Cytidylyltransferase Research Articles

Lysophosphatidylcholine and 1-O-Octadecyl-2-O-Methyl-rac- Glycero-3-Phosphocholine Inhibit the CDP-Choline Pathway of Phosphatidylcholine Synthesis at the CTP:Phosphocholine Cytidylyltransferase Step

The regulation of the CDP-choline pathway of phosphatidylcholine synthesis at the CTP:phosphocholine cytidylyltransferase (CT) step by lysophosphatidylcholine (LPC) and the nonhydrolyzable LPC analog, 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3), was investigated in a colony-stimulating factor 1-dependent murine macrophage cell line. LPC inhibited phosphatidylcholine synthesis in vivo and led to the accumulation of choline and phosphocholine coupled to the disappearance of CDP-choline pointing to CT as the intracellular target. LPC neither inhibited cell growth nor decreased the cellular content of CT or altered the distribution of CT between soluble and particulate subcellular fractions. The inhibition of phosphatidylcholine synthesis was specific for LPC since lysophospholipids lacking the choline headgroup were not inhibitors. ET-18-OCH3 was a more potent inhibitor of phosphatidylcholine synthesis than LPC and caused the translocation of CT from the soluble compartment to the particulate compartment. Both LPC and ET-18-OCH3 were inhibitors of CT activity in vitro and kinetic analysis showed competitive inhibition with respect to the lipid activator. These data point to LPC as a negative regulator of de novo phosphatidylcholine synthesis that acts at the CT step and establish the mechanism for the inhibition of phosphatidylcholine biosynthesis by antineoplastic phospholipids.

Identification of the nuclear localization signal of rat liver CTP:phosphocholine cytidylyltransferase.

CTP:phosphocholine cytidylyltransferase (CT) is a major regulatory enzyme in phosphatidylcholine synthesis in mammalian cells. CT is found in both soluble and particulate forms, both of which are nuclear. We report here the identification of a 21-residue sequence at the amino terminus of CT, 8KVNSRKRRKEVPGPNGATEED28, which was sufficient to direct beta-galactosidase into the cell nucleus. Further deletions from either end of this sequence greatly reduced the nuclear localization of beta-galactosidase. Deletions of amino acids within the nuclear localization signal or of the entire signal disrupted CT nuclear localization, but CT was not completely excluded from the nucleus. Clones of stable transfectants of the nuclear localization signal-deficient CT expressed in Chinese hamster ovary (CHO) 58 cells, which is temperature-sensitive for growth and CT activity, were isolated and characterized. The deletion mutants were active under the same conditions as the wild-type enzyme. Despite the difference in subcellular location from wild-type CT, the nuclear localization mutants were fully able to complement the CT-deficient cell line CHO 58 for both growth and choline incorporation into phosphatidylcholine at the nonpermissive temperature. The mobility of the mutant enzymes on SDS gels was altered relative to the mobility of wild-type CT; however, the extent of phosphorylation of the mutant enzymes was decreased only slightly. Thus, the distribution of CT in both cytoplasm and nucleus, rather than exclusively nucleus, has little effect on the ability of CT to function in growing CHO cells.

Cholecystokinin Stimulates the Down-regulation of CTP:Phosphocholine Cytidylyltransferase in Pancreatic Acinar Cells

Stimulation of rat pancreatic acinar cells with cholecystokinin (CCK) is known to result in a significant inhibition of CTP:phosphocholine cytidylyltransferase (CT), a rate-limiting enzyme in phosphatidylcholine biosynthesis. Immunoprecipitation of CT from 32P-labeled acinar cells revealed that CCK treatment also caused a marked reduction in CT phosphate levels. The effects of CCK were maximal over 60 min and dependent on concentration, exhibiting an EC50 of 800 pM. Other calcium mobilizing secretagogues such as carbamylcholine (100 microM) and bombesin (10 nM) also reduced CT phosphate levels to 20 and 39% of control, respectively. Treatment of cells with thapsigargin and/or 12-O-tetradecanoyl-phorbol-13-acetate established that a combination of increased intracellular Ca2+ and protein kinase C activation was necessary to decrease phosphorylated CT content. Conversely, secretin (10 nM) or 8-(4-chlorophenylthio)-cAMP (100 microM) added alone had no effects. Use of the compound JMV-180 indicated CCK was acting through the low affinity state of the CCKA receptor to reduce CT phosphate levels. Further, the decrease in phosphorylated CT caused by CCK was blocked by the phosphatase inhibitors okadaic acid (3 microM) and calyculin A (100 nM). Finally, immunoblotting from whole cell lysates revealed CT was partially degraded in response to CCK, providing a novel mechanism by which the inhibition of CT enzyme activity occurs in response to the hormone. Moreover, this degradation was also blocked by a phosphatase inhibitor. These data suggest that the dephosphorylation of either CT itself or some other regulatory molecule(s) which mediates the CCK-induced protease activation may play a central role in reducing CT enzyme levels in acinar cells.

Expression of the ceramide galactosyltransferase gene during myelination of the mouse nervous system. comparison with the genes encoding myelin basic proteins, choline kinase and CTP:phosphocholine cytidylyltransferase

The present study documents the patterns of mRNA expression for the ceramide galactosyltransferase (CGT), the CTP:phosphocholine cytidylyltransferase (CT), and the choline kinase (CK) during the myelination period of the mouse central nervous system (CNS) and peripheral nervous system (PNS). Using the Northern blot technique with densitometric analyses, we show that the CK gene is not developmentally regulated during the period studied, whereas a peak of expression of the CT gene is observed around day 10. On the other hand, the expression of the CGT gene is similar to that of the MBP gene in the CNS and the PNS. Therefore, the synthesis of the galactosylceramides during the myelination period seems to be controlled at the level of the expression of the CGT gene. These results were compared to those of a neurological mutant, the trembler mouse, whose PNS myelination is deficient. Our results clearly indicate that the deficit in the accumulation of the galactosylceramides documented for this mutant is well corelated to a reduced CGT gene expression.

Primary structure and expression of a human CTP: Phosphocholine cytidylyltransferase

Human CTP: phosphocholine cytidylyltransferase (CT) cDNAs were isolated by PCR amplification of a human erythroleukemic K562 cell library. Initially two degenerate oligonucleotide primers derived from the sequence of the rat liver CT cDNA were used to amplify a centrally located 230 bp fragment. Subsequently overlapping 5′ and 3′ fragments were amplified, each using one human CT primer and one vector-specific primer. Two cDNAs encoding the entire translated domain were also amplified. The human CT (HCT) has close homology at the nucleotide and amino acid level with other mammalian CTs (from rat liver, mouse testis or mouse B6SutA hemopoietic cells and Chinese hamster ovary). The region which deviates most from the rat liver CT sequence is near the C-terminus, where 7 changes are clustered within 34 residues (345–359), of the putative phosphorylation domain. The region of the proposed catalytic domain (residues 75–235) is 100% identical with the rat liver sequence. Significant homology was observed between the proposed catalytic domain of CT and the Saccharomyces cerevisiae MUQ1 gene product, and between the proposed amphipathic α-helical membrane binding domains of CT and soybean oleosin, a phospholipid-binding protein. There are several shared characteristics of these amphipathic helices. An approx. 42000 Da protein was over-expressed in COS cells using a pAX142 expression vector containing one of the full-length HCT cDNA clones. The specific activity of the HCT in COS cell homogenates was the same as that of analogously expressed rat liver CT. The activity of HCT was lipid dependent. The soluble form was activated 3 to 4-fold by anionic phospholipids and by oleic acid or diacylglycerol-containing PC vesicles.

Identification of a putative membrane-interacting domain of CTP: Phosphocholine cytidylyltransferase from rat liver

A putative membrane-interacting domain of CTP:phosphocholine cytidylyltransferase (CT) was identified using two peptide-specific antibodies. One antibody (SA2) was raised against the N-terminus of CT (amino acid residues 1–17) and the other antibody (SA209) against an α-helical domain of the enzyme (amino acid residues 247–257). Both antibodies quantitatively immunoprecipitated CT from rat liver cytosol and showed specificity towards CT when octylglucoside extracts of rat liver cytosol were assessed by Western blot analysis. However, further experiments revealed that the antibodies had different characteristics. Whereas the antibody directed against the N-terminus of CT (SA2) did not influence CT/membrane interaction, the new antibody (SA209) against the α-helical domain of the enzyme interfered with this interaction. Our results provide experimental evidence that the α-helical domain (amino acid residues 228–287) of CT may serve as a membrane-interacting domain.

Identification of phosphorylation sites in rat liver CTP: phosphocholine cytidylyltransferase.

CTP:phosphocholine cytidylyltransferase (CT) is an important regulatory enzyme in phosphatidylcholine biosynthesis. The enzyme exists as a soluble, inactive form that is highly phosphorylated; activation of the enzyme is accompanied by dephosphorylation and translocation to the membrane. We have used a recombinant baculovirus clone to obtain CT labeled in vivo with 32PO4. The tryptic phosphopeptide pattern of the baculovirus-expressed CT was the same as for CT expressed in mammalian cells, indicating that insect cells modify the same phosphorylation sites as do mammalian cells. 32PO4-labeled, baculovirus-expressed CT was digested with trypsin and the peptides purified by reversed-phase high performance liquid chromatography. Phosphoamino acid analysis of the complete protein as well as individual peptides revealed that only serine residues were phosphorylated. Sequence analysis of purified radioactive peptides revealed that phosphorylation of CT was confined to the carboxyl-terminal region and that all or nearly all Ser residues from Ser315 to the carboxyl terminus were labeled. Ser315, Ser319, Ser329, Ser323, Ser331, Ser343, and Ser347 all reside in potential sites for proline-directed kinases. Two other phosphorylated serine residues, Ser315 and Ser333, are found within protein kinase C consensus phosphorylation sites. Ser321, Ser322, Ser333, Ser345, Ser346, Ser350, Ser352, and Ser362 were also found to be phosphorylated. Serine362 resides within a putative casein kinase II phosphorylation site, and there are five potential sites for phosphorylation by glycogen synthase kinase 3. Identification of these sites will allow investigations that focus on the establishment of the physiological function of phosphorylation at each site.

Free cholesterol loading of macrophages stimulates phosphatidylcholine biosynthesis and up-regulation of CTP: phosphocholine cytidylyltransferase

Atheroma macrophages accumulate large amounts of free cholesterol (FC) as well as cholesteryl ester (CE). An important adaptive response to FC loading might be increased cellular phospholipid to accommodate the excess FC. To explore this idea, J774 macrophages were incubated for 48 h without lipid, with acetyl-low density lipoprotein to induce mostly CE loading, or with acetyl-low density lipoprotein plus an acyl-CoA:cholesterol O-acyltransferase inhibitor (58035) to induce marked FC loading. The total phospholipid content approximately doubled in FC-loaded versus control or CE-loaded macrophages, with phosphatidylcholine showing the largest increase (approximately 2.5-fold versus control). Electron micrographs revealed the presence of multiple intracellular membrane whorls in the FC-loaded macrophages but not in the control or CE-loaded macrophages. [3H]Choline incorporation into phosphatidylcholine was also greater in FC-loaded macrophages versus control or CE-loaded macrophages, whereas [3H]phosphatidylcholine degradation was similar in all of the macrophages. In these experiments and in others that used non-lipoprotein cholesterol, there was a very close correlation between cellular FC content and phosphatidylcholine biosynthesis. To determine the mechanism of increased phosphatidylcholine synthesis, FC-loaded and CE-loaded macrophages were pulsed with [3H]choline, then chased and assayed for labeled phosphatidylcholine biosynthetic precursors. The only major differences were a 2-fold greater disappearance of label from [3H]choline phosphate and a 5-fold greater appearance of label in CDP-[3H]choline in the FC-loaded macrophages. These data suggest a stimulation of CTP:phosphocholine cytidylyltransferase (CT), which was confirmed by microsomal CT assays. Further studies revealed that the increase in phosphatidylcholine biosynthesis in FC-loaded macrophages was: (a) reversible under conditions of high density lipoprotein3-mediated cellular cholesterol efflux; (b) not blocked by cycloheximide-induced protein synthesis inhibition; and (c) not associated with increased CT mRNA levels. Thus, FC loading of macrophages leads to an increase in phosphatidylcholine mass which is caused by increased phosphatidylcholine biosynthesis. The mechanism appears to be FC-mediated post-translational activation of CT. This adaptive response may be important for atheroma macrophage survival, and disruption of the response may lead to macrophage necrosis and lesion progression.

Coordination of membrane phospholipid synthesis with the cell cycle.

The formation of membrane phospholipid is coordinated with the cell cycle in a colony-stimulating factor 1-dependent macrophage cell line. Net phospholipid accumulation occurs as cells enter S phase and results from an interaction between cell cycle-dependent oscillations in the rates of phosphatidylcholine (PtdCho) biosynthesis and degradation. CTP:phosphocholine cytidylyltransferase (CT) is the rate-controlling step in PtdCho biosynthesis, and its activity is inhibited by cell cycle-dependent phosphorylation. CT phosphorylation is low in early G1, begins to rise in late G1, increases steadily through S and G2/M, and then declines precipitously as cells exit mitosis and reenter G1. CT activity increases in early G1 and then decreases steadily as the cells progress through late G1, S, and G2/M, reflecting the elevated phosphorylation state of the protein. Membrane phospholipid degradation is also periodic: the rate is rapid in G1, slows significantly during S phase, and accelerates again as cells reenter G1. The data support the hypothesis that CT dephosphorylation accelerates PtdCho synthesis in response to the increased membrane phospholipid degradation to maintain membrane phospholipid mass during G1 and that the periodic cessation of phospholipid degradation during S phase accounts for the transition to net membrane phospholipid accumulation. Inactivation of CT associated with hyperphosphorylation of the protein during G2/M likely plays a determining role in the cessation of net membrane accumulation prior to cell division.

Regulation of CTP: Phosphocholine cytidylyltransferase in HepG2 cells: Effect of choline depletion on phosphorylation, translocation and phosphatidylcholine levels

We studied the effect of choline depletion on the biosynthesis of phosphatidylcholine (PC) and the distribution and phosphorylation of cytidylyltransferase (CT) in HepG2 cells. Phosphocholine concentrations decreased within 24 h of choline depletion to values less than 2% of controls. The incorporation of [ 3H]glycerol into PC was reduced in choline-depleted (CD) cells. The apparent turnover of PC was similar in CD and choline-supplemented (CS) cells ( T 1 2 = 20 h ). The methylation pathway for PC synthesis increased nearly 10-fold in CD cells. Cell growth was similar in CD and CS cells. Over 95% of CT activity in CS cells was in the soluble pool. Choline depletion resulted in a progressive decrease in CT activity and immunodetected enzyme in the soluble pool and a corresponding increase in membrane CT over a 48-h period. Choline supplementation of CD cells caused a rapid release of membrane CT (complete release by 3 h). Two phosphorylated forms of CT were identified. One form contained a higher level of phosphorylation (HPCT) then the other form (LPCT). HPCT migrated slightly slower than LPCT on SDS gels. CD cells contained only LPCT in both soluble and membrane pools. CS cells contained only HPCT. During choline depletion PC content decreased nearly 20% but CT binding did not occur until LPCT was generated in cytosol. Conversely, choline supplementation released LPCT into cytosol and HPCT was formed only after the release. We conclude that both the induction of binding sites, perhaps by depletion of PC and dephosphorylation of HPCT to LPCT, are required for CT translocation to membranes. The release of CT from membranes is initiated by changes in membrane binding sites followed by trapping of the CT in the soluble pool by phosphorylation of LPCT to HPCT.

Comparative effects of aging process on phosphatidylcholine biosynthesis pathway: a key role for CTP-phosphocholine cytidylyltransferase?

The effects of aging on phosphatidylcholine (PtdCho) biosynthesis were investigated in liver and brain subcellular fractions of the rat, by studying the activity and regulation of CTP phosphocholine cytidylyltransferase (CT), the rate limiting enzyme in PtdCho biosyntheses. With both tissues, CT activity was present in cytosolic and microsomal fractions, but in brain, CT activity seemed to escape to an inhibiting feed back mechanism. In brain, CT activity was greater in the microsomal fraction, whilst in liver, a higher CT activity was seen in the cytosolic fraction. In liver fractions of aged animals, there was no significant change in CT activity or its sensitivity to negative feedback regulation, as compared to young animals. In contrast, a progressive age related decline in CT activity was observed in the brain microsomal fraction. Furthermore, the incorporation of newly formed CDPCho into PtdCho was also reduced in aged animals, and paralleled the decreased incorporation of choline in PtdCho. The age-related decrease in CT activity cannot be explained by product feed back inhibition or decreased diacylglycerol levels. Since PtdCho is a major membrane lipid, the reduction in CT activity may lead to a decreased membrane integrity and fluidity during the aging process, and these effects may be greater in neuronal cells.

Modulation of CTP: Phosphocholine cytidylyltransferase activity in choline-sensitive and choline-resistant tobacco cells

Microsomes of cultured tobacco cells exhibited a CTP:phosphocholine cytidylyltransferase (CT) activity which was involved in the regulation of phosphatidylcholine (PC) biosynthesis. Some properties of this cellular activity measured in vitro are reported, especially its changes over a 35-day culture period. The changes were modulated by the presence of choline in the culture medium and differed between choline-sensitive (ChS) and choline-resistant (ChR) cell lines. In ChS cells a high CT stimulation, which was partially cycloheximide insensitive, occurred during the lag phase and the first wave of cell divisions, and was followed by an abrupt drop during the final cell divisions. The total activity did not parallel the microsomal protein content of the cells. The early stimulation of CT activity was also found in ChS cells which were cultivated in the presence of choline but was not as high as in cells cultivated in the absence of choline. CT activity, as well as microsomal protein content of the cells, remained high in the stationary growth phase of ChS cells cultivated on a choline-supplemented medium. In marked contrast, in ChR cells, routinely cultivated on choline, CT activity was constant so that the total activity only varied in accordance with microsomal protein content during the exponential and stationary growth phases. A non-specific nucleotidase catalysing the hydrolysis of CDP-choline exhibited an activity opposite to the overall activity of CT. In conclusion, PC biosynthesis appears to be controlled either by de novo synthesis of CT either by a reversible association of this protein to membranes, or by variable levels of nucleotidase and PC precursors. Alternative pathways concerned with ethanolamine methylation should also be considered.

Immunological characterization, lipid dependence, and subcellular localization of CTP:phosphoethanolamine cytidylyltransferase purified from rat liver. Comparison with CTP:phosphocholine cytidylyltransferase.

CTP:phosphoethanolamine cytidylyltransferase (ET) (ethanolamine-phosphate cytidylyltransferase, EC 2.7.7.14), which is generally considered as the rate-regulatory enzyme of phosphatidylethanolamine synthesis via the CDP-ethanolamine pathway, was purified to homogeneity from a rat liver postmicrosomal supernatant. A polyclonal antibody was raised against the enzyme in rabbits and subsequently purified by affinity chromatography. The affinity-purified antibody recognized one single immunoreactive 49.6-kDa protein band on SDS-polyacrylamide gel. The enzyme showed an isoelectric point at a pH of 6.5 and was sensitive to various sulfhydryl reagents. Cross-reactivity experiments of ET and CTP:phosphocholine cytidylyltransferase (CT) (choline-phosphate cytidylyltransferase, EC 2.7.7.15) with their corresponding antibodies showed that these enzymes were immunologically distinct. In contrast with the well known lipid dependence of CT, the activities of both purified and cytosolic ETs were not affected by the presence of various phospholipid preparations. Differential centrifugation studies as well as release experiments with digitonin-permeabilized hepatocytes demonstrated that ET, unlike CT, is not associated with cellular organelles. However, amino acid analysis of ET revealed a high content of hydrophobic amino acids, suggesting a possible association of this enzyme with some kind of cellular structure in the hepatocyte.

Identification of a protein complex between choline-phosphate cytidylyltransferase and a 112-kDa protein in rat liver.

Antisera raised against purified cytidylyltransferase (CT) immunoprecipitated CT activity from liver cytosol and detected the M(r) 45,000 subunit of CT on Western blots. Antisera detected a M(r) 112,000 protein on Western blots of liver cytosol. This protein was not detected in purified CT and was not detected by preimmune serum. The 112-kDa antibodies, isolated by affinity chromatography, did not immunoprecipitate CT activity. Antiserum raised against an N-terminal sequence of CT and antibodies raised against an internal sequence of CT immunoprecipitated CT activity but did not detect the 112-kDa protein. These results showed that the 112-kDa protein was not a form of CT. We concluded that the antiserum probably contained anti-idiotypic antibodies that recognized CT binding sites on the 112-kDa protein. Purified CT that was conjugated to horseradish peroxidase bound to crude 112-kDa protein immobilized on nitrocellulose. The binding was competitively reduced by purified CT and by affinity-purified antibodies to the 112-kDa protein. CT and 112-kDa protein coeluted from DEAE-Sepharose. When the putative 112-kDa protein-CT complex was chromatographed on a second DEAE-Sepharose column or on a Bio-Gel A-1.5m column, CT activity and 112-kDa protein were eluted together. Chromatography of the complex on hydroxylapatite dissociated most of the complex, producing CT free of the 112-kDa protein. We conclude that the 112-kDa protein is a CT-binding protein. The formation and/or dissociation of this complex may be important in the regulation of CT.

Expression of Rat CTP:Phosphocholine Cytidylyltransferase in Insect Cells Using a Baculovirus Vector

CTP:phosphocholine cytidylyltransferase (CT) is a key regulatory enzyme in phosphatidylcholine biosynthesis. We constructed a recombinant baculovirus (bCT) containing rat CT cDNA under the control of the polyhedrin promoter. Crude cell extracts of Spodoptera frugiperda (Sf9) cells infected with bCT possessed 250-fold higher specific activities for CT compared to rat liver cytosol, and CT protein constituted 3-6% of the total cellular protein. The 42-kDa form of CT predicted from the cDNA sequence was the first immunoreactive CT protein detected at Day 2 after infection and this form continued to accumulate until Day 5. On Day 3 following infection, a 37-kDa protein immunologically related to CT began to accumulate, indicating that CT was being degraded. The active, 42-kDa form of CT was purified to homogeneity in a single step using hydroxyapatite chromatography. Antibodies raised against recombinant CT were employed to quantitatively extract and assay CT activity in mammalian cell lines. The baculovirus expression system is suitable for the preparation of large amounts of protein for investigating the structure, function, and regulation of CT.

On the mechanism of the okadaic acid-induced inhibition of phosphatidylcholine biosynthesis in isolated rat hepatocytes.

The mechanism of inhibition of phosphatidylcholine biosynthesis by okadaic acid was investigated in suspension cultures of isolated rat hepatocytes. Cells were pulsed with [methyl-3H]choline and chased in the absence or presence of 1 microM okadaic acid for up to 120 min. Phosphatidylcholine biosynthesis was inhibited after 15 min of chase. To see if okadaic acid altered the degree of phosphorylation of cytidylyltransferase (CT), hepatocytes were incubated with 32P(i) and chased in the absence or presence of okadaic acid. Okadaic acid caused a rapid (within 15 min) increase in the phosphorylation state of the cytosolic enzyme. Two-dimensional peptide map analysis revealed an increase in the phosphorylation of several peptides in okadaic acid-treated hepatocytes compared with controls. After 15 min of incubation of hepatocytes with okadaic acid, membrane CT activity was decreased and a corresponding increase in cytosolic CT activity was observed. In hepatocytes incubated with okadaic acid and oleate a correlation between membrane CT activity, diacylglycerol level, and phosphatidylcholine biosynthesis was observed. These data suggest that the concentration of diacylglycerol is responsible for the increase in membrane CT activity and subsequently phosphatidylcholine biosynthesis in oleate-treated cells. We postulate that the okadaic acid-induced decrease in phosphatidylcholine biosynthesis is due to an increase in the phosphorylation state of CT which promotes a translocation of CT activity from the membranes to the cytosol.

Chronical ingestion of oxidized oil in the rat: Effect on lipid composition and on cytidylyl transferase activity in various tissues

An oxidized oil with 370 mmol hydroperoxides by kg and 41% oxidized esters was mixed with a fat-free diet at a rate of 4%, alone or in equivalent weight with fresh oil, and given to rats for 4 weeks. The fat-free diet and a well-balanced food were used as references. The oxidized oil group had a one fourth reduction in the weight growth, superior to that of the fat-free group. In the stools, the concentration of the lipids was increased, with the presence of polar and high MW compounds in proportion to the ingested quantity of oxidized oil, which suggested an intestinal sorting out of the oxidized esters. For the oxidized oil and the fat-free groups, a liver steatosis was observed and, in their liver phospholipids, linoleic acid was reduced to one half, whereas monounsaturated FA were increased and eicosatrienoic acid reached respectively 3% and 14% against 1% in the control group. The cytidylyl transferase activity was increased in the 106000 g supernatant of the liver homogenates, linked to the monounsaturated FA variations. Arachidonic acid was conserved in the phospholipids of the oxidized oil group, with 29% in the liver against 26% and 12% for the control and the fat free groups, in consequence or a possible arachidonic acid preferential synthesis or of an eicosanoids biosynthesis clamping.

Colony-stimulating factor 1 regulates CTP: phosphocholine cytidylyltransferase mRNA levels.

Growth factor regulation of phosphatidylcholine (PtdCho) metabolism during the G1 stage of the cell cycle was investigated in the colony-stimulating factor 1 (CSF-1)-dependent murine macrophage cell-line BAC1.2F5. The transient removal of CSF-1 arrested the cells in G1. Incorporation of [3H]choline into PtdCho was stimulated significantly 1 h after growth factor addition to quiescent cells. Metabolic labeling experiments pointed to CTP:phosphocholine cytidylyltransferase (CT) as the rate-controlling enzyme for PtdCho biosynthesis in BAC1.2F5 cells. The amount of CT mRNA increased 4-fold within 15 min of CSF-1 addition and remained elevated for 2 h. The rise in CT mRNA levels was accompanied by a 50% increase in total CT specific activity in cell extracts within 4 h after the addition of CSF-1. CSF-1-dependent elevation of CT mRNA content was neither attenuated nor superinduced by the inhibition of protein synthesis with cycloheximide. The rate of CT mRNA turnover decreased in the presence of CSF-1 indicating that message stabilization was a key factor in determining the levels of CT mRNA. These data point to increased CT mRNA abundance as a component in growth factor-stimulated PtdCho synthesis.

The phospholipid analogue hexadecylphosphocholine inhibits phosphatidylcholine biosynthesis in Madin—Darby canine kidney cells

The influence of the phospholipid analogue hexadecylphosphocholine on phosphatidylcholine biosynthesis was investigated in Madin—Darby canine kidney (MDCK) cells. It inhibits the incorporation rate of [methyl-3H]choline into phosphatidylcholine at a concentration of 50 μM by about 50%. The radiolabelled precursor accumulates in the phosphocholine pool indicating that hexadecylphosphocholine inhibits the formation of phosphatidylcholine via the CDP-choline pathway at the level of the rate-limiting enzyme, CTP:phosphocholine cytidylyl transferase (EC 2.7.7.15). This was verified by the determination of the activity of the enzyme in vitro. In consequence of its inhibitory effect it could be shown that the treatment of MDGK cells for 24 h with 50 μM hexadecylphosphocholine induces alterations of the phospholipid composition. Whereas in treated cells the relative phosphatidylcholine content was decreased from the control level of 36.0±0.9% to 29.9±0.2%; in contrast, the relative content of phosphatidylethanolamine was increased from 19.3±0.9% to 24.3±0.9%.

Regulation of CTP:phosphocholine cytidylyltransferase by lipids. 1. Negative surface charge dependence for activation.

The activity of phosphocholine cytidylyltransferase (CT), the regulatory enzyme in phosphatidylcholine synthesis, is dependent on lipids. The enzyme, obtained from rat liver cytosol, was purified in the presence of Triton X-100 [Weinhold et al. (1986) J. Biol. Chem. 261, 5104]. The ability of lipids to activate CT when added as Triton mixed micelles was limited to anionic lipids. The relative effectiveness of the lipids tested suggested a dependence on the negative surface charge density of the micelles. The mole percent lipid in the Triton mixed micelle required for activation decreased as the net charge of the lipid varied from 0 to -2. Evidence for the physical association of CT with micelles and vesicles containing phosphatidylglycerol was obtained by gel filtration. The activation by micelles containing PG was influenced by the ionic strength of the medium, with a higher surface charge density required for activation at higher ionic strength. The micelle surface potential required for full activation of CT was calculated to be -43 mV. A specificity toward the structure of the polar group of the acidic lipids was not apparent. CT was activated by neutral lipids such as diacylglycerol or oleyl alcohol when included in an egg PC membrane, but the activities were reduced by dilution with as little as 10 mol % Triton. Thus Triton mixed micelles are not suitable for studying the activation of CT by these neutral lipid activators. We conclude that one way that lipid composition can control CT-membrane binding and activity is by changing the surface potential of the membrane. Other distinct mechanisms involved in the activation by neutral lipids are discussed.