224 I- LIEBERMAN, A. KORNBERG VOL. 12 (ig53) 



(3) COOH COOH 



+ H„0 



The enzyme that catalyzes reaction (i), the reversible reduction of orotic acid to di- 

 hydro-orotic acid, has been partially purified and named dihydro-orotic dehydrogenase. 

 The purpose of this report is to present the evidence for reaction (i). 



METHODS 



Isolation and growth of the organism. The organism used in these studies is an obligate anaerobe 

 isolated from San Francisco Bay mud by enrichment culture, by one of us (AK), in the laboratory 

 of Dr. H. a. Barker*. 



The growth medium consisted of 2% tryptone, 0.05% Difco yeast extract, 0.2% orotic acid, 

 and 0.05% sodium thioglycolate. For the preparation of stock test tube cultures, the medium was 

 adjusted to pH 7.0 with i M KOH prior to autoclaving (15 minutes at 15 pounds pressure). Anaerobic 

 conditions were maintained with a pyrogallol-NagCOg seal. Large cultures were grown in Erlenmeyer 

 flasks (i to 6 liters) without an anaerobic seal. After autoclaving for 20-30 minutes at 15 pounds 

 pressure, the medium was cooled, neutralized with a sterile 50% ¥iJZO^ solution and sterile water was 

 added to the neck of the flask. The inoculum (one or two fresh stock cultures) was added promptly. 

 Growth appeared to be complete in 16-20 hours at 30° C. 



Preparation of the cell-free extract. The cells were harvested in a Sharpies supercentrifuge and 

 resuspended in 0.0 1 M sodium orotate (7 ml per liter of culture), potassium phosphate buffer (i M, 

 pH 7.0, 0.4 ml per liter of culture) and cysteine (o.i M, pH 7.0, 0.4 ml per liter of culture). This 

 cell suspension was incubated in vacuo at 26° C for 20 minutes. More active extracts appeared to be 

 obtained when this step was included in the procedure. After centrifugation, the cells were suspended 

 in ice cold water (about 5 ml per liter of culture), and an aliquot of the suspension (about 6 ml) was 

 shaken with 6 g of glass beads(o. 10-0.15 mm diameter) **in a Mickle vibrator for 15 minutes at 2^ C. 

 The mixture was centrifuged in a Sorvall centrifuge (at ca. 10,000 g) and the precipitate was washed 

 once with cold water. The volume of the extract (combined supernatant solutions) was adjusted to 

 10 ml per liter of culture. If the purification procedure was not initiated at once, the extract was acidi- 

 fied to pH 6.5 with 2 M sodium acetate buffer (pH 6.0) and stored at — 10° C. 



Glucose dehydrogenase was purified as described by Strecker and Korkes^®. Lactic dehydrogenase 

 was prepared as previously described^''. 



2-^'^C-Orotic acid. Radioactive orotic acid was synthesized from "C-KCNO and DL-aspartic 

 acid as described by Nyc and Mitchell^^***. Non-isotopic material was a commercial preparation 

 whose absorption spectrum corresponded to that given by Mitchell and Nyc^^. 



Fusion product of maleic acid and urea. This compound, named dihydro-orotic acid by Bachstez 

 AND Cavallini^'', was prepared by their method. The recrystallized product gave the following 

 analysist when calculated for dihydro-orotic acid. 



CjHgO^Na Calculated C 37.97, H 3.79, N 17.72 

 Found C 38.15, H 4.08, N 17.77 



Diphosphopyridine nucleotide (DPN) was prepared as previously described^^. Reduced diphospho- 

 pyridine nucleotide (DPNH) was prepared by Ohlmeyer's method"^^. 



The name Zymobacterium oroticum has been j)rovisionally given to this organism by Dr. Barker. 

 Obtained from Minnesota Mining and Manufacturing Company, St. Paul, Minnesota. 

 * In the preparation of the intermediate5-(carboxymethylidene)-hydantoin, 4.28gbrominewere 

 used, rather than the 1.28 g stated in the Nye-Mitchell paper by typographical error. We are indebted 

 to S. K. Kornberg for this synthesis. 



t Microanalyses were performed by the Microanalytical Laboratory of the National Institutes of 

 Health under the supervision of Dr. W. C. Alford. 



References p. 234. 



