Pathway For Phosphatidylcholine Biosynthesis Research Articles

Functional redundancy of CDP-ethanolamine and CDP-choline pathway enzymes in phospholipid biosynthesis: ethanolamine-dependent effects on steady-state membrane phospholipid composition in Saccharomyces cerevisiae.

It has been established that yeast membrane phospholipid content is responsive to the inositol and choline content of the growth medium. Alterations in the levels of transcription of phospholipid biosynthetic enzymes contribute significantly to this response. We now describe conditions under which ethanolamine can exert significant influence on yeast membrane phospholipid composition. We demonstrate that mutations which block a defined subset of the reactions required for the biosynthesis of phosphatidylcholine (PC) via the CDP-choline pathway cause ethanolamine-dependent effects on the steady-state levels of bulk PC in yeast membranes. Such an ethanolamine-dependent reduction in bulk membrane PC content was observed for both choline kinase (cki) and choline phosphotransferase (cpt1) mutants, but it was not observed for mutants defective in cholinephosphate cytidylyltransferase, the enzyme that catalyzes the penultimate reaction of the CDP-choline pathway for PC biosynthesis. Moreover, the ethanolamine effect observed for cki and cpt1 mutants was independent of the choline content of the growth medium. Finally, we found that haploid yeast strains defective in the activity of both the choline and ethanolamine phosphotransferases experienced an ethanolamine-insensitive reduction in steady-state PC content, an effect which was not observed in strains defective in either one of these activities alone. The collective data indicate that specific enzymes of the CDP-ethanolamine pathway for phosphatidylethanolamine biosynthesis, while able to contribute to PC synthesis when yeast cells are grown under conditions of ethanolamine deprivation, do not do so when yeast cells are presented with this phospholipid headgroup precursor.

Lidocaine inhibits choline uptake and phosphatidylcholine biosynthesis in human leukemic monocyte-like U937 cells.

The effect of lidocaine on [3H]choline uptake and the incorporation of label into phosphatidylcholine (PC) in human monocyte-like U937 cells was investigated. Lidocaine inhibited the rate of choline uptake in a dose-dependent manner; at 3.2 mM it resulted in a drastic reduction, by as much as 65 per cent (n = 10; p < 0.0005) or 55 per cent (n = 10; p < 0.0006) in a 3- or 6-h incubation, respectively. Lidocaine also decreased the rate of choline incorporation into PC in a dose-dependent manner. At the highest dose, nearly 70 per cent or 45 per cent reduction was seen in a 3- or 6-h incubation, respectively. Analysis of choline-containing metabolites showed that the major label association with phosphocholine and PC was reduced to a similar extent which was also parallel to the inhibition of choline uptake. At 3.2 mM lidocaine, the reduction of choline uptake was shown to follow a competitive inhibition. In the case of [3H] choline incorporation into PC, the inhibitory pattern was shown to be of a mixed type. The pulse-chase study dissecting the effect on choline metabolism from that on total choline uptake indicated that lidocaine exerted an additionally inhibitory effect on intracellular choline metabolism into PC. In a separate protocol in which the labelled cells were first allowed to be chased until 3H-incorporation into PC reached a steady state, lidocaine no longer showed any effect. These results seem to exclude the possibility of enhanced PC breakdown and further suggest that the main inhibitory effect is on the CDP-choline pathway for PC biosynthesis. After a 3-h treatment, CTP: cholinephosphate cytidylyltransferase (CYT) in both the cytosolic and microsomal fractions was inhibited by approximately 20 per cent, while choline kinase (CK) and choline phosphotransferase (CPT) remain relatively unchanged. There was no evidence for translocation of CYT between cytosol and microsomes. Taken together, we have demonstrated a dual inhibitory function of lidocaine which inhibits PC biosynthesis in addition to its ability to block choline uptake profoundly in U937 cells.

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.

15] Choline/ethanolamine kinase from rat liver

Choline kinase is the initial enzyme in the CDPcholine pathway for phosphatidylcholine biosynthesis in mammalian cells. It catalyzes the Mg 2+ -ATP-dependent phosphorylation of choline. Choline kinase is rate limiting for phosphatidylcholine formation in several cells and tissues. The enzyme exists as at least two isoelectric forms in all tissues examined; a third form is inducible with polycyclic aromatic hydrocarbons and hepatoxic agents in liver. Both constitutive isoforms in liver have ethanolamine kinase activity, and kinetic and immunological data show that both kinase activities are catalyzed by the same enzymes. These results on the regulation and enzymatic activities of choline kinase indicate that the enzyme plays an important role in the long-term regulation of the CDPcholine pathway and may be involved in the coordinate regulation of phosphatidylethanolamine biosynthesis. To study choline and ethanolamine phosphorylation with a purified kinase preparation and to investigate the nature of the isoforms, a purification protocol for rat liver choline-ethanolamine kinase is developed. With the use of size-exclusion chromatography as the final step, a preparation of very high specific activity can be obtained. To obtain homogeneous, though less active, enzyme, chromatofocusing is used as the final step.

Channeling of intermediates in the CDP-choline pathway of phosphatidylcholine biosynthesis in cultured glioma cells is dependent on intracellular Ca2+

The major route of phosphatidylcholine (Ptd-choline) biosynthesis in mammalian cells is the CDP-choline pathway which involves stepwise conversion of choline to phosphocholine (P-choline), cytidine diphosphate choline (CDP-choline), and Ptd-choline. Our previous studies with electropermeabilized (EP) rat glioma (C6) cells have indicated that the intermediates of this pathway are not freely diffusible in the cell but are channeled toward synthesis of Ptd-choline (George, T.P., Morash, S.C., Cook, H.W., Byers, D.M., Palmer, F. B. St.C., and Spence, M.W. (1989) Biochim. Biophys. Acta 1004, 283-291). In this study, Ca(2+)-[ethylene-bis(oxyethylenenitrilo)]tetraacetic acid buffers were used to investigate the role of intracellular free Ca2+ levels in functional organization of this pathway in EP glioma cells. In EP cells reduction of free Ca2+ in the medium from 1.8 mM to less than 200 nM resulted in 2-3-fold stimulation of exogenous [3H]choline and [14C]P-choline incorporation into Ptd-choline whereas incorporation of exogenous CDP-[14C]choline was augmented 100-fold; there was no uptake or incorporation of labeled P-choline or CDP-choline in intact cells. In EP cells incubated at 1.8 mM Ca2+ the water-soluble products of choline metabolism (choline, P-choline, CDP-choline, and glycerophosphocholine) were retained at 37 degrees C; in contrast, in the presence of 100 nM Ca2+ there was uniform leakage of these metabolites. Experiments with hemicholinium-3, an inhibitor of choline transport, and EP cells at 100 nM Ca2+ show that linkage of choline transport and Ptd-choline biosynthesis is also dependent on Ca2+. These results suggest that channeling of intermediates in the CDP-choline pathway of Ptd-choline biosynthesis in glioma cells is mediated by intracellular Ca2+ levels that may coordinately regulate the steps involved in conversion of choline to Ptd-choline.

Mutations in the CDP-choline pathway for phospholipid biosynthesis bypass the requirement for an essential phospholipid transfer protein

SEC14p is the yeast phosphatidylinositol (PI)/phosphatidylcholine (PC) transfer protein, and it effects an essential stimulation of yeast Golgi secretory function. We now report that the SEC14p localizes to the yeast Golgi and that the SEC14p requirement can be specifically and efficiently bypassed by mutations in any one of at least six genes. One of these suppressor genes was the structural gene for yeast choline kinase ( CKI), disruption of which rendered the cell independent of the normally essential SEC14p requirement. The antagonistic action of the CKI gene product on SEC14p function revealed a previously unsuspected influence of biosynthetic activities of the CDP-choline pathway for PC biosynthesis on yeast Golgi function and indicated that SEC14p controls the phospholipid content of yeast Golgi membranes in vivo.

Phosphatidylcholine metabolism: masochistic enzymology, metabolic regulation, and lipoprotein assembly

Phosphatidylcholine is apparently essential for mammalian life, since there are no known inherited diseases in the biosynthesis of this lipid. One of its critical roles appears to be in the structure of the eucaryotic membranes. Why phosphatidylcholine is required and why other phospholipids will not substitute are unknown. The major pathway for the biosynthesis of phosphatidylcholine occurs via the CDP-choline pathway. Choline kinase, the initial enzyme in the sequence, has been purified to homogeneity from kidney and liver and also catalyzes the phosphorylation of ethanolamine. Most evidence suggests that the next enzyme in the pathway, CTP:phosphocholine cytidylyltransferase, catalyzes the rate-limiting and regulated step in phosphatidylcholine biosynthesis. This enzyme has also been completely purified from liver. Cytidylyltransferase appears to exist in the cytosol as an inactive reservoir of enzyme and as a membrane-bound form (largely associated with the endoplasmic reticulum), which is activated by the phospholipid environment. There is evidence that the activity of this enzyme and the rate of phosphatidylcholine biosynthesis are regulated by the reversible translocation of the cytidylyltransferase between membranes and cytosol. Three major mechanisms appear to govern the distribution and cellular activity of this enzyme. (i) The enzyme is phosphorylated by cAMP-dependent protein kinase, which results in release of the enzyme into the cytosol. Reactivation of cytidylyltransferase by binding to membranes can occur by the action of protein phosphatase 1 or 2A. (ii) Fatty acids added to cells in culture or in vitro causes the enzyme to bind to membranes, where it is activated. Removal of the fatty acids dissociates the enzyme from the membrane. (iii) Perhaps most importantly, the concentration of phosphatidylcholine in the endoplasmic reticulum feedback regulates the distribution of cytidylyltransferase. A decrease in the level of phosphatidylcholine causes the enzyme to be activated by binding to the membrane, whereas an increase in phosphatidylcholine mediates the release of enzyme into the cytosol. The third enzyme in the CDP-choline pathway, CDP-choline:1,2-diacylglycerol choline-phosphotransferase, has been cloned from yeast but never purified from any source. In liver an alternative pathway for phosphatidylcholine biosynthesis is the methylation of phosphatidylethanolamine by phosphatidylethanolamine N-methyltransferase. This enzyme is membrane bound and has been purified to homogeneity. It catalyzes all three methylation reactions involved in the conversion of phosphatidylethanolamine to phosphatidylcholine.(ABSTRACT TRUNCATED AT 400 WORDS)

Altered phosphatidylcholine metabolism in C3H10T1/2 cells transfected with the Harvey-ras oncogene.

The effect of expression of the Harvey-ras oncogene on phosphatidylcholine metabolism in C3H10T1/2 mouse fibroblast cells was examined. There were multiple changes in the CDP-choline pathway for phosphatidylcholine biosynthesis in the ras-expressing cells. The activity of the first enzyme in the pathway, choline kinase, was stimulated 1.9-fold, while the activity of the second enzyme, CTP:phosphocholine cytidylyltransferase, was decreased by one-half. High levels of intracellular phosphocholine measured in the ras cells were consistent with the altered activities of choline kinase and cytidylyltransferase. The overall rate of phosphatidylcholine synthesis appeared to be increased because the turnover rate of phosphocholine from the intracellular pool was higher in the ras-transfected cells. There also appeared to be an increased rate of phosphatidylcholine degradation in ras-expressing C3H10T1/2 cells. Very high levels of glycerophosphocholine (6-fold increased over control cells) suggested that phospholipase A was activated in these cells. These results indicate that the ras oncogene product directly or indirectly causes an increased turnover of phosphatidylcholine in C3H10T1/2 cells.

Purification and characterization of choline/ethanolamine kinase from rat liver.

Choline kinase, the first enzyme in the CDP-choline pathway for phosphatidylcholine biosynthesis, was purified 26,000-fold from rat liver to a specific activity of 143,000 nmol.min-1.mg-1 protein. The subunit molecular mass was 47 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, while the apparent native molecular mass was 160 kDa by size exclusion chromatography, suggesting a tetrameric structure. Two peaks of choline kinase activity were obtained by chromatofocusing. These isoforms eluted at pH 4.7 (CKI) and 4.5 (CKII). CKII appeared to be homogeneous by sodium dodecyl sulfate gel electrophoresis. Peptide mapping of two isoforms indicated a high degree of similarity, although there were peptides not common to both. Ethanolamine kinase activity copurified with both isoforms. The ratio of choline to ethanolamine kinase activity was 3.7 +/- 0.7 throughout the purification procedure. Choline and ethanolamine were mutually competitive inhibitors. The respective Km values, 0.013 and 1.2 mM, were similar to the Ki values of 0.014 and 2.2 mM. An antibody raised against CKII immunoprecipitated both choline and ethanolamine kinase activities from liver cytosol at the same titer. These data suggest that both activities reside on the same protein and occur at the same active site. Similarly, both activities were immunoprecipitated from brain, lung, and kidney cytosols. Western blot analysis showed both purified liver isoforms, as well as brain, lung and kidney enzymes, to have a molecular mass of 47 kDa.

Co-ordinate control of CDP-choline and phosphatidylethanolamine methyltransferase pathways for phosphatidylcholine biosynthesis occurs in response to change in diet fat

Brain microsomal and synaptic plasma membrane phosphatidylcholine composition and biosynthetic activity were examined in relation to the composition of diet fat fed. Phosphocholinetransferase and methyltransferase activities are shown to be modulated by the diet, and by changes in the membrane phospholipid content of long-chain polyunsaturated fatty acids. This physiological modulation is co-ordinated such that the rate of phosphatidylcholine synthesis via one route is inversely regulated with activity of the alternate pathway.

Phosphatidylcholine biosynthesis in castor bean endosperm. Mechanisms of regulation during the immediate postgermination period

Changes in phosphatidylcholine content, lipid precursor incorporation and lipid biosynthetic enzyme activities were observed in castor bean endosperms during a six day postgermination period. The metabolism of phosphatidylcholine was regulated independently of other phospholipids, as evidenced by the ratio of phosphatidylcholine to total phospholipid not being constant during development. The main pathway for phosphatidylcholine biosynthesis was the nucleotide pathway, although studies with labelled glycerol-3-phosphate suggested that the diacylglycerol for this pathway was not synthesized de novo, but probably obtained by degradation of pre-existing lipids. The control of the nucleotide pathway appears to reside in choline phosphate cytidylyltransferase and choline phosphotransferase, the activities of which were coordinated, but not in choline kinase. The cytidylyltransferase is regulated in part by translocation of the soluble form of the enzyme to the endoplasmic reticulum, but this regulation could not account for all of the observed changes in phosphatidylcholine synthesis.

Regulation of phosphatidylcholine biosynthesis in Plasmodium-infected erythrocytes

Plasmodium knowlesi-infected erythrocytes efficiently incorporated choline and metabolize it into phosphatidylcholine via the de novo Kennedy pathway. Nor formation of either betaine or acetylcholine was detected. At physiological concentrations of external choline, isotopic equilibrium between intracellular choline and phosphocholine was reached in less than 1 h, whereas labeled phosphatidylcholine accumulated constantly, until at least 210 min. During this time, intracellular CDP-choline remained quite low compared to phosphocholine, which suggests that choline-phosphate cytidylyltransferase (EC 2.7.7.15) is the rate-limiting step of the Kennedy pathway. However, this activity was probably not saturated in situ by phosphocholine, since the external choline concentration, up to 100 μM, can regulate phosphatidylcholine biosynthesis via the level of intracellular phosphocholine. This was corroborated by the respective velocities and affinity characteristics of the three enzymatic steps involved in the Kennedy pathway. These results, together with the localization of both choline metabolites and enzyme activities, provide a precise scheme of the dynamics of de novo phosphatidylcholine biosynthesis. Concerning the alternative pathway for phosphatidylcholine biosynthesis via the methylation of phosphatidylehanolamine, we show that an increase in de novo phosphatidylcholine biosynthesis could instigate a concomitant decrease in the steps of phosphatidylethanolamine methylation, indicating that the parasite is able to modulate its phosphatodylcholine biosyntheses.

Choline glycerophospholipid biosynthesis in the guinea pig heart.

The aims of this study were to (i) elucidate the biosynthetic pathways for the formation of plasmenylcholine in the mammalian heart and (ii) investigate whether the control of choline glycerophospholipid production is different in hearts with high plasmenylcholine content. Guinea pig hearts were used throughout this study, since 34% of the cardiac choline glycerophospholipids in this species is present in the plasmenylcholine form. By perfusion of the guinea pig heart in the Langendorff mode with labeled choline, we demonstrated that the majority of plasmenylcholine in the heart was synthesized via the CDP-choline pathway. The ability of the heart to form plasmenylcholine from CDP-choline and 1-alkenyl-2-acylglycerol was also shown. We postulate that 1-alkenyl-2-acylglycerol in the guinea pig heart might originate from the hydrolysis of plasmenylethanolamine. In mammalian liver and other tissues, the CDP-choline pathway is the major pathway for phosphatidylcholine biosynthesis and the rate-limiting step is catalyzed by CTP:phosphocholine cytidylyltransferase. The results obtained from the present study support this supposition. In addition, evidence was obtained indicating that phosphorylation of choline by choline kinase in the CDP-choline pathway may also be rate limiting. Although the involvement of choline kinase as a rate-limiting enzyme in the CDP-choline pathway has been shown in a number of cell cultures, the rate-limiting role of this enzyme in intact mammalian organs has not been previously reported. The rationale for the presence of more than one rate-limiting step in the CDP-choline pathway in the guinea pig heart remains undefined.

Choline Incorporation by Schistosoma mansoni: Distribution of Choline Metabolites during Development and after Sexual Differentiation

Choline metabolism was investigated in Schistosoma mansoni during the main phases of its development, namely, schistosomula, 11- and 15-day-old worms, and adults. At the physiological choline concentration used in the assay (20 microM), betaine was, along with phosphatidylcholine, one of the most abundant choline metabolites, revealing considerable choline oxidation activity. Very little radioactivity was associated with CDP-choline, whereas a sustained incorporation into phosphocholine occurred. These results provide good evidence that CTP:phosphocholine cytidylyltransferase (EC 2.7.7.15) plays a regulatory role in the de novo pathway of phosphatidylcholine biosynthesis. During development, the incorporation of choline into its various metabolites was maximal in 11-day-old worms. At this stage, the oxidative pathway predominated over the Kennedy pathway, whereas at all other stages the de novo phosphatidylcholine biosynthesis was predominant. Furthermore, choline incorporation into betaine was much more important in the adult female worm than in the male, indicating a major difference in choline incorporation and distribution between the 2 sexes of the adult worms.

Phosphocholinetransferase activity in plasma membrane: Effect of diet

The presence of phosphocholinetransferase, a component of the CDP-choline pathway for phosphatidylcholine biosynthesis is demonstrated in brain synaptic plasma membrane. Activity of phosphocholinetransferase is higher in weanling versus adult tissues, and responds to alterations in fat intake. The implications of phosphocholinetransferase activity in plasma membrane, and dietary manipulation of phosphatidylcholine biosynthesis via this pathway are discussed.

Phosphatidylcholine homeostasis in phosphatidylethanolamine-depleted Tetrahymena

The relative contributions of the two pathways of phosphatidylcholine biosynthesis, phosphatidylethanolamine N-methyltransferase (EC 2.1.1.17) and diacylglycerol:CDP-choline cholinephosphotransferase (EC 2.7.8.1), are altered in the ciliate protozoan Tetrahymena thermophila whose phospholipid composition has been modified by culturing the organism in the presence of one of several aminophosphonic acids, as determined by measuring the incorporation of [ methyl- 3H]choline and [ methyl- 14C]methionine into phosphatidylcholine in vivo. In control cells the phosphotransferase pathway provides about 40% of the phosphatidylcholine, while in cells grown with 2-aminoethylphosphonate (AEP), 3-aminopropylphosphonate (APP), and N,N,N-trimethylaminoethylphosphonate (TMAEP) the contribution of the phosphotransferase pathway to phosphatidylcholine formation is 75, 90, and 26%, respectively. In AEP- and APP-grown cells, in which 80% of the phosphatidylethanolamine has been replaced by the corresponding phosphonolipid, the methyltransferase is less active since the level of the substrate phosphatidylethanolamine is reduced and neither of the phosphonolipids is a substrate for the enzyme. In TMAEP-grown cells, TMAEP competes with and reduces the incorporation of phosphocholine by the phosphotransferase pathway, leading to a smaller contribution of the pathway to phosphatidylcholine biosynthesis. The relative amounts of the two different radioactive labels incorporated into diacylphosphatidylcholine vs alkylacylphosphatidylcholine are also altered in the phosphonate-grown cells. The exogenous AEP induces a change in the glyceryl ether content of the 2-aminoethylphosphonolipid—33% in the AEP-grown cells compared to 70% in the control cells—indicating that the exogenous AEP is entering the phospholipids by the ethanolamine-phosphotransferase pathway rather than by the route of the endogenous AEP.

The role of phosphatidylcholine biosynthesis in the secretion of lipoproteins from hepatocytes.

An investigation of the role of phospholipids in lipoprotein assembly and secretion is important since phospholipids, particularly phosphatidylcholine, are prominent components of all plasma lipoproteins. The fatty acid composition of phosphatidylcholine is virtually identical in human very low (VLDL), low, and high density lipoproteins, which supports the idea that phosphatidylcholine exchanges freely among plasma lipoproteins. However, the fatty acid composition of phosphatidylcholine from cultured rat hepatocytes is different from that in the secreted lipoproteins. In addition, the composition of molecular species of phosphatidylcholine is quite different in the rat liver, plasma, and red cells. Phosphatidylcholine is made in liver by two alternate pathways, by the CDP-choline pathway and by the methylation of phosphatidylethanolamine. Regulation of phosphatidylcholine biosynthesis by the CDP-choline pathway in rat liver is well established. In most instances, the rate of phosphatidylcholine synthesis is governed by the activity of CTP:phosphocholine cytidylyltransferase, which is present in the cytosol and also associated with microsomes. The cytosolic enzyme is inactive but can be reversibly translocated to the microsomes, where it is active. Translocation of this enzyme to the microsomes can be achieved either by a dephosphorylation reaction or by the presence of fatty acids in the cytosol. Once synthesized, how is phosphatidylcholine assembled into lipoprotein particles? The sequence of assembly of phospholipids into VLDL has been investigated in several studies. In a pulse-chase experiment, there was an initial labelling (within 15 min of the pulse) of phospholipids in secreted VLDL, which probably reflected the rapid movement of the phospholipids from their site of synthesis (the endoplasmic reticulum) to the Golgi. There appears to be a rapid exchange of phospholipid between the Golgi membranes and contents. There was also a delayed labelling (after 30 min) of the phospholipids and triacylglycerols (from [3H]glycerol) and the apoproteins (from [3H]leucine) in the secreted VLDL. This lag was attributed to the time taken for the nascent VLDL particles to move from the lumen of the endoplasmic reticulum to the Golgi and into the medium. Is phosphatidylcholine biosynthesis is required for lipoprotein secretion? This question was investigated in rats maintained on a choline-deficient diet for 10 days. The total amount of plasma phosphatidylcholine decreased by approximately 40% and the rats developed fatty livers.(ABSTRACT TRUNCATED AT 400 WORDS)

Regulation of phosphatidylcholine biosynthesis in Chinese hamster ovary cells by reversible membrane association of CTP: phosphocholine cytidylyltransferase.

Treatment of Chinese hamster ovary cells with phospholipase C was previously shown to stimulate the CDP-choline pathway for phosphatidylcholine biosynthesis, and to cause activation of the CTP:phosphocholine cytidylyltransferase with a concomitant change in subcellular location of the enzyme (Sleight, R., and Kent, C. (1983) J. Biol. Chem. 258, 831-835). This paper presents a detailed analysis of the early events in the phospholipase C treatment, and provides evidence that the increased cytidylyltransferase activity causes the increased flux through the pathway. The time courses for the increase in cytidylyltransferase activity, increase in amount of membrane-associated enzyme, decrease in phosphocholine levels, and increase in phosphatidylcholine synthesis were similar, with all changes occurring within 30 min after addition of phospholipase C. These events preceded a decrease in cellular choline levels which correlated with a decreased capacity for choline uptake. The rate at which radioactive label was lost from pulse-labeled phosphocholine was the same as the rate at which label was incorporated into phosphatidylcholine, and these rates were stimulated 2.2-fold by phospholipase C treatment. We have also shown that the association of cytidylyltransferase with membranes was rapidly reversible when phospholipase C was removed from the cultures, and that the rate of decrease in phosphatidylcholine synthesis paralleled the rate of decrease in cytidylyltransferase activity. Cytidylyltransferase became reassociated with membranes when phospholipase C was added back to cultures from which it was previously removed. These results represent the first detailed account of the time frame involved in regulating phosphatidylcholine synthesis by the reversible association of cytidylyltransferase with cellular membranes.

Coordinate regulation of phosphatidylserine decarboxylase activity and phospholipid N-methylation in yeast.

Membranes isolated from Saccharomyces cerevisiae, strain ATCC 26615, catalyze the decarboxylation of exogenous phosphatidylserine added as an aqueous dispersion in detergent. Active preparations of the decarboxylase can be obtained by extracting salt-washed membranes with 0.5% Cutscum . The properties of the phosphatidylserine decarboxylase activity associated with a particulate fraction and the detergent extracts have been characterized by assaying the enzymatic conversion of exogenous [14C]phosphatidylserine to [14C]phosphatidylethanolamine. The yeast decarboxylase does not require a divalent cation and is inhibited by hydroxylamine and p-hydroxymercuribenzoate. The rate of decarboxylation of exogenous phosphatidylserine catalyzed by membranes prepared from cells grown in the presence of choline is reduced by approximately 60% compared to membranes from cells grown in a choline-deficient medium. Relatively smaller reductions in phosphatidylserine decarboxylase activity are also seen in cells grown in the presence of mono- or dimethylethanolamine. In vitro incorporation studies with [14C]serine demonstrate that endogenous, prelabeled phosphatidylserine can be utilized for the biosynthesis of phosphatidylcholine by the coupled action of the hydroxylamine-sensitive decarboxylase and the phospholipid N-methyltransferases in the presence of 2 mM S-adenosylmethionine. A similar comparative enzymatic study shows that the rates of synthesis and decarboxylation of [14C]phosphatidylserine, as well as phospholipid N-methylation, are lower for membranes prepared from cells grown in the presence of choline relative to identical preparations from cells grown in the absence of choline. These studies describe the properties of particulate and detergent-solubilized phosphatidylserine decarboxylase activity in S. cerevisiae and provide evidence that its activity is regulated in coordination with other enzymes in the pathway for phosphatidylcholine biosynthesis involving N-methylation.

Regulation of phosphatidylcholine biosynthesis in mammalian cells. I. Effects of phospholipase C treatment on phosphatidylcholine metabolism in Chinese hamster ovary cells and LM mouse fibroblasts.

Addition of phospholipase C from Clostridium perfringens to cultures of Chinese hamster ovary (CHO) cells resulted in rapid degradation of cellular phosphatidylcholine with concomitant release of phosphocholine. The rate of incorporation of radiolabeled choline into lipids was increased 2-fold in phospholipase C-treated CHO cells as compared to untreated controls. The only enzyme in the pathway of phosphatidylcholine biosynthesis with increased activity in phospholipase C-treated cells was CTP:phosphocholine cytidylyltransferase, indicating that the cytidylyltransferase plays an important role in the stimulation of phosphatidylcholine biosynthesis. The phospholipase treatment was toxic to a CHO mutant cell line with abnormally low cytidylyltransferase activity. Mouse LM fibroblasts were resistant to enzymatic attack by phospholipase C, and cytidylyltransferase activity in LM cells did not change upon phospholipase C treatment.