)90452-9

✦ Synopsis

Blue Dextran-Sepharose and Cibacron Blue F3GA-Sepharose (Blue Sepharose) were found to act as affinity adsorbents for orotate phosphoribosyltransferase (PRTase) and orotidine 5'-monophosphate (OMP) decarboxylase from bakers' yeast. Experiments with columns of Blue Dextran-Sepharose and partially purified preparations of the PRTase and decarboxylase revealed that both enzymes were selectively eluted by a low concentration (0.1-2 mM) of their respective substrate or immediate product. On the other hand, a much higher concentration (50-400 mM) of NaCl was required to displace these two enzymes from the above columns. Larger scale experiments showed that OMP decarboxylase in crude extracts was purified about 5700-and 6600-fold on Blue Sepharose using 0.5 mM OMP and 2 mM uridine 5'-monophosphate (UMP) as the eluting ligand, respectively. In contrast, orotate PRTase did not bind to Blue Sepharose unless crude extracts were first subjected to gel filtration. The resulting preparation of orotate PRTase, purified about sixfold with respect to cell-free extracts, was purified an additional 200-and 40-fold when the enzyme was eluted from Blue Sepharose with 0.5 mM OMP and 1 mM 5-phosphoribosyl I-pyrophosphate (PP-ribose-P), respectively. Blue Dextran-Sepharose, on the other hand, was found to provide a lower degree of enzyme purification and exhibited a lower sample-binding capacity. Samples of the PRTase and decarboxylase that had been purified about 200-and 6000-fold, respectively, on Blue Sepharose displayed a major protein band and one or more minor bands when subjected to polyacrylamide gel electrophoresis. Enzyme activity coincided with the major band in all cases.