Cholinephosphate Cytidylyltransferase Activity Research Articles

Imaging Lipid Synthesis in Hepatocellular Carcinoma with [Methyl-11C]Choline: Correlation with In Vivo Metabolic Studies

PET with [methyl-(11)C]-choline (11C-choline) can be useful for detecting well-differentiated hepatocellular carcinoma (HCC) that is not 18F-FDG-avid. This study was designed to examine the relationship between choline metabolism and choline tracer uptake in HCC for PET with 11C-choline. Dynamic PET scans of 11C-choline were acquired using the woodchuck models of HCC. After imaging, [methyl-(14)C]-choline was injected, and metabolites from both HCC tissue samples and the surrounding hepatic tissues were extracted and analyzed by radio-high-performance liquid chromatography. The enzymatic activities of choline kinase and choline-phosphate cytidylyltransferase were assayed for correlation with the imaging and metabolism data. PET with 11C-choline showed an HCC detection rate of 9 of 10. The tumor-to-liver ratio for the 9 detected HCCs was 1.89±0.55. Hematoxylin-eosin staining confirmed that all spots with high tracer uptake were well-differentiated HCCs. Variation of radioactivity distribution within HCCs indicated a heterogeneous uptake of choline. The activities of both choline kinase and choline-phosphate cytidylyltransferase were found to be significantly higher in HCC than in the surrounding hepatic tissues. The major metabolites of 11C-choline were phosphocholine in HCC and betaine and choline in the surrounding hepatic tissues at 12 min after injection; in HCC, phosphocholine rapidly converted to phosphatidylcholine at 30 min after injection. HCCs display enhanced uptake of radiolabeled choline despite a moderate degree of physiologic uptake in the surrounding hepatic tissues. Initially, increased radiolabeled choline uptake in HCCs is associated with the transport and phosphorylation of choline; as time passes, the increased uptake of radiolabeled choline reflects increased phosphatidylcholine synthesis derived from radiolabeled cytidine 5'-diphosphocholine (CDP-choline) in HCCs. In contrast, the surrounding hepatic tissues exhibit extensive oxidation of radiolabeled choline via the phosphatidylethanolamine methylation pathway, a major contributor to the observed physiologic uptake.

Role of c- fos gene in vasoactive intestinal peptide promoted synthesis of pulmonary surfactant phospholipids

We previously reported that vasoactive intestinal peptide (VIP) promoted synthesis of phosphatidylcholine (PC) in alveolar type II (ATII) cells. But the intracellular mechanism for this effect was unknown. In this work, we investigated the intracellular signal transduction pathway for VIP promoted synthesis of PC, the major lipid component of pulmonary surfactant (PS), by using an antagonist of VIP receptors, inhibitor of protein kinase C (PKC) and antisense oligonucleotides (AS-ODN) for c- fos oncogene. Our results showed that: ① [D-P-Cl-Phe(6)-Leu(17)]-VIP (10 − 6 mol/l), an antagonist of VIP receptors, could decrease the quantity of [ 3H] choline incorporation, microsomal choline-phosphate cytidylyltransferase (CCT) mRNA expression and CCT activity induced by VIP (10 − 8 mol/l) in cultured lung explants to the control levels; ② VIP (10 − 8 mol/l) upregulated c-Fos protein expression in ATII cells. AS-ODN for c- fos oncogene (9 × 10 − 6 mol/l) could block the elevation of [ 3H] choline incorporation, microsomal CCT mRNA expression and CCT activity induced by VIP in cultured lung explants and in ATII cells; ③ H7 (10 − 5 mol/l), a PKC inhibitor could also reduce VIP induced [ 3H] choline incorporation, microsomal CCT mRNA expression and CCT activity in cultured lung explants and in ATII cells. These results demonstrated that VIP receptors, PKC and c-Fos protein played important roles in the signaling pathway through which VIP promoted the synthesis of PC.

Stachybotrys chartarum Alters Surfactant-Related Phospholipid Synthesis and CTP:Cholinephosphate Cytidylyltransferase Activity in Isolated Fetal Rat Type II Cells

Stachybotry chartarum, a fungal contaminant of water-damaged buildings commonly grows on damp cellulose-containing materials. It produces a complex array of mycotoxins. Their mechanisms of action on the pulmonary system are not entirely clear. Previous studies suggest spore products may depress formation of disaturated phosphatidylcholine (DSPC), the major surface-active component of pulmonary surfactant (PS). If S. chartarum can indeed affect formation of this phospholipid, then mold exposure may be a significant issue for pulmonary function in both mature lung and developing fetal lung. To address this possibility, fetal rat type II cells, the principal source of DSPC, were used to assess effects of S. chartarum extract on formation of DSPC. Isolated fetal rat lung type II cells prelabeled with 3H-choline and incubated with spore extract showed decreased incorporation of 3H-choline into DSPC. The activity of CTP:cholinephosphate cytidylyltransferase (CPCT), the rate-limiting enzyme in phosphatidylcholine synthesis was reduced by approximately 50% by a 1:10 dilution of spore extract. Two different S. chartarum extracts (isolates from S. chartarum (Cleveland) and S. chartarum (Hawaiian)) were used to compare activity of CPCT in the presence of phosphatidylglycerol (PG), a known activator. PG produced an approximate two-fold increase in CPCT activity. The spore isolate from Hawaii did not alter enzyme activity. S. chartarum (Cleveland) eliminated the PG-induced activation of CPCT. These results support previous observations that mold products alter PS metabolism and may pose a risk in developing lung, inhibiting surfactant synthesis. Different isolates of the same species of fungus are not equivalent in terms of potential exposure risks.

Modulation of phosphatidylcholine biosynthesis in celery by exogenous fatty acids.

The effects of C16 and C18 fatty acids on the synthesis of phosphatidylcholine were studied in Apium graveolens cell suspension cultures and postmitochondrial supernatants. When cells were exposed to exogenous oleic acid, the rate of phosphatidylcholine biosynthesis increased 1.4-fold within 5 min of the addition of the fatty acid to the culture medium. The sensitivity of microsomal CTP:cholinephosphate cytidylyltransferase (EC 2.7.7.15) to saturated and unsaturated fatty acids was monitored through the addition of unesterified fatty acids to postmitochondrial supernatants. The saturated fatty acids, palmitic and stearic, appeared to have little effect on CTP:cholinephosphate cytidylyltransferase activity, whereas exposure to oleic, linoleic and cis-vaccenic acids resulted in significant increases in enzyme activity. Optimal microsomal CTP:cholinephosphate cytidylyltransferase activities were achieved by the incubation of postmitochondrial supernatants with 500 microM oleate. The exogenous fatty acids were found to be incorporated into microsomal membranes in their unesterified form. Removal of unesterified fatty acids by incubation of microsomal membranes with defatted bovine serum albumin resulted in the reduction of microsomal CTP:cholinephosphate cytidylyltransferase activity; demonstrating that the enzyme requires unesterified unsaturated fatty acids.

Cell proliferation and CTP:cholinephosphate cytidylyltransferase activity in higher plants.

Cell proliferation and CTP:cholinephosphate cytidylyltransferase activity in higher plants.

Oleic acid stimulates rapid translocation of cholinephosphate cytidylyltransferase in type II cells

Activity of cholinephosphate cytidylyltransferase, the rate-limiting enzyme in phosphatidylcholine synthesis, increases rapidly in the lung after birth predominantly due to an increase in membrane-associated activity. Although there is strong evidence that enzyme translocation is a major regulatory mechanism in other cells, this mechanism has not been conclusively demonstrated in intact alveolar type II cells. In this study, we show that oleic acid stimulates rapid translocation of cytidylyltransferase activity and protein from cytosol to microsomes in both primary cultured fetal and adult type II cells and MLE12 cells, a cell line derived from murine distal respiratory epithelial cells. Shifts in subcellular distribution occurred within 5 min of exposure to 200 μM oleic acid. The magnitude of the increases in microsomal enzyme activity and immunoreactive protein levels was several-fold greater in d21 fetal cells than adult type II cells. Oleic acid-induced translocation was confirmed in in vitro translocation experiments. After incubating MLE12 cell postmitochondrial supernatants at 37°C with oleic acid and separation of enzyme isoforms on glycerol density gradients, enzyme activity was decreased in gradient fractions corresponding to both cytosolic isoforms and microsomal activity increased 7.9-fold compared to the distribution of enzyme activity in postmitochondrial supernatants incubated at 4°C without oleic acid. The increase in microsomal activity was associated with an increased incorporation of [ 14C]oleic acid in the membrane free fatty acid fraction. Developmental changes in type II cell membrane lipid composition may induce the rapid translocation/activation of cytidylyltransferase in the lung after birth.

Control of membrane phosphatidylcholine biosynthesis by diacylglycerol levels in neuronal cells undergoing neurite outgrowth.

Phospholipids are the major components of cell membranes and are required for cellular growth. We studied membrane phosphatidylcholine (PtdCho) biosynthesis in neuronal cells undergoing neurite outgrowth, by using PC12 cells as a model system. When neurite outgrowth was induced by exposing PC12 cells to nerve growth factor for 2 and 4 days, the amounts of [14C]choline incorporated into [14C]phosphatidylcholine per cell (i.e., per DNA) increased approximately 5- and 10-fold, respectively, as compared with control cells, reflecting increases in the rate of PtdCho biosynthesis. [14C]choline uptake was not affected. Analysis of the three major PtdCho biosynthetic enzymes showed that the activity of CDPcholine:1,2-diacylglycerol cholinephosphotransferase was increased by approximately 50% after nerve growth factor treatment, but the activities of choline kinase or choline-phosphate cytidylyltransferase were unaltered; the cholinephosphotransferase displayed a high Km value ( approximately 1,200 microM) for diacylglycerol. Moreover, free cellular diacylglycerol levels increased by approximately 1.5- and 4-fold on the second and fourth days, respectively. These data indicate that PtdCho biosynthesis is enhanced when PC12 cells sprout neurites, and the enhancement is mediated primarily by changes in cholinephosphotransferase activity and its saturation with diacylglycerol. This suggests a novel regulatory role for diacylglycerol in membrane phospholipid biosynthesis.

Construction of a plasmid carrying both CTP synthetase and a fused gene formed from cholinephosphate cytidylyltransferase and choline kinase genes and its application to industrial CDP-choline production: enzymatic production of CDP-choline from orotic acid (Part II).

A new method for enzymatic production of cytidine diphosphate choline (CDP-choline) from orotic acid and choline chloride was developed. To establish an industrial manufacturing process, we constructed a plasmid, pCKG55, which simultaneously expressed in Escherichia coli the three following enzymes; CTP synthetase (encoded by the pyrG gene from E. coli), cholinephosphate cytidylyltransferase (encoded by the CCT gene from Saccharomyces cerevisiae), and choline kinase (encoded by the CKI gene from S. cerevisiae). CCT and CKI genes on pCKG55 were designed to be expressed as a single CCT/CKI fused protein. This CCT/CKI fused protein retained both activities and the thermal stability of its cholinephosphate cytidylyltransferase activity was nearly the same as the native CCT enzyme. Corynebacterium ammoniagenes KY13505 and E. coli MM294/pCKG55 were cultured in 5-liter jar fermentor independently. Equal volumes of each broth were mixed in a 2-liter jar fermentor, and then the enzymatic reaction was done using 47 mM orotic acid and 60 mM choline chloride as substrates. After 23 h of the reaction at 32 degrees C, 21.5 mM (11 g/liter) of CDP-choline was accumulated.

The effect of edelfosine on CTP: Cholinephosphate cytidylyltransferase activity in leukemic cell lines

Analogs of ether phospholipids have been shown to have selective anti-neoplastic activity. The compounds are known to inhibit phospholipid biosynthesis. This paper examines the effect of the alkyl-lysophospholipid, edelfosine, on the rate-limiting enzyme, CTP:choline-phosphate cytidylyltransferase, in de novo phosphatidylcholine synthesis in sensitive and resistant leukemic cell lines. Enzyme activity was measured by the incorporation of 14C-phosphocholine into CDP-choline by lysates of HL60 and K562; cells demonstrated inhibition of incorporation of 14C-phosphocholine in HL60 cell lysates but no inhibition in K562 lysates. Partial purification of cytidylyltransferase by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting demonstrated similarity between the enzyme isolated from each cell line. Cloning and sequencing of cytidylyltransferase cDNA of HL60 cells was accomplished using a probe encoding the entire protein sequence of the K562 cytidylyltransferase gene. A substitution at nucleotide 751 from A in the HL60 cell cDNA clone to G in the K562 cDNA clone resulted in a change in amino acid number 251 from lysine (positively charged) in the HL60 enzyme to glutamic acid (negatively charged) in the K562 enzyme. This negative charge in the lipid-binding domain of the K562 enzyme may result in a weaker binding of edelfosine and the observed decrease in activity, as evidenced by resistance to edelfosine by K562 cells.

Investigation of a choline phosphate synthesis pathway in Streptococcus pneumoniae: evidence for choline phosphate cytidylyltransferase activity.

In this study we have demonstrated the activity of a choline phosphate cytidylyltransferase in cell free extracts of Streptococcus pneumoniae. Southern blot analysis of restricted S. pneumoniae genomic DNA probed with the gene coding for the choline phosphate cytidylyltransferase of Saccharomyces cerevisiae demonstrated that there is homology between the S. cerevisiae cct gene and genomic DNA of S. pneumoniae. We believe that this enzyme is involved in the biosynthesis of the choline containing cell wall antigens, teichoic acid and lipoteichoic acid, catalysing the activation of choline phosphate to CDP-choline which is then incorporated into the polysaccharide moiety.

Betamethasone modulation of sphingomyelin hydrolysis up-regulates CTP:cholinephosphate cytidylyltransferase activity in adult rat lung.

Glucocorticoids appear to play an integral role in stimulating surfactant synthesis by activating the rate-regulatory enzyme for phosphatidylcholine synthesis, CTP:cholinephosphate cytidylyltransferase (CT). The activity of liver CT, in vitro, has been shown to be inhibited by the sphingomyelin hydrolysis product, sphingosine. In order to investigate the mechanisms by which glucocorticoids alter CT activity, in vivo, we administered betamethasone (1 mg/kg intraperitoneally) sequentially to adult male rats for 5 days. Betamethasone increased CT activity 2-fold relative to control in whole lung. The hormone also increased membrane-bound activity, but did not affect cytosolic enzyme activity. Betamethasone modestly increased CT mRNA as determined by the reverse-transcription PCR and Southern analysis of PCR products, but did not alter the levels of immunoreactive enzyme in lung membranes as demonstrated by Western blotting. The hormone did, however, produce a nearly 3-fold increase in membrane-associated sphingomyelin, and co-ordinately a substantial decrease in the levels of sphingosine in lung membranes. Sphingosine, but not sphinganine, was a competitive, reversible inhibitor of lung CT with respect to the enzyme activator, phosphatidylglycerol. Betamethasone decreased the activities of the sphingomyelin hydrolases: acid sphingomyelinase by 33% and of alkaline ceramidase by 21%. The hormone also inhibited the generation of sphingosine from lysosphingomyelin in lung membranes. There was no significant effect of the hormone on serine palmitoyltransferase activity, the first committed enzyme for sphingolipid biosynthesis. Further, administration of L-cycloserine, an inhibitor of sphingosine formation, was shown to stimulate CT activity by 74% and increase disaturated phosphatidylcholine in alveolar lavage by 52% relative to control. These observations suggest that glucocorticoids up-regulate surfactant synthesis at the level of a key regulatory enzyme by significantly altering the availability of inhibitory metabolites resulting from sphingomyelin hydrolysis.

Higher postpartum hepatic triacylglycerol concentrations in dairy cows with free rather than restricted access to feed during the dry period are associated with lower activities of hepatic glycerolphosphate acyltransferase.

We investigated the activities of hepatic glycerolipid synthesizing enzymes during postpartum fatty liver development in 10 high-producing dairy cows that had free access to feed during the dry period; a parallel group of 8 control cows was fed according to recommended energy requirements. After calving, both test and control cows had free access to feed. In the period of 10-14 wk before calving, voluntary dry matter intake of the test cows was 20.6 kg/d (SEM 0.42); the restricted control cows received 7 kg/d. Postpartum triacylglycerol concentrations in liver biopsies were one- to twofold higher in the test than in the control cows. The higher plasma nonesterified fatty acid (NEFA) concentrations after parturition in the test vs. the control group were probably caused by a more negative energy balance in the test cows, which was associated with a slightly lower postpartum dry matter intake. After calving, hepatic mitochondrial glycerolphosphate acyltransferase (GPAT) activities were significantly lower in the test than in the restricted control cows. A low GPAT activity may divert fatty acids from esterification to beta-oxidation to protect the hepatocytes against further accumulation of triacylglycerols. The activities of hepatic phosphatidate phosphohydrolase, diacylglycerol acyltransferase, and cholinephosphate cytidylyltransferase were not different in the two groups. This study indicates that in cows given free instead of restricted access to feed during the dry period have a postpartum hepatic triacylglycerol accumulation that is mainly determined by a raised hepatic uptake of plasma NEFA.

Time Trends of Plasma Lipids and Enzymes Synthesizing Hepatic Triacylglycerol During Postpartum Development of Fatty Liver in Dairy Cows

We studied development of fatty liver in high producing dairy cows with free access to feed during the dry period and thus showed the combined effects of parturition and prepartum overfeeding. Postpartum liver triacylglycerol concentrations at 1 wk postpartum, as measured in liver biopsies, had increased more than 6-fold, which was preceded or accompanied by an increase in plasma NEFA concentrations. Concentrations of hepatic phospholipid changed only slightly. The amounts of total lipids in serum, very low density lipoproteins, and high density lipoproteins significantly decreased by .5 wk after parturition, and concentrations of high density lipoproteins rose steadily. The pattern was similar for concentrations of total cholesterol and phospholipid in serum. Total lipid concentrations in low density lipoproteins were not altered after parturition. The activity of microsomal phosphatidate phosphohydrolase in the liver showed a transient increase at .5 wk after calving, but activity of microsomal glycerolphosphate acyltransferase remained relatively constant. The activities of diacylglycerol acyltransferase had increased about twice at 1 wk after calving and remained at this high level until at least 4 wk after parturition. The rise in activity of diacylglycerol acyltransferase was probably a response to the extra influx of fatty acids to channel them into triacylglycerol. Activities of microsomal cholinephosphate cytidylyltransferase initially increased after calving and then decreased slightly. Activities of hepatic choline kinase had increased after calving. This study indicates that hepatic triacylglycerol accumulates because of the increased hepatic uptake of NEFA and the simultaneous increase in activity of diacylglycerol acyltransferase.

The Saccharomyces cerevisiae phosphatidylinositol-transfer protein effects a ligand-dependent inhibition of choline-phosphate cytidylyltransferase activity.

The Saccharomyces cerevisiae protein SEC14p is required for Golgi function and cell viability in vivo. This requirement is obviated by mutations that specifically inactivate the CDP-choline pathway for phosphatidylcholine biosynthesis. The biochemical basis for the in vivo relationship between SEC14p function and the CDP-choline pathway has remained obscure. We now report that SEC14p effects an in vivo depression of CDP-choline pathway activity by inhibiting choline-phosphate cytidylyltransferase (CCTase; EC 2.7.7.15), the rate-determining enzyme of the CDP-choline pathway. Moreover, this SEC14p-mediated inhibition of CCTase was recapitulated in vitro and was saturable. Finally, whereas the SEC14p-dependent inhibition of CCTase in vitro was markedly reduced under assay conditions that were expected to increase levels of phosphatidylinositol-bound SEC14p, assay conditions expected to increase levels of phosphatidylcholine-bound SEC14p resulted in significant potentiation of CCTase inhibition. The collective data suggest that the phosphatidylcholine-bound form of SEC14p effects an essential repression of CDP-choline pathway activity in Golgi membranes by inhibiting CCTase and that the phospholipid-binding/exchange activity of SEC14p represents a mechanism by which the regulatory activity of SEC14p is itself controlled.

Fatty-acid synthase activity and mRNA level in hypertrophic type II cells from silica-treated rats.

Silica instillation causes a massive increase in lung surfactant. Two populations of type II pneumocytes can be isolated from rats administered silica by intratracheal injection: type IIA cells similar to type II cells from normal rats and type IIB cells, which are larger and contain elevated levels of surfactant protein A and phospholipid. Activities of choline-phosphate cytidylyltransferase, a rate-regulatory enzyme in phosphatidylcholine biosynthesis, and fatty-acid synthase (FAS) are increased in type IIB cells isolated from rats 14 days after silica injection. In the present study, we examined the increase in FAS and cytidylyltransferase activities in type IIB cells as a function of time after silica administration. FAS activity increased rapidly, was approximately threefold elevated 1 day after silica administration and has reached close to the maximum increase by 3 days. Cytidylyltransferase activity was not increased on day 1, was significantly increased on day 3 but was not maximally increased until day 7. Inhibition of de novo fatty-acid biosynthesis, by in vivo injection of hydroxycitric acid and inclusion of agaric acid in the type II cell culture medium, abolished the increase in cytidylyltransferase activity on day 3 but not FAS and had no effect on activities of two other enzymes of phospholipid synthesis. FAS mRNA levels were not increased in type IIB cells isolated 1-14 days after silica injection. These data show that the increase in FAS activity in type IIB cells is an early response to silica, that it mediates the increase in cytidylyltransferase activity, and that it is not due to enhanced FAS gene expression.

Developmental changes in cholinephosphate cytidylyltransferase activity and microsomal phospholipid fatty acid composition in alveolar type II cells

Cholinephosphate cytidylyltransferase is the rate-limiting enzyme in the choline pathway of phosphatidylcholine synthesis in fetal and adult lung. To examine the developmental changes in cytidylyltransferase activity, subcellular fractions were prepared from freshly isolated type II cells from 19 and 21 d gestation fetal rats, newborn (0 h, 3 h, 3–12 h, +1, +2, +7 postnatal day), and adult rats and the fractions assayed for cytidylyltransferase activity. Cytidylyltransferase activity per cell was low during late fetal gestation, but rose rapidly during the first 3 h after birth, predominantly due to an increase in microsomal enzyme activity. Microsomal and cytosolic enzyme specific activity increased 6 and 3.9 fold, respectively, from birth (0 h) to +1 postnatal day. The subcellular distribution of enzyme activity changed from 40% microsomal in fetal type II cells to 85% microsomal at 3–12 h, and 60% in this compartment in adult type II cells. Although evidence suggests that membrane lipid may affect enzyme activity, developmental changes in type II cell membrane phospholipid fatty acid composition have not been previously studied. Capillary gas chromatography analysis of phospholipid fatty acids extracted from microsome fractions revealed a 3-fold increase in both total saturated and unsaturated phospholipid fatty acids from day 19 to day 21 gestation. There was a further 62% increase in total saturated fatty acids during the first postnatal day, concomitant with the peak in microsomal cytidylyltransferase activity.

Evidence that CTP:choline-phosphate cytidylyltransferase is regulated at a pretranslational level in rat liver after partial hepatectomy.

Regulation of CTP:choline-phosphate cytidylyltransferase activity was studied in regenerating rat liver. The formation of phosphatidylcholine from [14C]choline in hepatocytes isolated from regenerating liver at 22 h after surgery was increased 1.9-fold when compared with hepatocytes from sham-operated animals. This effect was accompanied by a 1.4-fold increase in cytosolic cytidylyltransferase activity as well as by a 1.5-fold increase in the amount of immunoreactive cytidylyltransferase protein, and a 1.7-fold increase in [35S]methionine incorporation into cytidylyltransferase protein. Northern blot analysis of cytidylyltransferase mRNA showed two signals at 1.5 and 5.0 kb. Partial hepatectomy caused a significant 2-3-fold increase in the 1.5-kb and 5.0-kb messengers at 12 h after surgery. During the next 10 h after partial hepatectomy cytidylyltransferase mRNA levels slightly decreased, although they were still elevated in comparison with sham-operated rats 20-22 h after surgery. In contrast to the elevated cytidylyltransferase mRNA levels, the amount of acetyl-CoA carboxylase mRNA did not increase between 12 and 22 h after surgery, which is in line with the unchanged activity of this enzyme. In conclusion, our data demonstrate that in regenerating liver phosphatidylcholine biosynthesis and cytidylyltransferase activity are regulated at a pretranslational level.

Hexadecylphosphocholine inhibits translocation of CTP:choline-phosphate cytidylyltransferase in Madin-Darby canine kidney cells.

The mechanism of the inhibition of phosphatidylcholine biosynthesis by the phospholipid analogue, hexadecylphosphocholine, was investigated in Madin-Darby canine kidney cells. In the presence of 50 mumol/liter hexadecylphosphocholine, there was a translocation of CTP:choline-phosphate cytidylyltransferase (EC 22.7.7.15) activity from the membranes to the cytosol of the cells. Since we recently demonstrated that hexadecylphosphocholine also inhibits protein kinase C in vitro, [methyl-3H]choline labeling experiments were repeated with phorbol ester-desensitized cells. In these cells the same inhibitory effect of hexadecylphosphocholine was measured. As a consequence of inhibition, the [methyl-3H]choline incorporation into the phosphocholine pool was increased time-dependently. In addition, there was no evidence for a difference between the choline uptake of control and hexadecylphosphocholine-treated cells. Likewise, the amount of diacylglycerol, a known activator of the translocation process, was not reduced. Finally, we showed that the inhibitory effect of hexadecylphosphocholine on CTP:choline-phosphate cytidylyltransferase translocation cannot be explained by the detergent properties of this phospholipid analogue. Therefore, we suggest a direct inhibitory effect of hexadecylphosphocholine on the translocation of CTP:choline-phosphate cytidylyltransferase.

29] Choline-phosphate cytidylyltransferase

Publisher Summary Choline-phosphate cytidylyltransferase (CTP:cholinephosphate cytidylyltransferase) catalyzes a major rate-determining step in the biosynthesis of phosphatidylcholine in mammalian cells. Both cytosolic and membrane fractions contain choline-phosphate cytidylyltransferase activity. Enzyme activity is determined by measuring the formation of radioactive CDPcholine from phospho[methyl- 14 C]choline. Two methods have been used to separate CDPcholine from phosphocholine: adsorption of CDPcholine by charcoal or separation of CDPcholine from phosphocholine by thin-layer chromatography. The charcoal adsorption method is rapid, sensitive, and reproducible. The ability to perform many assays (50–100) per day is an additional advantage. A variety of lipids have been used by investigators for the assay of cytidylyltransferase. It has been a common practice to include lipid in assays involving soluble forms of cytidylyltransferase. The molecular and kinetic properties of cytidylyltransferase are discussed this chapter.

Phosphatidylcholine biosynthesis in celery cell suspension cultures with altered sterol compositions

Cell suspension cultures of celery were treated with the plant growth regulator, paclobutrazol. Lipid analysis revealed that use of this xenobiotic had little effect on the quantity or acyl quality of the major phospholipid classes or on the actual amounts of free sterol present in the cell. It did however, cause dramatic changes in the free sterol profile exhibited by treated cultures. In this respect, an increase in 14α‐methylsterols was observed.The synthesis of phosphatidylcholine in celery cell suspension cultures with altered free sterol compositions was studied using two radiolabelled biosynthetic precursors, [3H‐methyl]choline and [3H‐methyl]methionine. The studies showed that the rate of phosphatidylcholine biosynthesis via the CDP‐base pathway proceeded at a slower rate in paclobutrazol treated cultures. Accumulation of label phosphocholine was observed arising from reduced CTP:cholinephosphatecytidylyltransferase activity. In contrast, phosphatidylcholine biosynthesis via the sequential methylation of ethanolamine derivatives appeared to be enhanced in cells that had an unusually high 14α‐methylsterol content. From these investigations it may be postulated that the biosynthesis of phosphatidylcholine in Apium graveolens suspension cultures may be regulated by membrane sterol composition.