Table V. Conversion Of Cdp-Choline And Cdp-Ethanolamine To Phospholipides

Each tube contained 10 pmoles of MgCl2, 50 pmoles of phosphate buffer of pH 7.4, and 0.5 ml. of either a suspension of lyophilized particles from rat liver or fresh whole homogenate of rat liver. 20 mpmoles of CDP-choline labeled with P-choline-1,2-C14 (100,000 c.p.m. per micromole) or 20 mpmoles of CDP-ethanolamine labeled with P-ethanolamine-P32 (113,000 c.p.m. per micromole) were added in the tubes indicated. The final volume of the system was 1.0 ml. The tubes were incubated at 37° for 1 hour.

The possibility was considered that the CDP-ethanokmine may be enzymatically converted to CDP-choline before being incorporated into phospholipide. This is not the case, since, after hydrolysis of the enzymatically labeled lipide by the procedure of Dawson (14), glycerophos-phorylethanolamine was the only labeled product found. The glycero-phosphorylcholine had no detectable radioactivity.

It is to be expected by analogy that CDP-serine might be a precursor of phosphatidylserine. It has not yet been possible to synthesize CDP-serine by the carbodiimide procedure, since the unprotected amino and carboxyl groups of P-serine participate in side reactions leading to a complex mixture of products. It should be possible to synthesize this compound after suitably protecting these functional groups.

Isolation of CDP-choline and CDP-ethanolamine from Liver and Yeast- Detectable steady state levels of CDP-choline and CDP-ethanolamine must exist in tissues such as liver which are carrying out an active synthesis of phospholipide, if these compounds are precursors of lecithin and phosphatidylethanolamine in vivo. A determination of the concentration of CDP-choline and CDP-ethanolamine in rat liver was made by the following procedure.

Adult female albino rats were decapitated, and the livers removed as quickly as possible and homogenized at once in 10 volumes of 66 per cent ethanol in a Waring blendor at room temperature. A total of 58.9 gm. of rat liver was extracted in this manner, and the extract was clarified by centrifugation. The precipitate was washed twice with a total volume of 200 ml. of 66 per cent ethanol. Small amounts of labeled CDP-choline (0.16 pmole, 16,600 c.p.m.) and CDP-ethanolamine (0.30 µmole, 17,400 c.p.m.) were added to the combined extract and washes. The solution was concentrated to about 250 ml. and extracted with an equal volume of ether. The ether phase was discarded, traces of ether being removed from the aqueous phase by warming slightly under a jet of air. The pH of the solution was adjusted to 8 to 9 with 0.5 n KOH, and the solution passed over a column of Dowex 1 formate (20 cm. high) and chromatographed by using the formic acid system.

Fractions of 11.4 ml. each were collected. The radioactivity was recovered as a single peak in Tubes 21 to 26. No separation of CDP-choline and CDP-ethanolamine was achieved in this system. The material in Tubes 21 to 26 was pooled, neutralized, and rechromatographed by using the ammonium formate system. The volume of each fraction was 16 ml. CDP-choline was recovered in Tubes 6 to 8 and CDP-ethanolamine in Tubes 13 to 19. An unidentified non-radioactive cytidine compound (CDP-X) with 2 atoms of P per mole of cytosine appeared in Tubes 20 to 25.

The isolated nucleotides had the spectrum of pure cytidine compounds. The peaks of radioactivity, due to the small amount of added synthetic radioactive CDP-choline and CDP-ethanolamine, and of absorbancy at 280 n, due almost entirely to the liver nucleotides, were exactly coincident. The specific activity of successive tubes in each band was constant, indicating the identity and purity of the isolated compounds.

From the specific activity of the added and the reisolated CDP-choline and CDP-ethanolamine, the concentrations of these nucleotides in rat liver may be calculated (Table VI).

The concentrations of CDP-choline and CDP-ethanolamine in rat fiver are sufficiently high so that they may be easily detected without the aid of isotope tracer techniques. Hecht and Potter,' in an analysis of the nucleotides of rat liver, using chromatography on Dowex 1 formate, observed the presence of cytidine nucleotides which are eluted with dilute formic acid more rapidly than CMP. These compounds were found to possess 2 atoms of P per mole of cytosine but were not further identified. It is probable that CDP-choline and CDP-ethanolamine are present in these fractions.

Hevesy and Hahn (16) have shown that the phospholipides of the hen's egg are not synthesized in the ovary, but in the liver. The liver of a laying hen must therefore be carrying out a considerable net synthesis of phospholipides, and it was of interest to determine the concentration of CDPcholine and CDP-ethanolamine in this tissue. The levels of these nucleotides in the liver of the laying hen were found to be about 6 times higher than in the liver of the rat (Table VI). Although a firm conclusion cannot be drawn from the scanty data available, this finding is consistent with the view that the content of CDP-choline and CDP-ethanolamine is high in tissues which are carrying out the biosynthesis of phospholipides at rapid rates.

Table VI. Concentration Of Cdp-Choline And Cdp-Ethanolamine In Liver Of Rat And Hen

Details of the experiment with rat liver are given in the text. In the experiment with hen liver, 1.7 µmoles of CDP-choline and 0.50 pmole of CDP-ethanolamine of the specific activities shown were added to the extract derived from 49 gm. of liver.

CDP-choline and CDP-ethanolamine have also been detected in a preparation of crude nucleotides from brewers' yeast (Pabst lot No. X-54). More recently a crystalline nucleotide has been isolated in considerable quantity from yeast by workers at the Sigma Chemical Company. This compound has been conclusively identified as CDP-choline by Dr. I. Lieber-man.4 Samples of pure synthetic CDP-choline and of the crystalline substance isolated from yeast have been compared in detail both by Lieberman and in our own laboratory. The compounds are identical in chromatography on ion exchange resins, on paper in several different solvent systems, and in behavior in isolated enzyme systems.

33 Personal communication.

4 Dr. I. Lieberman, personal communication.

Enzymatic Synthesis of CDP-choline and CDP-ethanolamine-Enzymes have been found widely distributed in nature which catalyze Reaction a, Fig. 3, and also the reaction (2) CTP + P-ethanolamine ⇋ CDP-ethanolamine + P-O-P

In the terminology proposed by Kalckar and Klenow (3), these enzymes may be described as cytidyl transferases. Separate enzymes appear to be required for these reactions; it is proposed to refer to the enzyme catalyzing Reaction a (Fig. 3) as PC-cytidyl transferase, and to the enzyme catalyzing Equation 2 as PE-cytidyl transferase.

An enzyme extract containing both PC-cytidyl transferase and PE-cytidyl transferase was prepared by the following procedure, all the operations being carried out at 0-5°. 12.5 gm. of rat liver were homogenized for 1 minute in a Waring blendor in 50 ml. of 0.02 m K2HPO4 containing 0.001 m Versene. The homogenate was centrifuged at 20,000 X g for 40 minutes. The supernatant fluid, containing a considerable amount of particulate material, was diluted to 100 ml. with water, and solid ammonium sulfate was added to 0.40 saturation. The precipitate was collected by centrifugation, suspended in the Tris-Versene solution used for extraction, and dialyzed against the same solution.