De novo CDP-choline-dependent glycerophosphorylcholine synthesis and its involvement as an intermediate in phosphatidylcholine synthesis

The activity of CDP-choline-dependent glycerophosphorylcholine synthetase (CDP-choline:sn-3-glycerophosphate cholinetransferase), a newly discovered enzyme involved in the recently proposed pathways of acyl-specific phosphatidylcholine synthesis, is reported in rat liver. Endogenous CDP-choline, synthesized via the CMP-driven back reaction of phosphorylcholine transferase, is also shown to be a choline donor for this glycerophosphorylcholine synthetase. The function of glycerophosphorylcholine as an intermediate in phosphatidylcholine synthesis is demonstrated by specific isotope trapping whereby unlabelled glycerophosphorylcholine inhibited label incorporation from sn-[14C]glycerol-3-phosphate into phosphatidylcholine in mouse gastrocnemius, a tissue that is essentially devoid of the cytidine pathway for phosphatidylcholine synthesis and uses a non-allelic glycerophosphorylcholine synthetase (exogenous PC:sn-3-glycerophosphate cholinetransferase) in the synthesis of glycerophosphorylcholine.     

Impaired glycerophosphorylcholine synthesis in murine muscular dystrophy

A test of some of the tenets of a proposed hypothesis on muscle phospholipid synthesis, and its possible defect in murine muscular dystrophy, shows that the cytidine pathways for the synthesis of phosphatidylcholine and phosphatidylethanolamine have a negligible flux in differentiated mouse gastrocnemius, while that of the respective proposed de novo glycerophosphodiester pathways is normally high in this muscle. Evidence is presented that de novo glycerophosphorylcholine synthesis in dystrophic mouse gastrocnemius is about half that of the wild type homozygotes, while that of the heterozygotes is near the mean of the two homozygous groups. No significant differences in rates of glycerophosphorylcholine or glycerophosphorylethanolamine synthesis were observed in brain and liver tissues among the three genotypes. These results suggest that defective de novo synthesis of glycerophosphorylcholine may be the primary biochemical lesion in murine muscular dystrophy.     

Synthesis of phosphatidylcholine under possible primitive Earth conditions

Summary Using a primitive Earth evaporating pond model, the synthesis of phosphatidylcholine was accomplished when a reaction mixture of choline chloride and disodium phosphatidate, in the presence of cyanamide and traccs of acid, was evaporated and heated at temperatures ranging from 25° to 100°C for 7 hours. Optimum yields of about 15% were obtained at 80°C. Phosphatidylcholine was identified by chromatographic, chemical and enzymatic degradation methods. On enzymatic hydrolysis with phospholipase A₂ and phospholipase C, lysophosphatidylcholine and phosphorylcholine were formed, respectively. Alkaline hydrolysis gave glycerophosphorylcholine. The synthesis of phosphatidylcholine as the major compound was accompanied by the formation of lysophosphatidylcholine in smaller amounts. Cyanamide was found to be essential for the formation of phosphatidylcholine, and only traces of HCl, of the order of that required to convert the disodium phosphatidate to free phosphatidic acid were found necessary for the synthesis. This work suggests that phosphatidylcholine, which is an essential component of most biological membranes, could have been synthesized on the primitive Earth.     

Examination of the potential role of the glycerophosphorylcholine (GPC) pathway in the biosynthesis of phosphatidylcholine by liver and lung

The potential involvement of the glycerophosphorylcholine (GPC) pathway for the synthesis of phosphatidylcholine (PC) has been examined in rat liver and lung and in a human line, the A549 cell which possesses characteristics representative of mature alveolar type II epithelial cells. Although mitochondrial and microsomal fractions from the above sources readily incorporated radioactive glycerophosphate into lipids, the only incorporation observed with radioactive GPC was a small variable labelling with the mitochondrial and microsomal fractions from rat lung. Even with these fractions, no radioactivity from GPC was incorporated into PC or lysoPC. Attempts to increase the incorporation of GPC into lipids by manipulating the incubation conditions were unsuccessful. It was concluded that the occurrence of the GPC pathway in liver and lung is unlikely.     

1,2‐Diacylglycerol choline phosphotransferase catalyzes the final step in the unique Treponema denticola phosphatidylcholine biosynthesis pathway

Treponema denticola synthesizes phosphatidylcholine through a licCA‐dependent CDP‐choline pathway identified only in the genus Treponema. However, the mechanism of conversion of CDP‐choline to phosphatidylcholine remained unclear. We report here characterization of TDE0021 (herein designated cpt) encoding a 1,2‐diacylglycerol choline phosphotransferase homologous to choline phosphotransferases that catalyze the final step of the highly conserved Kennedy pathway for phosphatidylcholine synthesis in eukaryotes. T. denticola Cpt catalyzed in vitro phosphatidylcholine formation from CDP‐choline and diacylglycerol, and full activity required divalent manganese. Allelic replacement mutagenesis of cpt in T. denticola resulted in abrogation of phosphatidylcholine synthesis. T. denticola Cpt complemented a Saccharomyces cerevisiae CPT1 mutant, and expression of the entire T. denticola LicCA‐Cpt pathway in E. coli resulted in phosphatidylcholine biosynthesis. Our findings show that T. denticola possesses a unique phosphatidylcholine synthesis pathway combining conserved prokaryotic choline kinase and CTP:phosphocholine cytidylyltransferase activities with a 1,2‐diacylglycerol choline phosphotransferase that is common in eukaryotes. Other than in a subset of mammalian host‐associated Treponema that includes T. pallidum, this pathway is found in neither bacteria nor Archaea. Molecular dating analysis of the Cpt gene family suggests that a horizontal gene transfer event introduced this gene into an ancestral Treponema well after its divergence from other spirochetes.     

Relationships Between Phosphatidylcholine, Phosphatidylethanolamine, and Sphingomyelin Metabolism in Cultured Oligodendrocytes

Abstract: In most cell types the major pathway of sphingomyelin synthesis is the direct transfer of the phosphocholine head group from phosphatidylcholine to ceramide catalyzed by the enzyme l -acylsphingosine:phosphatidylcholine phosphocholinetransferase (SM synthase; EC 2.7.8.-). Although this pathway has been demonstrated in brain tissue, its quantitative importance has been questioned. An alternative biosynthetic pathway for sphingomyelin synthesis in brain tissue has been proposed, viz., the direct transfer of phosphoethanolamine from phosphatidylethanolamine to ceramide, followed by methylation of the ethanolamine moiety to a choline group. We have evaluated various possible biosynthetic pathways of sphingomyelin synthesis in rat spinal cord oligodendrocytes, the myelin-forming cells of the CNS, by labeling cells in culture with radiolabeled choline, ethanolamine, or serine. Our results indicate that, in oligodendrocytes, most of the phosphocholine for the biosynthesis of sphingomyelin is provided by phosphatidylcholine, which is predominantly derived from de novo synthesis. No evidence was found for the operation of the alternative pathway via ceramide-phosphoethanolamine. Furthermore, our results indicate that a small pool of phosphatidylcholine is provided by methylation of phosphatidylethanolamine, which in turn is formed preferentially by decarboxylation of phosphatidylserine.     

The regulation of the fatty-acid composition of the triacylglycerols in microsomal preparations from avocado mesocarp and the developing cotyledons of safflower

The utilisation of [¹⁴C]glycerol 3-phosphate and [¹⁴C]linoleoyl-CoA in the synthesis of triacylglycerol has been studied in the microsomal preparations of developing cotyledons of safflower seed. The results confirm that the glycerol backbone, which flows towards triacylglycerol from phosphatidic acid through the Kennedy pathway, can enter phosphatidylcholine from diacylglycerol. The equilibration between diacylglycerol and phosphatidylcholine offers a mechanism for the return of oleate to phosphatidylcholine for desaturation to linoleate. We have established that the oleate entering position 1 of sn-phosphatidylcholine from diacylglycerol is desaturated in situ to linoleate. The results indicate that the diacylglycerol phosphatidylcholine interconvertion coupled to the acyl exchange between acyl-CoA and position 2 of sn-phosphatidylcholine brings about the continuous enrichment of the glycerol backbone with C₁₈-polyunsaturated fatty acids and hence these enzymes are of major importance in regulating the acyl quality of the accumulating triacylglycerols. Microsomal preparations from avocado mesocarp, however, did not have detectable acyl exchange between acyl-CoA and phosphatidylcholine or diacylglycerol phosphatidylcholine interconversion despite the high activity of the enzymes of the Kennedy pathway. A scheme is presented which incorporates many of the observations on triacylglycerol synthesis and provides a working model for the regulation of acyl quality in linoleate-rich vegetable oils.Abbreviation BSA bovine serum albumin     

Quantitation of glycerophosphorylcholine by flow injection analysis using immobilized enzymes

A method for quantitating glycerophosphorylcholine by flow injection analysis is reported in the present paper. Glycerophosphorylcholine phosphodiesterase and choline oxidase, immobilized on controlled porosity glass beads, are packed in a small reactor inserted in a flow injection manifold. When samples containing glycerophosphorylcholine are injected, glycerophosphorylcholine is hydrolyzed into choline and sn-glycerol-3-phosphate. The free choline produced in this reaction is oxidized to betain and hydrogen peroxide. Hydrogen peroxide is detected amperometrically.Quantitation of glycerophosphorylcholine in samples containing choline and phosphorylcholine is obtained inserting ahead of the reactor a small column packed with a mixed bed ion exchange resin. The time needed for each determination does not exceed one minute.The present method, applied to quantitate glycerophosphorylcholine in samples of seminal plasma, gave results comparable with those obtained using the standard enzymatic- spectrophotometric procedure.An alternative procedure, making use of co-immobilized glycerophosphorylcholine phosphodiesterase and glycerol-3-phosphate oxidase for quantitating glycerophosphorylcholine, glycerophosphorylethanolamine and glycerophosphorylserine is also described.Abbreviations GPC sn-glycerol-3-phosphorylcholine - GPE sn-glycerol-3-phosphorylethanolamine - GPS sn-glycerol-3-phosphorylserine - GPA sn-glycerol-3-phosphoric acid - PDE glycerophosphorylcholine-phosphodiesterase - GPA-Ox glycerophosphate oxidase - Cho-Ox choline oxidase     

Turnover of phosphatidylcholine in Saccharomyces cerevisiae. The role of the CDP-choline pathway

The regulation of phosphatidylcholine degradation as a function of the route of phosphatidylcholine (PC) synthesis and changing environmental conditions has been investigated in the yeast Saccharomyces cerevisiae. In the wild-type strains studied, deacylation of phosphatidylcholine to glycerophosphocholine is induced when choline is supplied to the culture medium and, also, when the culture temperature is raised from 30 to 37 degrees C. In strains bearing mutations in any of the genes encoding enzymes of the CDP-choline pathway for phosphatidylcholine biosynthesis (CKI1, choline kinase; CPT1, 1, 2-diacylglycerol choline phosphotransferase; PCT1, CTP:phosphocholine cytidylyltransferase), no induction of phosphatidylcholine turnover and glycerophosphocholine production is seen in response to choline availability or elevated temperature. In contrast, the induction of phosphatidylcholine deacylation does occur in a strain bearing mutations in genes encoding enzymes of the methylation pathway for phosphatidylcholine biosynthesis (i.e. CHO2/PEM1 and OPI3/PEM2). Whereas the synthesis of PC via CDP-choline is accelerated when shifted from 30 to 37 degrees C, synthesis of PC via the methylation pathway is largely unaffected by the temperature shift. These results suggest that the deacylation of PC to GroPC requires an active CDP-choline pathway for PC biosynthesis but not an active methylation pathway. Furthermore, the data indicate that the synthesis and turnover of CDP-choline-derived PC, but not methylation pathway-derived PC, are accelerated by the stress of elevated temperature.     

Import of lyso-phosphatidylcholine into chloroplasts likely at the origin of eukaryotic plastidial lipids

Plastids rely on the import of extraplastidial precursor for the synthesis of their own lipids. This key phenomenon in the formation of plastidial phosphatidylcholine (PC) and of the most abundant lipids on earth, namely galactolipids, is poorly understood. Various suggestions have been made on the nature of the precursor molecule(s) transferred to plastids, but despite general agreement that PC or a close metabolite plays a central role, there is no clear-cut answer to this question because of a lack of conclusive experimental data. We therefore designed experiments to discriminate between a transfer of PC, 1-acylglycero phosphorylcholine (lyso-PC), or glycerophosphorylcholine. After pulse-chase experiments with glycerol and acetate, plastids of leek (Allium porrum L.) seedlings were purified. The labels of the glycerol moiety and the sn-1- and sn-2-bound fatty acids of plastidial lipids were determined and compared with those associated with the extraplastidial PC. After import, plastid lipids contained the glycerol moiety and the fatty acids esterified to the sn-1 position originating from the extraplastidial PC; no import of sn-2-bound fatty acid was detected. These results rule out a transfer of PC or glycerophosphorylcholine, and are totally explained by an import of lyso-PC molecules used subsequently as precursor for the synthesis of eukaryotic plastid lipids.     

A new crystalline phase of L-alpha-dipalmitoyl phosphatidylcholine monohydrate.

A new phase transition of L-alpha-dipalmitoyl phosphatidylcholine (DPPC) monohydrate from the "biaxial" phase to a crystalline phase (C phase) has been found at 71 degrees C by means of infrared attenuated total reflection (IR-ATR) spectroscopy. The transition is characterized by drastic conformational changes in the glycerophosphorylcholine moiety, which led on the one hand to an alignment of the turn near the ester group in the hydrocarbon chain at glycerol C(2) position. On the other hand a uniform conformation of the glycerophosphorylcholine moiety is found to be typical for the C phase, in contrast to nonuniform head group conformations of DPPC in other regions of the DPPC/water phase diagram investigated so far.     

Lung surface-active fraction as a model system for macromolecular ultrastructural studies with Crotalus atrox venom

The dog lung surface-active fraction and phosphatidylcholine constituents were subjected to hydrolysis by Crotalus atrox phospholipase A(2). Relative rates of hydrolysis were: dipalmitoyl glycerophosphorylcholine > phosphatidylcholine isolated from the surface-active fraction > phosphatidylcholine as an integral component of the intact surface-active macromolecular structure. Cholesterol markedly inhibited, whereas tripalmitin increased, the rate of hydrolysis with both pure phosphatidylcholine substrates. The effect of temperature on the velocity indicated the enzyme was most active when the substrates were in the gel state. These kinetic results, in conjunction with surface chemistry studies, can be interpreted to indicate that the phosphatidylcholine in the intact surface-active macromolecular particle is liquid crystalline due to molecular interactions with other constituents. Gas-liquid chromatographic analysis of the 2-lysophosphatidylcholines and fatty acids produced from the enzymatic hydrolysis of the intact surface-active fraction indicated that palmitoyl residues were more accessible to the enzyme, perhaps because they occupied positions near the surface of the particle.     

Uncoupling farnesol-induced apoptosis from its inhibition of phosphatidylcholine synthesis

Genetic inactivation of the synthesis of phosphatidylcholine, the most abundant membrane lipid in eukaryotic cells, induces apoptosis. Administration of farnesol, a catabolite within the isoprenoid/cholesterol pathway, also induces apoptosis. The mechanism by which farnesol induces apoptosis is currently believed to be by direct competitive inhibition with diacylglycerol for cholinephosphotransferase, the final step in the phosphatidylcholine biosynthetic pathway. Our recent isolation of the first mammalian cholinephosphotransferase cDNA has enabled us to more precisely assess how farnesol affects phosphatidylcholine synthesis and the induction of apoptosis. Induced over-expression of cholinephosphotransferase in Chinese hamster ovary cells prevented the block in phosphatidylcholine biosynthesis associated with exposure to farnesol. However, induced over-expression of cholinephosphotransferase was not sufficient for the prevention of farnesol-induced apoptosis. In addition, exogenous administration of diacylglycerol prevented farnesol-induced apoptosis but did not relieve the farnesol-induced block in phosphatidylcholine synthesis. We also developed an in vitro lipid mixed micelle cholinephosphotransferase enzyme assay, as opposed to the delivery of the diacylglycerol substrate in a detergent emulsion, and demonstrated that there was no direct inhibition of cholinephosphotransferase by farnesol or its phosphorylated metabolites. The execution of apoptosis by farnesol appears to be a separate and distinct event from farnesol-induced inhibition of phosphatidylcholine biosynthesis and instead likely occurs through a diacylglycerol-mediated process that is downstream of phosphatidylcholine synthesis.     

THE SYNTHESIS OF CHOLINE PHOSPHOGLYCERIDES IN THE GIANT FIBRE SYSTEM OF THE SQUID

The mechanisms and pathways of synthesis of phosphatidylcholine in the giant fibre system of the squid (Loligo vulgaris) have been examined by incubating the stellate ganglion-nerve preparation or its separated compartments in an artificial bathing solution with labelled choline. Other experiments were done by dissecting the whole stellate ganglion into axoplasm, axon sheath, giant fibre lobe, small fibres and ganglion residue, after incubation. The initial rate of choline incorporation into choline phosphoglycerides was severalfold higher in the lobe than in the axon. Higher lipid radioactivity was recovered in the axon sheath as compared to the axoplasm, and in the small fibres as compared to the ganglion residue which contains its cell bodies. The production of phosphorylcholine and CDP-choline in the intact ganglion-nerve preparation during incubation with choline points to the occurrence of the net synthesis pathway for phosphatidylcholine in this material. Base-exchange activity was also observed in the axon and giant fibre lobe preparations in vitro, but no indication can yet be given whether it also takes place in intact preparations. Electrical stimulation and‘depolarizing’conditions enhance choline phosphorylation in the squid axon and lobe, but decrease phosphatidylcholine labelling.     

Identification and partial characterization of phospholipid methylation in rat small-intestinal brush-border membranes

An earlier study (Biochim. Biophys. Acta 46 (1961) 205-216) failed to detect the enzymatic synthesis of phosphatidylcholine (PC) from phosphatidylethanolamine (PE) via a transmethylation pathway in rat small-intestinal microsomal membranes. This pathway was therefore assumed to be absent from this organ. Recently, however, in our laboratory it has been demonstrated that this pathway for the synthesis of phosphatidylcholine is present in rat colonic brush-border and basolateral membranes. It was therefore of interest to examine whether phospholipid methylation activity was present in rat small-intestinal brush-border membranes. The results of the present experiments demonstrate for the first time that this pathway for the synthesis of phosphatidylcholine exists in these plasma membranes. Evidence to support the enzymatic nature of this reaction include: loss of activity by heat denaturation and at 0 degree C, significant inhibition by S-adenosyl-L-homocysteine and saturation kinetics. The predominant product of this brush-border membrane phospholipid methyltransferase is phosphatidyl-N-monomethylethanolamine. This enzymatic activity has an apparent Km for S-adenosyl-L-methionine of 40 microM, a Vmax of 8.4 pmol/mg protein per 5 min, and a pH optimum of 8.0.     

Effects of aging on cholinephosphotransferase activity on guinea pig lung mitochondria and microsomes

It is known that the composition of phospholipids in lung changes with age. The final step in thede novo synthesis of phosphatidylcholine, a major component of lung surfactant, by the CDP-choline pathway, requires the enzyme cholinephosphotransferase (CPT). Even though CPT has earlier been proposed to be located exclusively in the endoplasmic reticulum, we have recently demonstrated its presence also in the mitochondria. We have earlier reported a gestational variation of CPT activity in fetal mitochondria and microsomes. In the present study we examined the subcellular distribution of CPT activity in lung as a function of age. After birth, the microsomal CPT activity continued to increase until adulthood (24 wks of age), thereafter it gradually decreased. On the otherhand, the CPT activity of mitochondria continued to increase with the advancement of age and beyond 72 wks of age, it was approximately 2-fold higher than that of the microsomal fraction.     

Synthesis of phosphatidylcholine by two distinct methyltransferases in rat colonic brush-border membranes: evidence for extrinsic and intrinsic membrane activities

The enzymatic synthesis of phosphatidylcholine from phosphatidylethanolamine via a transmethylation pathway has not been shown to occur in the small intestine and has been assumed to be absent from the entire gut. The existence of this pathway, however, has not been investigated in the large intestine. Utilizing a recently developed method for the isolation of brush-border membranes from rat colonocytes, the present studies were designed to determine whether phospholipid methylation activity was present in the large intestine. The results demonstrate that this pathway for synthesis of phosphatidylcholine exists in rat colonic plasma membranes and involves at least two distinct methyltransferases. The predominant product of the first enzyme (methyltransferase I) is phosphatidyl-N-monomethylethanolamine; phosphatidylcholine and phosphatidyl-N-monomethylethanolamine are the principal products of the second enzyme (methyltransferase II). Methyltransferase I has an apparent Km for S-adenosyl-L-methionine of 100.0 microM and a pH optimum of 8.0, while methyltransferase II has an apparent Km of 0.3 microM and a pH optimum of 6.0. Additional evidence to support the presence of two distinct enzymes includes the differential effects of ATP, Triton X-100, trypsin treatment, and temperature on their activities.     

Actions of dietary orotic acid on liver synthesis of phosphatidylcholine and phosphatidylethanolamine in rats

The in vivo rates of the reactions of the cytidine pathways of liver phosphatidylcholine and phosphatidylethanolamine synthesis were measured in rats after 1 day of feeding on a semisynthetic diet containing 1% orotic acid. The calculations were made from the specific and total radioactivity versus time curves of the precursors and products following intraportal injection of [1,2-14C]choline, [2-14C]ethanolamine, and [2-3H]glycerol. The liver CTP level was increased twofold and the rates of CDP-choline and phosphatidylcholine synthesis were stimulated 4.5-fold in the rats fed orotic acid. The rate of CDP-ethanolamine synthesis was increased but could not be accurately quantified because of its extreme rapidity. No change occurred in the rate of the ethanolaminephosphotransferase reaction and the overall rate of phosphatidylethanolamine synthesis was unchanged by orotic acid feeding. The catalytic activities of the enzymes of the cytidine pathways of phosphatidylcholine and phosphatidylethanolamine synthesis were not affected by feeding orotic acid for 1 day. Similar findings were obtained 3 h following intragastric administration of 100 mg of orotic acid. The results suggest the possibility that changes in the levels of liver CTP may play a role in regulation of the cytidine pathway of liver phosphatidylcholine synthesis but not of phosphatidylethanolamine synthesis, because the latter pathway appears to be tightly controlled at the ethanolaminephosphotransferase step.