VOL. 4 (1950) BIOCHIMICA ET BIOPHYSICA ACTA 205 



PARTIAL PURIFICATION OF ISOCITRIC DEHYDROGENASE AND 

 OXALOSUCCINIC CARBOXYLASE* 



by 



ALLAN L. GRAFFLIN** and SEVERO OCHOA 



Department of Pharmacology, New York University College of Medicine, 

 New York {U.S.A.) 



It has been shown^'^ that the over-all reversible Reaction i, catalysed by enzymes 

 present in a number of tissues, involves two steps (Reactions 2 and 3). 



1. rf-Isocitrate + TPN^^ ^^^^ a-ketoglutarate + CO2 + TPN,ed 



2. (^-Isocitrate + TPN^^ v "* oxalosuccinate + TPN^ed 



3. Oxalosuccinate "* a-ketoglutarate + COg 



Crude enzyme solutions from heart muscle^, liver^ and higher plants'* catalyse 

 Reaction i in either direction, as well as the decarboxylation of oxalosuccinate (Reac- 

 tion 3), in the presence of added manganous ions. Reaction 2 can be shown to occur in 

 either direction with the same enzyme solutions when Mn++ is excluded^. 



Partial purification of isocitric dehydrogenase, as tested by Reaction i, was pre- 

 viously reported^. A four-fold purification of the activity exhibited by extracts of ace- 

 tone-dried pig heart, with very low yield, was obtained at that time. Lynen and 

 ScHERER^ have recently reported the synthesis of oxalosuccinic acid and the catalysis 

 of the decarboxylation of this compound by enzymes from various sources. Their work, 

 carried out without knowledge of the work of this laboratory, led essentially to the same 

 results. They also reported partial purification of the oxalosuccinic carboxylase activity 

 (Reaction 3) of horse liver. 



A somewhat improved method of purification of the isocitric dehydrogenase and 

 oxalosuccinic carboxylase activities of pig heart, as determined according to Reactions 

 I and 3, is described in this paper. A six-fold purification of the activity of the extracts 

 with a yield of about 15% has been obtained. There was no separation of activities as 

 tested by Reactions i and 3, but both these activities were increased with respect to 

 malic dehydrogenase. Thus, the question whether Reactions 2 and 3 are catalysed by 

 distinct enzymes (isocitric dehydrogenase and oxalosuccinic carboxylase respectively), 

 or by a single enzyme, still remains unsettled. 



* Aided by grants from the United States Public Health Service, the American Cancer Society (re- 

 commended by the Committee on Growth of the National Research Council), the Office of Naval Research 

 and the Lederle Laboratories Division, American Cyanamid Company. 



Senior Fellow in Cancer Research, American Cancer Society, upon recommendation of the 

 Committee on Growth of the National Research Council. Present address, Department of Anatomy, 

 School of Medicine, The Johns Hopkins University, Baltimore, Maryland. 



References p. 210. 



