It has been found that surface-active agents, such as bile salts, digitonin, or Tween 20 (polyoxyethylene sorbitan monolaurate), promote the reversible dissociation of a,β-diglyceride from the enzyme surface. Of these, Tween 20 appears to be the most convenient. When rat liver particles are incubated with CDP-choline in the presence of 0.5 per cent Tween 20, the formation of radioactive lecithin is stimulated 10- to 20-fold by the addition of a mixture of D-a,β-diglycerides derived from egg lecithin (Fig. 8). The stimulatory effect of D-a,β-diglycerides is consistent and quite specific; the egg lecithin from which the D-a,β-diglyceride was derived is much less effective, as is a mixture of naturally occurring triglycerides (corn oil).

Discussion

Workers in several laboratories (10, 14, 18) have observed the presence in a number of tissues of enzyme systems which are active in the synthesis of phosphatidic acids. The concentration of phosphatidic acids in mammalian tissues is so low as to escape detection by ordinary methods. Considerable speculation has arisen as to the physiological significance of this rapid enzymatic synthesis of phosphatidic acids. Evidence has been presented in this paper that D-a,β-diglycerides are precursors of the a,β-di-glyceride portion of the lecithin molecule. The dephosphorylation in vivo of L-phosphatidic acids would yield D-a,β-diglycerides, which then may be converted to glycerophosphatides. Preliminary experiments in our laboratory have shown that there is a rapid release of inorganic phosphate when phosphatidic acids are incubated with enzymes from rat liver.

The pathways for the enzymatic synthesis of lecithin and phosphatidylethanolamine are essentially similar, suggesting that the mechanisms involved may be applied to the biosynthesis of glycerophosphatides in general, including the serine phosphatides and acetal phospholipides. Sphingomyelin, although not a glycerophosphatide, has a phosphorylcholine moiety like that of lecithin and it is possible that CDP-choline may be a precursor of this portion of the sphingomyelin molecule.

It is noteworthy that the enzymatic reactions in Fig. 3 are widely distributed in nature. Further, enzymes derived from widely different sources, such as fiver and yeast, show the same high specificity for cytidine nucleotides. It appears that the fundamental mechanisms involved in the biosynthesis of phospholipides are much the same in many types of living cells.

The brilliant work of Leloir and his collaborators (19-21) has led to the discovery of the uridine coenzymes and of some of the functions of these compounds in the metabolism of carbohydrates. The cytidine compounds described in the present paper have a structure (cytidine-P-P-base) closely similar to the uridine compounds (uridine-P-P-sugar). However, the cytidine coenzymes participate in a type of group transfer reaction which is quite different from any that has been described involving the uridine nucleotides.

The enzymatic synthesis of CDP-choline and of CDP-ethanolamine is similar to the synthesis of uridine diphosphate glucose described by Munch-Petersen et al. (22).

(3) UTP + a-glucose-1 -phosphate ⇋uridine diphosphate glucose + P-O-P

Both reactions are examples of a fundamentally important mode of synthesis of nucleotide pyrophosphates discovered by Kornberg in his studies on the biosynthesis of DPN (17).

(4) ATP + nicotinamide ribotide ⇋ DPN + P-O-P

Many of the metabolic transformations of glucose, such as conversion to sucrose (21) or oxidation to glucuronic acid (23), do not occur unless the sugar is first converted to uridine diphosphate glucose. The finding that CDP-choline and CDP-ethanolamine are naturally occurring "activated" forms of choline and ethanolamine raises the question as to whether these compounds undergo reactions other than those leading to the formation of phospholipides. If the analogy with the uridine compounds holds true, then many of the metabolic reactions of choline and ethanolamine may possibly involve CDP-choline or CDP-ethanolamine rather than the free bases themselves.

The presence of adenosine as an essential component of coenzymes has long been known. The work of Leloir and his collaborators on the uridine coenzymes has already been mentioned. Sanadi et al. (24) and Kurahashi and Utter (25) have shown that guanosine nucleotides may also participate as cofactors in enzymatic reactions. With the discovery of the cytidine coenzymes, it is now clear that all four of the ribotides which are present in ribonucleic acid are essential components of coenzymes. The underlying biological significance of this fact is not apparent at present, but it must be taken into consideration in any comprehensive theory of the function of ribonucleic acid in living cells.

The authors are indebted to Sylvia Wagner Smith for assistance in many of these experiments.

Summary

1. The enzymatic synthesis of lecithin and of phosphatidylethanolamine has been found to be mediated by cytidine coenzymes. Cytidine diphosphate choline and cytidine diphosphate ethanolamine, "activated" forms of phosphorylcholine and phosphorylethanolamine, are precursors of lecithin and phosphatidylethanolamine, respectively, in these enzyme systems.

2. The levels of cytidine diphosphate choline and cytidine diphosphate ethanolamine have been measured in the livers of the rat and of the hen. These compounds have also been detected in yeast.

3. Enzymes have been found widely distributed in nature which carry out the synthesis of cytidine diphosphate choline and cytidine diphosphate ethanolamine according to the following equations:

CTP + phosphorylcholine ⇋ cytidine diphosphate choline + inorganic pyrophosphate CTP + phosphorylethanolamine ⇋ cytidine diphosphate ethanolamine + inorganic pyrophosphate

The names PC-cytidyl and PE-cytidyl transferases have been suggested for these enzymes.

4. Cytidine diphosphate choline is converted by an enzyme (PC-glyceride transferase) to lecithin, and cytidine diphosphate ethanolamine is converted by a separate enzyme (PE-glyceride transferase) to phosphatidylethanolamine. When tested in the presence of surface-active agents, the PC-glyceride reaction is stimulated 10- to 20-fold by the addition of D-a, β-diglycerides.

5. These results are discussed and a reaction mechanism is presented to account for the function of cytidine coenzymes in the biosynthesis of phospholipides.

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