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.     

Biosynthesis of retinal phospholipids: incorporation of radioactivity from labeled phosphorylcholine and cytidine diphosphate choline

Phosphorylcholine-1,2-(14)C and choline-1,2-(14)C-labeled cytidine diphosphate choline are incorporated into lecithin by whole homogenates and particulate fractions of rat retina with optimal incorporation of label by the microsomal fraction. The soluble fraction contains a factor(s) which stimulates incorporation of label with release of inorganic phosphate. Mg(++) is required for optimal incorporation of intermediates into lecithin in the presence of added diglycerides; without added diglycerides, incorporation of phosphorylcholine or cytidine diphosphate choline was moderately stimulated by preincubating the system in the absence of Mg(++) with added phosphatidic acid and by adding this mixture to fresh enzyme and the complete incubation mixture (including Mg(++)). The results show that the retina is capable of de novo synthesis of phosphatides and suggest that the rod outer segments depend on the pigment epithelium and(or) the inner rod segments for a source of phospholipids. Coenzyme A and ATP added to whole homogenate of retina did not significantly increase the incorporation of CDP-choline-1,2-(14)C into lecithin but slightly increased the radioactivity found in lysolecithin and sphingomyelin. Rats with hereditary retinitis pigmentosa have an abnormally high lipid phosphorus content of the retina, but they do not incorporate labeled CDP-choline into lecithin of retina at a higher rate than do normal animals.     

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.     

Effect of cytidine diphosphate choline (CDP-choline) on ischemia-induced alterations of brain lipid in the gerbil

Brain ischemia was produced in gerbils (Meriones unguiculatus) by the bilateral ligation of the carotid arteries. Definite changes in the energy status of brain demonstrated that carotid occlusion was effective. Five minutes before ligation, an intraventricular injection of either saline or cytidine diphosphate choline (CDP-choline, 0.6 mol/brain, 3l) was given to groups of animals. Control animals, with and without CDP-choline, together with the ischemic groups, were decapitated directly into liquid nitrogen; 10 min after arterial ligation. Brain free fatty acids, neutral lipids and phospholipids, which were labeled in vivo by the intraventricular injection of [1-¹⁴C] arachidonic acid (0.4–0.6 Ci, 6–9 nmol) 2 hr prior to ligation, were extracted, purified, and separated by thin-layer chromatographic procedures. The CDP-choline treatment noticeably corrected the increase of total and individual fatty acids due to ischemia and the increase of their radioactivity content. The changes in neutral lipids, particularly in the diacyl glycerol fraction, were also corrected by the injection of the nucleotide. CDP-choline partially reversed the decrease of brain phosphatidylcholine and of its labeling, which was due to ischemia. All the data indicate that the prior injection of CDP-choline stimulates the choline phosphotransferase reaction of brain towards synthesis of phosphatidylcholine and prevents the release of free fatty acids, particularly of arachidonic acid, associated with ischemia.     

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.     

Immobilization of yeasts on ceramic supports

Selected yeast strains were tested for their affinity for a ceramic support. It is shown that the degree of affinity and strength of adsorption are characteristic of the strain and, furthermore that the population of adsorbed cells have a higher rate of respiration than the free cells.