G. ROBERT GREENBERG 



then followed by the purification of one enzyme from A in the 

 presence of fraction B, and then B is purified in the presence of 

 the purified A. Several difficulties arise: (7) the two enzymes 

 may be very similar in physical characteristics and both are not 

 easily separated in good yield; (2) the system may consist of 

 more that two enzymes which increases the diflficulties enor- 

 mously; (3) one of the enzymes is either very labile under the 

 conditions required for fractionating the other enzyme or is 

 limiting because of concentration or activity. 



Some of these difficulties may be overcome by looking for 

 another source of one of the enzymes in question. Romberg and 

 co-workers (18) have used this technique beautifully. Pigeon 

 liver contains an enzyme system which converts orotic acid to 

 uridylic acid at a slow rate. 



The reaction mechanism (18) is now known to be 



R-5-P + ATP — ^ PRPP + AMP 



2 



PRPP + orotic acid > orotidylic acid + P-P 



3 



Orotidylic acid > uridylic acid + CO2 



By adding yeast extracts to the pigeon liver system these 

 authors found very rapid synthesis of UMP. Reaction (1) 

 was found to be present in liver in good activity but (2) and (3) 

 were limiting and were supplied by the yeast system. Therefore 

 enzyme 1 could be purified by using 2 and 3 to analyze for the 

 products and then 2 and 3 could be fractionated. 



Purification of one system against another is not always so 

 straightforward. Ratner and co-workers (23) have studied the 

 condensation of aspartic acid with citrulline to form argininosuc- 

 cinic acid by an ATP-dependent reaction. In an effort to gain 

 insight into the mechanism of this reaction these workers have 

 separated the system into two parts, one from liver and the other 

 from yeast. It was desirable to employ another source such as 

 yeast for one of the enzymes, since the enzyme from liver was un- 

 stable. However, whereas both the yeast and the stable liver 

 enzymes could be fractionated to a rather good state of purity, 



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