Phosphatidylcholine biosynthesis and choline transport in the anaerobic protozoon Entodinium caudatum.
ABSTRACT: Choline accumulation and phosphatidylcholine biosynthesis were investigated in the choline-requiring anaerobic protozoon Entodinium caudatum by incubating whole cells or subcellular fractions with [14C] choline, phosphoryl [14C] choline and CDP-[14C] choline. 2. All membrane fractions contained choline kinase (EC 126.96.36.199) and CDP-choline-1,2-diacylglycerol cholinephosphotransferase (EC 188.8.131.52), although the specific activities were less in the cell-envelope fraction. Choline phosphate cytidylyltransferase (EC 184.108.40.206) was limited to the supernatant, and this enzyme was rate-limiting for phosphatidylcholine synthesis in the whole cell. 3. Synthesis of phosphatidylcholine from free choline by membranes was only possible in the presence of supernatant. Such reconstituted systems required ATP (2.5 mM), CTP (1 mM) and Mg2+ (5 mM) for maximum synthesis of the phospholipid. CTP and Mg2+ were absolute requirements. 4. Hemicholinium-3 prevented choline uptake by the cells and was strongly inhibitory towards choline kinase; the other enzymes involved in phosphatidylcholine synthesis were minimally affected. 5. Ca2+ ions (0.5 mM) substantially inhibited CDP-choline-1,2-diacylglycerol cholinephosphotransferase in the presence of 15 mM-Mg2+, but choline phosphate cytidylyltransferase and choline kinase were less affected. 6. No free choline could be detected intact cells even after short (10-180s) incubations or at temperatures down to 10 degrees C. The [14C] choline entering was mainly present as phosphorylcholine and to a lesser extent as phosphatidylcholine. 7. It is suggested that choline kinase effectively traps any choline within the cell, thus ensuring a supply of the base for future growth. At low choline concentrations the activity of choline kinase is rate-limiting for choline uptake, and the enzyme might possibly play an active role in the transport phenomenon. Thus the choline uptake by intact cells and choline kinase have similar Km values and show similar responses to temperature and hemicholinium-3.
Project description:The incorporation of [14C]choline chloride and [14C]glycerol into segments taken from rye (Secale cereale L., cv. Rheidal) roots was greater in segments from roots grown at 5 degrees C than in segments taken from roots growing at 20 degrees C. The incorporation was measured at the temperature at which the root had been growing. Measurements in vitro of the enzymes of the nucleotide pathway showed activity of choline kinase (EC 220.127.116.11), choline-phosphate cytidylyltransferase (EC 18.104.22.168) and cholinephosphotransferase (EC 22.214.171.124) to be higher in homogenates from the cooler roots when assayed at 5 degrees C than the activities assayed at 20 degrees C in the 20 degrees C-root homogenates. Changes in vivo in the pool sizes of the CDP-base intermediates with temperature, relative differences in nucleotide-pathway-enzyme activities and a pulse-chase experiment with [14C]choline indicated that the rate-limiting step for phosphatidylcholine biosynthesis in this tissue, at both temperatures, was the reaction catalysed by cytidylyltransferase.
Project description:1. Incorporation of [Me-14C]choline and [2-14C]ethanolamine into lipids was studied in germinating soya bean (Glycine max L.) seeds. The precursors are only incorporated into phosphatidylcholine and into phosphatidylethanolamine respectively. 2. Base-labelling via a phospholipase-D type of reaction was eliminated as a significant factor. 3. Cyclo heximide inhibited labelling of phosphatidylcholine from [Me-14C]choline but did not affect labelling of the aqueous choline pool. It had no effect on [2-14C]ethanolamine uptake or incorporation into phosphatidylethanolamine. 4. Hemicholinium-15 at 10mM concentrations decreased uptake and lipid labelling from the both bases. 5. There was no evidence for base competition. 6. The endogenous pool of choline was much larger than that of ethanolamine, which resulted in higher specific radioactivities for phosphatidyl-ethanolamine than for phosphatidylcholine. 7. The results can be interpreted as indicating that the kinase and phosphoryltransferase enzymes of the CDP-base pathways are separate for each phospholipid.
Project description:The control of phosphatidylcholine biosynthesis in the hamster liver was examined. Livers of hamsters fasted for 24 and 48 h were perfused with labelled choline. Under both fasting conditions, the incorporation of labelled choline into phosphatidylcholine was reduced. After 48 h of fasting, the 52% reduction in phosphatidylcholine biosynthesis was caused by changes in several factors including a diminishing rate of choline uptake and severe reductions in the pool sizes of ATP and CTP (to 33-37% control values) which resulted in a decrease in the pools of choline-containing metabolites. The activation of cytidylyltransferase after 48 h of fasting might be regarded as a compensatory mechanism for the maintenance of phosphatidylcholine biosynthesis. After 24 h of fasting, a 25% reduction in phosphatidylcholine biosynthesis was observed. The ATP and CTP levels were decreased but the reduction was not severe enough to affect the choline uptake or the labelling of the phosphocholine fraction. The activities of the cytidylyltransferase remained unchanged but an accumulation of labelled CDP-choline was detected. Although choline-phosphotransferase activity was not changed in the microsomes, the enzyme activity was attenuated in the postmitochondrial fraction. Further analysis revealed that cholinephosphotransferase in the liver was inhibited by an endogenous inhibitor in the cytosol which was later identified as argininosuccinate. The level of argininosuccinate was elevated during fasting and the change quantitatively accounted for the attenuation of cholinephosphotransferase activity. The inhibition of choline-phosphotransferase by argininosuccinate, coupled with a substantial decrease in the diacylglycerol level, would provide the hamster liver with an immediate mechanism for the transient modulation of phosphatidylcholine biosynthesis during short-term fasting.
Project description:Indol-3-ylacetic acid stimulated stem elongation within 1 h of treatment of Pisum sativum L. cv. Feltham First stem sections. This elongation was accompanied by an increase in the endogenous level of phosphocholine and a decrease in that of CDP-choline. Measurements in vitro of the CDP-base pathway enzymes showed an increase in choline phosphotransferase and a decrease in cytidylyltransferase activity on hormone treatment. These results indicate that the decrease in phosphatidylcholine labelling from [14C]choline that is observed on indol-3-ylacetic acid treatment of pea stem sections is caused by the decrease in cytidylyltransferase activity.
Project description:1. Microsomal membranes from rat liver were incubated with ATP, CoA, Mg2+, [14C]palmitate, F- and sn-glycerol 3-phosphate in order to label them with [14C]phosphatidate. These membranes were isolated and used in a second incubation in which [3H]CTP was present, and the simultaneous synthesis of [14C]diacylglycerol and [3H]CDP-diacylglycerol was measured. 2. The addition of phosphatidate phosphohydrolase, which had been partially purified from the particle-free supernatant, supplemented the activity of the endogenous phosphohydrolase, but it did not alter the rate of CDP-diacylglycerol formation. 3. Adding EDTA inhibited phosphatidate cytidylyl-transferase activity and stimulated the activity of the phosphohydrolases by removing excess of Mg2+. 4. Increasing the concentration of Mg2+, norfenfluramine or chlorpromazine in the assay system stimulated cytidylyltransferase activity, but decreased the activities of both phosphohydrolases. 5. The mechanism for the stimulation of cytidylyl=transferase activity by the cationic drugs and Mg2+ was investigated with emulsions of phosphatidate and the microsomal fraction of rat liver. 6. There was a threshold concentration of about 5mM-MgCl2 below which no cytidylyltransferase activity was detected in the presence or absence of norfenfluramine. Just above this threshold concentration norfenfluramine stimulated cytidylyltransferase activity, but this stimulation disappeared as the Mg2+ concentration was raised to its optimum of 20mM. Norfenfluramine therefore partially replaced the bivalent-cation requirement. 7. At 30 mM-MgCl2 amphiphilic cationic drugs inhibited cytidylyltransferase activity at relatively high concentrations in a non-competitive manner with respect to phosphatidate. 8. The implications of these results are discussed with respect to the regulation of the synthesis of the acidic phospholipids compared with the synthesis of phosphatidylcholine, phosphatidylethanolamine and triacylglycerol.
Project description:When type II pneumonocytes from adult rats were maintained in a medium that lacked choline, the incorporation of [14C]glycerol into phosphatidylcholine was not greatly diminished during the period that the cells displayed characteristics of type II pneumonocytes. Cells that were maintained in choline-free medium that contained choline oxidase and catalase, however, became depleted of choline and subsequent synthesis of phosphatidylcholine by these cells was responsive to choline in the extracellular medium. Incorporation of [14C]glycerol into phosphatidylcholine by choline-depleted cells was stimulated maximally (approx. 6-fold) by extracellular choline at a concentration (0.05 mM) that also supported the greatest incorporation into phosphatidylglycerol. The incorporation of [14C]glycerol into other glycerophospholipids by choline-depleted cells was not increased by extracellular choline. When cells were incubated in the presence of [3H]cytidine, the choline-dependent stimulation of the synthesis of phosphatidylcholine and phosphatidylglycerol was accompanied by an increased recovery of [3H]CMP. This increased recovery of [3H]CMP reflected an increase in the intracellular amount of CMP from 48 +/- 9 to 76 +/- 16 pmol/10(6) cells. Choline-depleted cells that were exposed to [3H]choline contained [3H]CDP-choline as the principal water-soluble choline derivative. As the extracellular concentration of choline was increase, however, the amount of 3H in phosphocholine greatly exceeded that in all other water-soluble derivatives. Choline-depletion of cells resulted in an increase in the specific activity of CTP:phosphocholine cytidylyltransferase in cell homogenates (from 0.40 +/- 0.15 to 1.31 +/- 0.20 nmol X min-1 X mg of protein-1). These data are indicative that the biosynthesis of phosphatidylcholine is integrated with that of phosphatidylglycerol and are consistent with the proposed involvement of CMP in this integration. The choline-depleted type II pneumonocyte provides a new model for investigating the regulation of CTP:phosphocholine cytidylyltransferase activity.
Project description:Treatment of murine adult MLE-12 type-II and fetal-rat type-II cells with fetal-rat-fibroblast-conditioned medium (FFCM) resulted in a 2-fold stimulation of [14C]choline incorporation into phosphatidylcholine. Soluble CTP:phosphocholine cytidylyltransferase (CT) activity was increased approx. 3-fold in FFCM-treated fetal-rat type-II cells but was not changed in MLE-12 cells. Neither choline kinase nor cholinephosphotransferase activities were affected by treatment of MLE-12 cells with FFCM. Long-term labelling of MLE-12 cells with [14C]choline, followed by a 14-18 h chase with FFCM, resulted in a 2.5-fold decrease in [14C]phosphocholine levels relative to controls, suggesting that CT was being activated. In contrast, oleate treatment increased CT activity in the particulate fraction in both cells. Western blots indicate that soluble CT undergoes dephosphorylation in response to FFCM, but no translocation to the particulate fraction was noted. Treatment with oleate stimulated a marked translocation. Tryptic phosphopeptide maps from FFCM-treated cells revealed only minor alterations in the phosphorylation pattern. It is concluded that FFCM and oleate activate CT through different mechanisms. The results are consistent with FFCM activating CT in MLE-12 as well as fetal type-II cells. However, the reason why this activation cannot be detected in vitro is not known.
Project description:The addition of 1 microM-vasopressin or -angiotensin to isolated rat hepatocytes induced a fast transient inhibition of the rate of incorporation of [Me-3H]choline into phosphatidylcholine. The cationophore A23187 induced a similar inhibition of phosphatidylcholine synthesis. The addition of micromolar Ca2+ to rat liver microsomes inhibited the activity of CDP-choline: 1,2-diacylglycerol cholinephosphotransferase. This inhibition is due a decrease in the Vmax. of the enzyme without affecting the Km for CDP-choline. It is concluded that Ca2+ regulates phosphatidylcholine synthesis in rat liver.
Project description:Treponema denticola synthesizes phosphatidylcholine through a licCA-dependent CDP-choline pathway identified only in the genus Treponema. However, the mechanism of conversion of CDP-choline to phosphatidylcholine remained unclear. We report here characterization of TDE0021 (herein designated cpt) encoding a 1,2-diacylglycerol choline phosphotransferase homologous to choline phosphotransferases that catalyze the final step of the highly conserved Kennedy pathway for phosphatidylcholine synthesis in eukaryotes. T. denticola Cpt catalyzed in vitro phosphatidylcholine formation from CDP-choline and diacylglycerol, and full activity required divalent manganese. Allelic replacement mutagenesis of cpt in T. denticola resulted in abrogation of phosphatidylcholine synthesis. T. denticola Cpt complemented a Saccharomyces cerevisiae CPT1 mutant, and expression of the entire T. denticola LicCA-Cpt pathway in E. coli resulted in phosphatidylcholine biosynthesis. Our findings show that T. denticola possesses a unique phosphatidylcholine synthesis pathway combining conserved prokaryotic choline kinase and CTP:phosphocholine cytidylyltransferase activities with a 1,2-diacylglycerol choline phosphotransferase that is common in eukaryotes. Other than in a subset of mammalian host-associated Treponema that includes T. pallidum, this pathway is found in neither bacteria nor Archaea. Molecular dating analysis of the Cpt gene family suggests that a horizontal gene transfer event introduced this gene into an ancestral Treponema well after its divergence from other spirochetes.
Project description:The effects of different 1,2-diacyl-sn-glycerols on the kinetic properties of CDP-choline:1,2-diacylglycerol cholinephosphotransferase (EC 126.96.36.199) from mouse liver microsomes have been studied. Initial-velocity experiments were carried out with various concentrations of several species of diacylglycerol at different fixed concentrations of CDP-choline. Kinetic analysis of these data showed a family of intersecting lines consistent with a sequential kinetic mechanism of catalysis. The Km and Vmax. values derived from rate data revealed a pronounced effect of diacylglycerol species utilization on the Km value for CDP-choline. There was a biphasic relationship between diacylglycerol chain length and the Km for CDP-choline. Substitution of an unsaturated fatty acid in the sn-2 position of distearin also dramatically increased the CDP-choline Km value as well as the Vmax. 1,2-Dipalmitoyl-sn-glycerol was the preferred substrate over other disaturated species, but 1,2-dihexanoyl-sn-glycerol could not be utilized. These results demonstrate the kinetic mechanism of in vitro catalysis and suggest a regulatory role for CDP-choline concentration in the diacylglycerol species selectivity of cholinephosphotransferase resulting in the de novo biosynthesis of different molecular species of phosphatidylcholine.