Enzymatic synthesis of cytidine diphosphate diglyceride

Evidence is presented for the enzymatic formation of cytidine diphosphate diglyceride in microsomal preparations from guinea pig liver according to the reaction: CTP + phosphatidic acid right harpoon over left harpoon CDP-diglyceride + p-O-P. Conditions have been found in which the incorporation of labeled CTP into CDP-diglyceride is almost entirely dependent upon added phosphatidic acid. The incorporation of CMP into lipid is very slight. A substantial net synthesis of CDP-diglyceride takes place under these conditions. Some properties of the enzyme system are described.     

Isolation and characterization of cytidine diphosphate diglyceride from beef liver.

Cytidine diphosphate diglyceride was isolated from beef liver by a combination of silicic acid column, DEAE-cellulose column, and this layer chromatography. The product (5.8 to 17.4 mumol/kg of liver) contained cytidine/phosphate/fatty acids in the molar proportions 1.05/2.0/2.05 (theoretical, 1.0/2.0/2.0) (average for three preparations). The liponucleotide was split quantitatively by a partially purified hydrolase from Escherichia coli, specific for CDP-diglyceride, (Raetz, C. R. H., Hirschberg, C. B., Dowhan, W., Wickner, W. T., and Kennedy, E. P. (1972) J. Biol. Chem. 247, 2245-2247) into phosphatidic acid and a water-soluble nucleotide that was chromatographically identical with CMP. No dCMP was located in these hydrolysates. The liver liponucleotide was more effective than a synthetic preparation of CDP-diglyceride in promoting the formation of phosphatidylinositol with guinea pig brain microsomes. The fatty acid composition of CDP-diglyceride was compared with metabolically related phospholipids from beef liver. The liponucleotide had a similar composition to phosphatidylinositol, characterized by a high level of stearate and with arachidonate as the major unsaturated fatty acid. The content of arachidonate in both lipids was significantly higher than that in phosphatidic acid. The profile of fatty acids of cardiolipin was quite unlike that of CDP-diglyceride. These findings suggest several alternatives for the metabolic origins of beef liver CDP-diglyceride: (a) CDP-diglyceride is formed from an atypical pool of phosphatidic acid, (b) the enzyme is selective for arachidonoyl-containing species of phosphatidic acid, (c) the liponucleotide may also be derived from phosphatidylinositol by the back-reaction of CDP-diglyceride: inositol phosphatidyltransferase.     

Biophysical properties of cytidine diphosphate diacylglycerol in solution

The physical properties of CDP diacylglycerol derived from egg phosphatidylcholine are very different from those of the common glycerophospholipids, such as phosphatidylcholine. Gently dispersed in buffer (5 mM phosphate, 0.15 M NaCl, pH 7.4), the liponucleotide initially forms an opalescent suspension of spherical vesicles, up to 50 micron in diameter, which appear to be unilamellar. These large vesicles are unstable and, independently of initial concentration, unstirred suspensions are no longer turbid after being incubated for about 1 h at room temperature. The passage of samples through Sepharose and Sephadex at increasing time intervals after the first hour reveals a continuing but slow diminution in size until, at about two days, a final peak is obtained which remains invariant for longer times. Chromatography of these ultimate stable micelles on Sephadex G-200 gives a Stokes radius of 4.2 nm. Their sedimentation coefficient extrapolated to zero concentration is 6.1 S. These numbers, combined with a partial specific volume of 0.835 ml X g-1, give an anhydrous mass of 155 000 Da and an aggregation number of 158. Although the data suggest the particles to be spherical, other compact forms cannot be excluded. Proton NMR at 220 MHz shows time-dependent spectral changes which are consistent with the slow structural transformation observed by gel-filtration chromatography, and indicate that the sugar and cytosine groups in the ultimate micelles apparently are motionally restricted. The critical micelle concentration is near 6 microM, but micelle-free molecule equilibration requires at least 7 days at a total concentration of 89 microM. Sonication considerably decreases the time required for the vesicle-micelle transformation and the micelle-free molecule equilibration. Some implications for enzymology are discussed.