208 



A. L. GRAFFLIN, S. OCHOA 



VOL. 4 (1950) 



TABLE II 



PARTIAL PURIFICATION OF ISOCITRIC DEHYDROGENASE AND OXALOSUCCINIC CARBOXYLASE 

 800 gm OF POWDER OF WASHED, ACETONE-DRIED, PIG HEART 



* Over-all reaction isocitrate -f TPN,, 

 ** Specific activity (units/mg protein) 



a-ketoglutarate -f COj + TPNjed 



These preparations are very unstable and lose activity rather rapidly even when 

 stored at 0°. If dried from the frozen state, 30 to 40% of the activity is lost but, on the 

 other hand, the remaining activity persists unchanged for many months when the dry 

 powder is stored in the cold over calcium chloride. The preparations contain no aconitase 

 and only traces of lactic dehydrogenase. 



The results of a typical fractionation are summarized in Table II. 



Occasionally the purification obtained after ammonium sulphate and ethanol 

 fractionation may be lower than that reported in Table II. The purity of these prepa- 

 rations can be increased about 1.5 times, with a yield of 60% or better, by adsorption 

 on calcium phosphate gel. For this purpose the enzyme solution is diluted with o.oi M 

 phosphate buffer pjj 7.4 to give a protein concentration of about 1%. The adsorption 

 is carried out successively with small amounts of the gel, until all the activity has been 

 removed from solution, and the sediments are separately eluted with o.i M phosphate 

 buffer Ph 7.4. The eluates are tested separately and the best ones are combined. The 

 calcium phosphate gel was prepared following the directions of Keilin and Hartree^^. 



COMPARISON OF MANOMETRIC AND OPTICAL DETERMINATION OF OXALOSUCCINIC 



CARBOXYLASE ACTIVITY 



The specific oxalosuccinic carboxylase activity of the extract of acetone-dried pig 

 heart, as determined manometrically, has been previously reported^. The determinations 

 were carried out at pn 5-6 and 15°, in the presence of 0.0014 M MnClg and 0.0065 M 

 oxalosuccinate, and the COg evolution due to spontaneous decarboxylation was sub- 

 tracted from the total to obtain the enzyme-catalysed decarboxylation rate. Pig heart 

 extract catalysed the evolution of 70 /ul of COg during the first 5 minutes per mg of 

 protein. The activity of pig liver extract was about one tenth of this value. 



The manometric specific activity of 70 corresponds to an optical specific activity 

 of 462 (cf. Table II). Thus, the activity of the ethanol fraction of Table II is 2,860-70/462 

 or 435 1^^ of CO2 in 5 minutes per mg of protein (at 15°). The best fraction of Lynen 

 AND Scherer^ had a specific activity of 100 jul CO2 (corrected for spontaneous decarboxy- 

 References p. 210. 



