Purification of an Enzyme with Two Catalytic Activities

The last two steps in the biosynthesis of the mononu- cl cotide uridine 5 -monophosphate (UMP) are catalyzed by (1) orotate phosphoribosyltransferase (OPRTase) and (2) orotate 5 -monophosphate decarboxylase (OMPDase). 

Mary Ellen Jones and her colleagues set out to purify the enzyme or enzymes involved in these two reactions. Their main goal was to determine whether the two reactions are carried out by one protein or more than one. Their findings indicated that the two reactions were both catalyzed by the same enzyme, consisting of a single polypeptide chain. To demonstrate this fact, it was necessary to monitor both enzyme activities at each step in the purification and show that both activities copurified. For this purpose, Jones used specific enzyme assays for both enzyme activities. All fractions were assayed for both enzymatic activities at each stage of the purification. 

The main data associated with the purification are summarized in table 6.4. This table indicates the total protein obtained in each step, the number of enzyme units3 for each enzyme, and the ratio of enzyme units to total protein, called the specific activity. In the absence of enzyme inactivation, the specific activity should be directly proportional to the enrichment. The percent recovery refers to the amount of enzyme activity in the indicated fraction, as compared with the amount present in fraction 1. This number is usually less than 100%. The apparent losses may reflect actual losses of enzyme during purification, or they may reflect inactivation (usually due to unknown causes) of the enzyme during purification. 

The nine steps involved in the purification of UMP synthase from starting tissue are outlined in figure 6.8. All steps were carried out at 0-5°C. About 200 g of Ehrlich ascites cells, a mammalian tumor rich in the desired enzymes, was suspended in buffer and processed in a tissue homogenizer, which mechanically breaks down the tissue 

Outline of Purification of UMP Synthase from Ehrlich s Ascites Carcinoma 

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