VOL. 12 (1953) ENZYMIC SYNTHESIS AND BREAKDOWN OF OROTIC ACID 



225 



DETERMINATIONS 



Assay of dihydro-orotic dehydrogenase. DPNH, which is essential to the first step in the break- 

 down of orotic acid, was generated by the addition of glucose dehydrogenase, glucose, and DPN. 

 The test system contained o.i ml of MgClg (0.15 M), o.i ml of potassium phosphate buffer (i M, 

 pH 6.1), 0.04 ml of sodium orotate (o.oi M), 0.2 ml of cysteine (0.1 M, pH 7.0), 0.03 ml of DPN 

 (o.ooi M), 0.2 ml of glucose (i J\I), 250 units of glucose dehydrogenase, and the enzyme preparation 

 in a volume of 3.0 ml. All the components except the glucose dehydrogenase were mixed and incubated 

 at room temperature for 5 minutes. The glucose dehydrogenase was then added and the rate of 

 orotate removal was followed in the Beckman DU spectrophotometer by the decrease in optical 

 density at 280 m/<. A unit of enzyme was defined as the amount producing an optical density decrease 

 of o.ioo in a 6 minute interval. In general, not more than 4 units of activity were used for the assay. 

 Specific activity was defined as units of activity per mg of protein. 



Under the conditions of the assay, the rate of orotate reduction was proportional to the amount 

 of enzyme. Thus the decrease in optical density at 280 mjx in 6 minutes was found to be 0.000, 0.080, 

 0.137, 0-17. and 0.296 with 0.00, 0.03, 0.05, 0.07, and o.io ml of enzyme solution, respectively. In 

 the absence of glucose, glucose dehydrogenase, or DPN, no removal of orotate was observed. 



G/z^cose was estimated spectrophotometrically with hexokinase*^' (freed of glucose by dialysis 

 against o.oi M sodium acetate solution, pH 5.4) and Zwischenfernienfi'^. The assay was carried out as 

 described^^ for the estimation of adenosine triphosphate (ATP) except that excess ATP (0.2 micro- 

 mole) was added. Glucose (0.05-0.10 micromole) was added last to initiate the reaction. 



Protein was determined by the method of Lowry et al.^^. 



^*C-containing samples were in general plated as infinitely thin layers on aluminium discs and 

 measured in a gas flow counter. 



RESULTS 



Purification of dihydro-orotic dehydrogenase 



Protamine fraction. Purification of the enzyme was carried out at 0-2° C. 100 ml 

 of freshly prepared cell-free extract were diluted with an equal volume of water and 

 15 ml of a 1% solution of protamine sulfate (Eli Lilly) were added with stirring. After 

 5 minutes, the precipitate was collected by centrifugation and the supernatant solution 

 discarded. 100 ml of sodium citrate buffer (0.5 M, pH 6.0) were added to the hard and 

 difficultly soluble precipitate. After 12-24 hours the softened precipitate was dissolved 

 to a considerable extent by homogenization with a glass pestle, 200 ml of water were 

 added with stirring, and the resultant stringy precipitate was discarded after centrifuga- 

 tion. The supernatant solution (Protamine, Table I) was essentially free of nucleic acid 

 as indicated by the ratio of optical densities at 280 and 260 m/x (0.98). 



TABLE I 



PURIFICATION OF DIHYDRO-OROTIC DEHYDROGENASE 



Enzyme fraction 



Cell-free extract 

 Protamine 

 Ammonium sulfate 

 Acid ammonium sulfate 



For details of the fractionation procedure see the text. 



Ammonium sulfate fraction. To the protamine fraction were now added with 

 stirring 100 g of ammonium sulfate. After 5 minutes, the precipitate was removed by 



* Kindly supplied by Dr. F. E. Hunter, Jr. 

 References p. 234. 



