Abstract
It has been demonstrated that slices of rat liver have caused the in vitro incorporation of radioactive orotic acid into the pyrimidine nucleotides of nucleic acid and not into the purines.
Three milligrams of orotic acid (labeled in the 4 position) were incubated with about 6 g of slices of rat liver for 5 hours at 37°C in a Krebs saline-phosphate buffer of pH 7.4. The solution was saturated with a 95% C02: 5% C02 mixture at the beginning and nothing was used to diminish bacterial action. The slices were homogenized after preliminary washing and the homogenate precipitated with 10% trichloroacetic acid. The extraction of lipids was then carried out with a 3:1 alcohol and ether solution. The nucleic acid was extracted from the protein with hot 10% NaCl and precipitated with alcohol. All of the dried impure nucleic acid (35 mg containing both r.n.a. and d.n.a.) was hydrolyzed with 0.75 cc of N HCl in a boiling water bath for one hour. This hydrolyzed the Purina nucleotides and left the pyramiding nucleotides unchanged. 0.04-0.06 cc were placed on each of 4 filter paper strips 12 cm wide and run with ascending columns of tertiary butyl alcohol (70% made 0.8 N with HCl, 30% water) (1). After drying, the papers were viewed with an ultraviolet Mineralite. The separated bands on paper were eluted with 0.01 N HO, and the solutions read with a Beckman ultraviolet spectrophotometer. The maximum absorptions and the ratios of densities (278-262 mμ for cytidylic and uridvlic acids; 248-262 for guanine and adenine) were determined in order to identify and quantitate the bases. The separate solutions were evaporated on plates and radioactivity determined with a windowless counter.
Approximately three-fourths of the hydrolysate from the above experiment containing Purina bases and pyrimidine nucleotides was adjusted to pH 8 and the guanine precipitate removed by centrifugation.
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