248 Charles Heidelberger and Van R. Potter 



prevent further oxidation of the a-ketoglutarate, which is 

 isolated and purified as the dinitrophenylhydrazone. Malo- 

 nate is also added to the homogenate to eliminate the possi- 

 bility of any recycling. After determination of the specific 

 activity, the compound is oxidized with acid permanganate 



COg + CH^COCOOH 



HOoScHpCO + (-CH9COX) 



COOH '^ 



I 65^ j 



BOOCCH2COHCH2COOH 



Rat Kidney 

 Homogenate I. 



COOH 



Rat Kidney Homogenate II. 



HOC5COCH2CH2COOH 



KMnOi^ 



CO2 + HOOCCH2CH2COOH 

 lOOJg <.01Jg 



Fig. 3. Distribution of CO2 fixed into citrate and a-keto- 

 glutarate (Potter and Heidelberger, 1949). 



to CO2 and succinic acid, and the radioactivity in each com- 

 pound is measured. 



Our experiment was confirmed and extended by the elegant 

 work of Lorber, Utter, Rudney and Cook (1950) as indicated 

 in Fig. 4. They used oxaloacetate as the labelled substrate, 

 partially purified enzyme preparations, and degraded the 

 citric acid chemically to distinguish between the radioactivity 

 in the primary and the tertiary carboxyl groups. We have 

 adopted the latter practice in our more recent experiments. 



In order to illustrate more fully the stereochemical implica- 

 tions of this work a consideration of Fig. 5 will be helpful. 

 Ogston pointed out the necessity of assuming that there is at 



