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.     

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.     

Non-enzymatic synthesis of the coenzymes,uridine diphosphate glucose and cytidine diphosphate choline,and other phosphorylated metabolic intermediates

The synthesis of uridine diphosphate glucose (UDPG), cytidine diphosphate choline (CDP-choline), glucose-1-phosphate (G1P) and glucose-6-phosphate (G6P) has been accomplished under simulated prebiotic conditions using urea and cyanamide, two condensing agents considered to have been present on the primitive Earth. The synthesis of UDPG was carried out by reacting G1P and UTP at 70 °C for 24 hours in the presence of the condensing agents in an aqueous medium. CDP-choline was obtained under the same conditions by reacting choline phosphate and CTP. G1P and G6P were synthesized from glucose and inorganic phosphate at 70 °C for 16 hours. Separation and identification of the reaction products have been performed by paper chromatography, thin layer chromatography, enzymatic analysis and ion pair reverse phase high performance liquid chromatography. These results suggest that metabolic intermediates could have been synthesized on the primitive Earth from simple precursors by means of prebiotic condensing agents.     

Synthesis of the coenzymes adenosine diphosphate glucose,guanosine diphosphate glucose,and cytidine diphosphoethanolamine under primitive earth conditions

The nonenzymatic synthesis of the coenzymes adenosine diphosphate glucose (ADPG), guanosine diphosphate glucose (GDPG), and cytidine diphosphoethanolamine (CDP-ethanolamine) has been carried out under conditions considered to have been prevalent on the early Earth. The production of these compounds was performed by allowing simple precursor molecules to react under aqueous solutions, at moderate temperatures and short periods of time, with mediation by cyanamide or urea. These two condensing agents are considered to have been present in significant amounts on the primitive Earth and have been previously used in the nonenzymatic synthesis of several other important biochemical compounds. In our experiments, ADPG was obtained by heating glucose-1-phosphate (G1P) and ATP in the presence of cyanamide for 24 h at 70 degrees C. The reaction of G1P and GTP under the same conditions yielded GDPG. The cyanamide-mediated production of CDP-ethanolamine was carried out by reacting a mixture of ethanolamine phosphate and CTP for 24 h at 70 degrees C. The separation and identification of the reaction products was carried out by paper chromatography, thin-layer chromatography, high performance thin-layer chromatography, high performance liquid chromatography, both normal and reverse-phase, UV spectroscopy, enzymatic assays, and acid hydrolysis. Due to the mild conditions employed, and to the relative ease of these reactions, these studies offer a simple attractive system for the nonenzymatic synthesis of phosphorylated high-energy metabolic intermediates under conditions considered to have been prevalent on the ancient Earth.     

Separation of cytidine diphosphate reductase from rat Yoshida ascites sarcoma

CDP reductase was separated from the cytosol of rat Yoshida ascites sarcoma. The precipitate, which resulted from the acidification of the cytosol by acetic acid at pH 5.2, catalyzed specifically the reduction of CDP, whereas the concurrently resulted supernatant catalyzed those of UDP, ADP and GDP. The CDP reductase showed a single peak in the pattern of the enzyme activity in DEAE-cellulose and also in Sepharose 4B column chromatography with adequate recovery of the activity.     

Synthesis, characterization and some properties of dideoxynucleoside analogs of cytidine diphosphate diacylglycerol.

Phospholipid conjugates of antiretroviral nucleoside analogs have been proposed to have several advantageous features when compared to the parent drugs (Hostetler, K.Y. et al. (1990) J. Biol. Chem. 265, 6112-6117). Here we report on the synthesis of one such type of lipid conjugates, i.e., nucleosides diphosphate diacylglycerols. The syntheses of 3'-azido-3'-deoxythymidine diphosphate diacylglycerol, 3'-deoxythymidine diphosphate diacylglycerol and 2',3'-dideoxycytidine diphosphate diacylglycerol (with different acyl chains) were performed starting from phosphatidic acid and the antiviral nucleoside. A high-performance liquid chromatography procedure for a single step purification of the compounds is presented. The compounds were characterized biochemically, using rat liver enzymes and chemically by phosphorus, fatty acid, ultraviolet, IR and 1H-NMR analyses. Preliminary data on the behaviour in aqueous solution of some of the compounds are presented.     

Binding and structural studies of the complexes of type 1 ribosome inactivating protein from Momordica balsamina with cytosine,cytidine, and cytidine diphosphate

The type 1 ribosome inactivating protein from Momordica balsamina (MbRIP1) has been shown to interact with purine bases, adenine and guanine of RNA/DNA. We report here the binding and structural studies of MbRIP1 with a pyrimidine base, cytosine; cytosine containing nucleoside, cytidine; and cytosine containing nucleotide, cytidine diphosphate. All three compounds bound to MbRIP1 at the active site with dissociation constants of 10^?4 M–10^?7 M. As reported earlier, in the structure of native MbRIP1, there are 10 water molecules in the substrate binding site. Upon binding of cytosine to MbRIP1, four water molecules were dislodged from the substrate binding site while five water molecules were dislodged when cytidine bound to MbRIP1. Seven water molecules were dislocated when cytidine diphosphate bound to MbRIP1. This showed that cytidine diphosphate occupied a larger space in the substrate binding site enhancing the buried surface area thus making it a relatively better inhibitor of MbRIP1 as compared to cytosine and cytidine. The key residues involved in the recognition of cytosine, cytidine and cytidine diphosphate were Ile71, Glu85, Tyr111 and Arg163. The orientation of cytosine in the cleft is different from that of adenine or guanine indicating a notable difference in the modes of binding of purine and pyrimidine bases. Since adenine containing nucleosides/nucleotides are suitable substrates, the cytosine containing nucleosides/nucleotides may act as inhibitors.     

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.