Asymmetry of the site of choline incorporation into phosphatidylcholine of rat liver microsomes.

[14C]Choline was incorporated into microsomal membranes in vivo, and from CDP-[14C]choline in vitro, and the site of incorporation determined by hydrolysis of the outer leaflet of the membrane bilayer using phospholipase C from Clostridium welchii. Labelled phosphatidylcholine was found to be concentrated in the outer leaflet of the membrane bilayer with a specific activity approximately three times that of the inner leaflet. During incorporation of CDP-choline and treatment with phospholipase C the vesicles retained labelled-protein contents indicating that they remained intact. When the microsomes were opened with taurocholate after incorporation of [14C]choline in vivo, the labelled phosphatidylcholine behaved as a single pool. Selective hydrolysis of labelled phosphatidylcholine in intact vesicles is not, therefore, a consequence of specificity of phospholipase C. These results indicate that the phosphatidylcholine of the outer leaflet of the microsomal membrane bilayer is preferentially labelled by the choline-phosphotransferase pathway and that this pool of phospholipid does not equilibrate with that of the inner leaflet.     

Cholesterol movement between human skin fibroblasts and phosphatidylcholine vesicles

Cholesterol readily exchanges between human skin fibroblasts and unilamellar phospholipid vesicles. Only a fraction of the exchangeable cholesterol and only 10–15% of the total cellular free cholesterol is available for net movement or depletion to cholesterol-free phosphatidylcholine vesicles. [¹⁴C]Cholesterol introduced into the fibroblast plasma membrane by exchange from lipid vesicles does not readily equilibrate with fibroblast cholesterol labelled endogenously from [³H]mevalonic acid. While endogenously-synthesized [³H]cholesterol readily becomes incorporated into a pool of esterified cholesterol, little, if any, of the [¹⁴C]cholesterol introduced into the fibroblast plasma membrane by exchange from lipid vesicles becomes available for esterification. We interpret these findings as suggesting that: (1) net cholesterol movement from fibroblasts to an acceptor membrane is limited to a small percentage of the plasma membrane cholesterol, and (2) separate pools of cholesterol exist in human skin fibroblasts, one associated with the plasma membrane and the second associated with intracellular membranes, and equilibration of cholesterol between the two pools is a very limited process.     

Increased catabolism of phosphatidylcholine to betaine regulates the liver phosphatidylcholine content in rats fed orotic acid

Feeding a semi-synthetic diet containing 1% orotic acid to rats for one day stimulates the CDPcholine pathway of liver phosphatidylcholine synthesis 4.5-fold without significantly increasing the liver phosphatidylcholine level. The liver betaine level increases 1.6-fold. The present experiments were performed to investigate the source of the increased liver betaine. Orotic acid feeding did not alter the rate of oxidation of 1,2[14C] choline to betaine. After liver phosphatidylcholine was labelled in vivo with 2[14-C]-ethanolamine, over 90% of the choline-derived radioactivity was recovered in liver betaine and this was consistently increased in rats fed orotic acid. It is concluded that the increased synthesis of liver phosphatidylcholine caused by dietary orotic acid is accompanied by an increased rate of liver phosphatidylcholine catabolism, with betaine as the major end-product of the choline moiety.     

The rate-limiting reaction in phosphatidylcholine synthesis by alveolar type II cells isolated from fetal rat lung

The rate-limiting reaction in the formation of phosphatidylcholine by type II cells isolated from fetal rat lung was examined. Studies on the uptake of [Me-3H]choline and its incorporation into its metabolites indicated that in these cells the choline phosphate pool was much larger than both the choline and CDPcholine pools. Chemical measurements of the pool sizes showed that the choline phosphate pool was indeed much larger than the intracellular choline and CDPcholine pools. Pulse-chase studies with [Me-3H]choline revealed that labelled choline taken up by the cells was rapidly phosphorylated to choline phosphate and that the radioactivity lost from choline phosphate during the chase period appeared in phosphatidylcholine. Little change was observed in the labelling of CDPcholine during the chase period. These results indicate that cholinephosphate cytidylyltransferase catalyzes a rate-limiting reaction in phosphatidylcholine formation by fetal rat lung type II cells.     

Use of phospholipid exchange protein to measure inside-outside transposition in phosphatidylcholine liposomes

The exchange of phosphatidylcholine between [³²P]phosphatidylcholine liposomes and unlabeled mitochondria was catalyzed by a purified phospholipid exchange protein from bovine heart cytosol. The loss of [³²P]phosphatidylcholine from the liposomes appeared to proceed in two stages: with 100 units of phospholipid exchange protein per ml the half-time of initial stage was about 10 min and that of the final stage 4 days or greater. Agarose-gel chromatography of the liposomes showed an elution compatible with a homogeneous pool of small single walled vesicles. Treatment of phosphatidyl [¹⁴C]choline liposomes with phospholipase D (phosphatidylcholine phosphatidohydrolase) showed that labeled phospholipid removable during the rapid exchange phase was subject to hydrolysis by the phospholipase, but that the labeled phospholipid left after the rapid exchange was completed could not be hydrolyzed by phospholipase D. It is proposed that the rapidly exchanging phosphatidylcholine constitutes the outer layer of the liposome bilayer. The long half-lives of 4 days or more probably represent the transposition of Phosphatidylcholine from the inner to the outer layer of the liposome bilayer.     

Anabolic action of prolactin on phosphatidylcholine metabolism in guinea pig adrenocorticocytes]

Isolated adrenocortical cells of guinea pigs whom injected with prolactin (PRL) during 3 days incorporated [3H] choline into phosphatidylcholine more intensively than those cells of animals in control. Labelling of intracellular pools of choline and phosphorylcholine remained unchanged, though a part of radioactivity represented by the water-soluble precursors decreased due to PRL influence. The rate of disappearance of labelled phosphatidylcholine in adrenocortical cells prelabelled with [3H] choline was lower in cells obtained from PRL-treated animals. The discharge of [3N] choline accumulated during prelabeling accelerated simultaneously. Rate of the phosphorylcholine radioactivity fall remained unchanged. The obtained data showed that prolonged influence of PRL caused a shift of the phosphatidylcholine metabolism to anabolism. That effect might be a part of the mechanism of proliferative PRL action in the adrenal cortex.     

A CDP-choline pathway for phosphatidylcholine biosynthesis in Treponema denticola

The genomes of Treponema denticola and Treponema pallidum contain a gene, licCA, which is predicted to encode a fusion protein containing choline kinase and CTP:phosphocholine cytidylyltransferase activities. Because both organisms have been reported to contain phosphatidylcholine, this raises the possibility that they use a CDP-choline pathway for the biosynthesis of phosphatidylcholine. This report shows that phosphatidylcholine is a major phospholipid in T. denticola, accounting for 35-40% of total phospholipid. This organism readily incorporated [14C]choline into phosphatidylcholine, indicating the presence of a choline-dependent biosynthetic pathway. The licCA gene was cloned, and recombinant LicCA had choline kinase and CTP:phosphocholine cytidylyltransferase activity. The licCA gene was disrupted in T. denticola by erythromycin cassette mutagenesis, resulting in a viable mutant. This disruption completely blocked incorporation of either [14C]choline or 32Pi into phosphatidylcholine. The rate of production of another phospholipid in T. denticola, phosphatidylethanolamine, was elevated considerably in the licCA mutant, suggesting that the elevated level of this lipid compensated for the loss of phosphatidylcholine in the membranes. Thus it appears that T. denticola does contain a licCA-dependent CDP-choline pathway for phosphatidylcholine biosynthesis.     

The phosphatidylcholine transfer protein from bovine liver discriminates between phosphatidylcholine isomers. A monolayer study

The activity of the phosphatidylcholine transfer protein from bovine liver toward phosphatidylcholine isomers carrying a long and a short fatty acyl chain on either the sn-1- or sn-2-position was determined by way of the monolayer-vesicle assay. In this assay equimolar mixtures of the isomers were spread at the air/water interface and their transfer measured to the vesicles in the subphase initiated by addition of the transfer protein. The following isomers were tested: 1-decanoyl-2-[3H]oleoyl-sn-glycero-3-phosphocholine (C10:0/[3H]C18:1-PC) and 1-oleoyl-2-decanoyl-sn-glycero-3-phospho[14C]choline (C18:1/C10:0-[14C]PC); 1-lauroyl-2-[3H]oleoyl-sn-glycero-3-phosphocholine (C12:0/[3H]C18:1-PC) and 1-oleoyl-2-[14C]lauroyl-sn-glycero-3-phosphocholine (C18:1/[14C]C12:0-PC); 1-myristoyl-2-[3H]oleoyl-sn-glycero-3-phosphocholine (C14:0/[3H]C18:1-PC) and 1-oleoyl,2-myristoyl-sn-glycero-3-phospho[14C]choline (C18:1/C14:0-[14C]PC). It was found that the protein transferred C10:0/[3H]C18:1-PC twice as fast as C18:1/C10:0-[14C]PC. Similar differences in rate were observed for C12:0/[3H]C18:1-Pc and C18:1/[14C]C12:0-PC but not for the isomers carrying myristic acid. We propose that the transfer protein can discriminate between PC isomers due to the presence of distinct binding sites for the sn-1- and sn-2-acyl chain (Berkhout et al. (1984) Biochemistry, 23, 1505-1513).     

The synthesis of phosphatidylcholine by adult rat lung alveolar type II epithelial cells in primary culture

1. The formation of phosphatidylcholine from radioactive precursors was studied in adult rat lung alveolar type II epithelial cells in primary culture. 2. The incorporation of [Me-14C]choline into total lipids and phosphatidylcholine was stimulated by addition of palmitate, whereas the incorporation of [U-14C]glucose into phosphatidylcholine and disaturated phosphatidylcholine was stimulated by addition of choline. Addition of glucose decreased the absolute rate of incorporation of [1(3)-3H]glycerol into total lipids, phosphatidylcholine and disaturated phosphatidylcholine, decreased the percentage [1(3)-3H]glycerol recovered in phosphatidylcholine, but increased the percentage phosphatidylcholine label in the disaturated species. 3. At saturating substrate concentrations, the percentages of phosphatidylcholine radioactivity found in disaturated phosphatidylcholine after incubation with [1-(14)C]acetate (in the presence of glucose) [1-(14)C]palmitate (in the presence of glucose), [Me-14C]choline (in the presence of glucose and palmitate) and [U-14C]glucose (in the presence of choline and palmitate) were 78, 75, 74 and 90%, respectively. 4. Fatty acids stimulated the incorporation of [U-14C]glucose into the glycerol moiety of phosphatidylcholine. The degree of unsaturation of the added fatty acids was reflected in the distribution of [U-14C]glucose label among the different molecular species of phosphatidylcholine. It is suggested that the glucose concentration in the blood as related to the amount of available fatty acids and their degree of unsaturation may be factors governing the synthesis of surfactant lipids.     

Insulin stimulates turnover of phosphatidylcholine in rat adipocytes

The present study investigated the effect of insulin on phosphatidylcholine turnover in rat adipocytes labelled to equilibrium with [¹⁴C]-choline. Insulin induced a rapid turnover of this major phospholipid that was maximal by 1 min and transient in nature. Following a 1 min stimulation of the cells with insulin at a maximally effective concentration (7 nM), a 4–6% decrease in the percentage of total cellular choline associated with this phospholipid was observed. This reflected a significant transient increase in the percentage of total cellular choline associated with phosphorylcholine, which together with diacylglycerol are the phospholipase C cleavage products of phosphatidylcholine. These effects were observed over a physiological range of insulin concentrations. No effect of insulin on any other choline phospholipid or metabolite (sphingomyelin, lysophophatidylcholine, glycerophosphocholine or choline) was seen. These results suggest that insulin stimulates a phospholipase C-mediated turnover of phosphatidylcholine in rat adipocytes. The rapid nature of this turnover suggests a potential role in signal transduction.     

Rapid transport of phosphatidylcholine occurring simultaneously with protein transport in the frog sciatic nerve

1. Either l-[4,5-(3)H]leucine or [Me-(3)H]choline, or both l-[U-(14)C]leucine and [Me-(3)H]-choline, were injected into the ninth dorsal root ganglion of the frog, and peripheral transport of labelled proteins and/or phospholipids, mostly phosphatidylcholine, was studied by analysis of consecutive segments of the sciatic nerve. 2. At 25 degrees C, approx. 5% of the (3)H-labelled protein was transported at the rate of 152mm/day. The rate was temperature-dependent with the Q(10) value of 2.6. The flow was completely blocked by the local application of colchicine, but was unaffected by cytochalasin D. 3. [Me-(3)H]-Choline was incorporated into phosphatidylcholine at a comparatively slow rate, but was transported in the nerve at a rate equivalent to that for (3)H-labelled proteins. 4. The simultaneous transport of phosphatidylcholine and the protein was further supported in the double-labelling experiments by an identical transport rate of (3)H-labelled phosphatidylcholine and (14)C-labelled proteins, by their identical temperature dependence, by simultaneous blockade with colchicine, and also by the parallel distribution of the two labels in subcellular fractions. Specific radioactivities on a protein basis of both (3)H and (14)C labels were highest in microsomal subfractions enriched with Na(+)-plus-K(+)-stimulated adenosine triphosphatase and acetylcholinesterase. It is suggested that (3)H-labelled phosphatidylcholine and (14)C-labelled proteins transported in the nerve reside in the same structural entity, most probably a membrane component.     

Effect of hyperoxia on phosphatidylcholine synthesis, secretion, uptake and stability in the newborn rabbit lung

The effects of breathing greater than 95% oxygen from birth for 48 h of life on surfactant phosphatidylcholine synthesis and secretion, as well as uptake and stability of exogenous phosphatidylcholine were studied using rabbit lung tissue slices. Lung slices from animals breathing greater than 95% oxygen for 48 h exhibited a decreased rate of [14C]phosphatidylcholine release (30%) in comparison to lung slices from air-exposed controls. In vitro incorporation of [14C]choline into phosphatidylcholine was decreased by a similar amount in lung slices from pups exposed to greater than 95% oxygen. Uptake of exogenous [14C]phosphatidylcholine by lung slices from hyperoxic-exposed and control groups was not different, and the stability of extracellular phosphatidylcholine was likewise unaffected by hyperoxia. Turnover of labelled phosphatidylcholine taken up by tissue slices from medium was apparently decreased in association with hyperoxic exposure. These results are consistent with multiple sites of effect of hyperoxia on the pulmonary surfactant system in the newborn. These effects of hyperoxia on the lung surfactant system occur at a time of critical adaption to extrauterine life, and thus may have major consequences on lung function and ultimate survival of the premature infant with respiratory distress syndrome.     

Synthesis of phosphatidylcholine by rat lung during choline deficiency

The effect of choline deficiency on the de novo pathway for phosphatidylcholine (PC) synthesis in the lung was investigated in rats fed a washed soy protein (lipotrophic) diet deficient in choline and methionine for 2-3 wk. Lungs from lipotrophic rats showed a decreased content of choline and choline-phosphate (P less than 0.05) compared with control but no change in content of cytidine 5'-diphosphocholine or PC. Isolated perfused lungs from lipotrophic rats were evaluated for choline and fatty acid utilization for PC synthesis. Lipotrophic lungs perfused with 5 microM [14C-methyl]-choline chloride showed increased incorporation into PC while there was no significant effect at saturating levels of choline (100 microM). There was increased incorporation of [1-14C]-palmitic acid into PC and diglyceride and increased incorporation of D-[U-14C]glucose into fatty acids of PC. Increased choline and glucose incorporation was not due to alteration of intracellular specific activity of these substrates. This study indicates the utilization of choline and fatty acid for PC synthesis is stimulated as a result of choline deficiency while lung CDP-choline concentration is maintained, possibly through regulation of choline phosphate cytidyl transferase activity. These mechanisms compensate for decreased choline availability to maintain the PC content of lungs.     

Biosynthesis of phosphatidylcholine from a phosphocholine precursor pool derived from the late endosomal/lysosomal degradation of sphingomyelin

Previous studies suggest that the steps of the CDP- choline pathway of phosphatidylcholine synthesis are tightly linked in a so-called metabolon. Evidence has been presented that only choline that enters cells through the choline transporter, and not phosphocholine administered to cells by membrane permeabilization, is incorporated into phosphatidylcholine. Here, we show that [(14)C]phosphocholine derived from the lysosomal degradation of [(14)C]choline-labeled sphingomyelin is incorporated as such into phosphatidylcholine in human and mouse fibroblasts. Low density lipoprotein receptor-mediated endocytosis was used to specifically direct [(14)C]sphingomyelin to the lysosomal degradation pathway. Free labeled choline was not found either intracellularly or in the medium, not even when the cells were energy-depleted. Deficiency of lysosomal acid phosphatases in mouse or alkaline phosphatase in human fibroblasts did not affect the incorporation of lysosomal [(14)C]sphingomyelin-derived [(14)C]phosphocholine into phosphatidylcholine, supporting our finding that phosphocholine is not degraded to choline prior to its incorporation into phosphatidylcholine. Inhibition studies and analysis of molecular species showed that exogenous [(3)H]choline and sphingomyelin-derived [(14)C]phosphocholine are incorporated into phosphatidylcholine via a common pathway of synthesis. Our findings provide evidence that, in fibroblasts, phosphocholine derived from sphingomyelin is transported out of the lysosome and subsequently incorporated into phosphatidylcholine without prior hydrolysis of phosphocholine to choline. The findings do not support the existence of a phosphatidylcholine synthesis metabolon in fibroblasts.     

The roles of phospholipase D and a GTP-binding protein in guanosine 5''-[gamma-thio]triphosphate-stimulated hydrolysis of phosphatidylcholine in rat liver plasma membranes.

1. Guanosine 5'-[gamma-thio]triphosphate (GTP[S]) stimulated by 50% the rate of release of [3H]choline and [3H]phosphorylcholine in rat liver plasma membranes labelled with [3H]choline. About 70% of the radioactivity released in the presence of GTP[S] was [3H]choline and 30% was [3H]phosphorylcholine. 2. The hydrolysis of phosphorylcholine to choline and the conversion of choline to phosphorylcholine did not contribute to the formation of [3H]choline and [3H]phosphorylcholine respectively. 3. The release of [3H]choline from membranes was inhibited by low concentrations of SDS or Triton X-100. Considerably higher concentrations of the detergents were required to inhibit the release of [3H]phosphorylcholine. 4. Guanosine 5'-[beta gamma-imido]triphosphate and guanosine 5'-[alpha beta-methylene]triphosphate, but not adenosine 5'-[gamma-thio]-triphosphate, stimulated [3H]choline release to the same extent as did GTP[S]. The GTP[S]-stimulated [3H]choline release was inhibited by guanosine 5'-[beta-thio]diphosphate, GDP and GTP but not by GMP. 5. It is concluded that, in rat liver plasma membranes, (a) GTP[S]-stimulated hydrolysis of phosphatidylcholine is catalysed predominantly by phospholipase D with some contribution from phospholipase C, and (b) the stimulation of phosphatidylcholine hydrolysis by GTP[s] occurs via a GTP-binding regulatory protein.     

Choline derived from the phosphatidylcholine specific phospholipase D is not directly available for the CDP choline pathway in phorbol ester- treated C3H10T1/2 C18 fibroblasts

We have shown that 12-O-tetradecanoylphorbol 13-acetate (TPA) increases protein kinase C (PKC)-mediated choline transport, incorporation of choline into phosphatidylcholine (PtdCho) and PtdCho degradation by phospholipase D (PLD) in C3H10T1/2 Cl 8 cells. Dual prelabeling experiment using [³H]/[¹⁴C]choline indicated that intracellular choline generated from the PLD reaction was not directly recycled to PtdCho synthesis within the cell, and that a large fraction of the choline was transported out of the TPA-treated cells. In contrast, medium derived choline was preferably channeled to PtdCho synthesis. These results indicate that in TPA-treated cells, the choline derived from the PKC-mediated increased PLD activity and the choline newly taken up by the cell behave as two distinctly different metabolic pools.     

Effect of 1,25-dihydroxyvitamin D3 on phospholipid metabolism in chick duodenal mucosal cell. Relationship to its mechanism of action

Pretreatment of the D-deficient chick with 1,25-dihydroxyvitamin D3 increases de novo synthesis of phosphatidylcholine by a stimulation of CDP-choline: sn-1,2-diacylglycerol choline-phosphotransferase reaction. The time course of change in the incorporation of [3H]choline and [14C]ethanolamine into the brush border lipid fraction after 1,25-dihydroxyvitamin D3 treatment correlates closely with the time course of change in calcium uptake into the brush border membrane vesicles. Prior treatment with cycloheximide does not block this increase in phosphatidylcholine synthesis. In addition, 1,25-dihydroxyvitamin D3 administration increases the incorporation of [3H]arachidonic acid into the phosphatidylcholine fraction of the brush border to a great extent but does not increase the incorporation of [3H]palmitic acid into the phosphatidylcholine fraction. The incorporation of these 3H labeled fatty acids into diacylglycerol is not changed by 1,25-dihydroxyvitamin D3. These data indicate that 1,25-dihydroxyvitamin D3 enhances the synthesis of phosphatidylcholine independent of new protein synthesis, and also increases the incorporation of unsaturated fatty acids into phosphatidylcholine. From these results we suggest that changes in phospholipid metabolism in the enterocyte are the mechanisms by which 1,25-dihydroxyvitamin D3 acts to enhance calcium entry across the brush border membrane.     

Choline release and inhibition of phosphatidylcholine synthesis precede excitotoxic neuronal death but not neurotoxicity induced by serum deprivation

N-Methyl-d-aspartate (NMDA) receptor overactivation has been proposed to induce excitotoxic neuronal death by enhancing membrane phospholipid degradation. In previous studies, we have shown that NMDA releases choline and reduces membrane phosphatidylcholine in vivo. We now observed that glutamate and NMDA induce choline release in primary neuronal cortical cell cultures. This effect is Ca(2+)-dependent and is blocked by MK-801 ((+)-5-methyl-10, 11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate). In cortical neurons, the NMDA receptor-mediated choline release precedes excitotoxic cell death but not neuronal death induced by either osmotic lysis or serum deprivation. Glutamate, at concentrations that release arachidonic acid, does not release choline in cerebellar granule cells, unless these cells are rendered susceptible to excitotoxic death by energy deprivation. The NMDA-evoked release of choline is not mediated by phospholipases A(2) or C. Moreover, NMDA does not activate phospholipase D in cortical cells. However, NMDA inhibits incorporation of [methyl-(3)H]choline into both membrane phosphatidylcholine and sphingomyelin. These results show that the increase in extracellular choline induced by NMDA receptor activation is directly related with excitotoxic cell death and indicate that choline release is an early event of the excitotoxic process produced by inhibition of phosphatidylcholine synthesis and not by activation of membrane phospholipid degradation.     

Muscarinic receptor activation of phosphatidylcholine hydrolysis. Relationship to phosphoinositide hydrolysis and diacylglycerol metabolism

We examined the relationship between phosphatidylcholine (PC) hydrolysis, phosphoinositide hydrolysis, and diacylglycerol (DAG) formation in response to muscarinic acetylcholine receptor (mAChR) stimulation in 1321N1 astrocytoma cells. Carbachol increases the release of [3H]choline and [3H]phosphorylcholine ([3H]Pchol) from cells containing [3H]choline-labeled PC. The production of Pchol is rapid and transient, while choline production continues for at least 30 min. mAChR-stimulated release of Pchol is reduced in cells that have been depleted of intracellular Ca2+ stores by ionomycin pretreatment, whereas choline release is unaffected by this pretreatment. Phorbol 12-myristate 13-acetate (PMA) increases the release of choline, but not Pchol, from 1321N1 cells, and down-regulation of protein kinase C blocks the ability of carbachol to stimulate choline production. Taken together, these results suggest that Ca2+ mobilization is involved in mAChR-mediated hydrolysis of PC by a phospholipase C, whereas protein kinase C activation is required for mAChR-stimulated hydrolysis of PC by a phospholipase D. Both carbachol and PMA rapidly increase the formation of [3H]phosphatidic acid ([3H]PA) in cells containing [3H]myristate-labeled PC. [3H]Diacylglycerol ([3H]DAG) levels increase more slowly, suggesting that the predominant pathway for PC hydrolysis is via phospholipase D. When cells are labeled with [3H]myristate and [14C]arachidonate such that there is a much greater 3H/14C ratio in PC compared with the phosphoinositides, the 3H/14C ratio in DAG and PA increases with PMA treatment but decreases in response to carbachol. By analyzing the increase in 3H versus 14C in DAG, we estimate that the DAG that is formed in response to PMA arises largely from PC. Muscarinic receptor activation also causes formation of DAG from PC, but approximately 20% of carbachol-stimulated DAG appears to arise from hydrolysis of the phosphoinositides.     

Topological Biosynthesis of Phosphatidylcholine in Brain Microsomes

The sidedness of the biosynthesis of phosphatidylcholine and its transbilayer movement in brain microsomes were investigated. Microsomes were labelled in vitro or in vivo either through Kennedy's pathway or by the base-exchange reaction. The vesicles were treated with phospholipase C under conditions where only the phospholipids present in the external leaflet were hydrolyzed. The incubation of microsomes with CDP-[14C]choline or [14C]choline showed that most of the newly synthesized phosphatidylcholine molecules were localized in the external leaflet. With time a few molecules were transferred into the inner leaflet. When phosphatidylcholine was labelled in vivo by intraventricular injection of [3H]choline the specific activities of the phosphatidylcholine in the outer leaflet were higher than those in the inner leaflet after short times of labelling but became similar after long times of labelling. The results suggest that in brain microsomes the synthesis of phosphatidylcholine through Kennedy's pathway or by the base-exchange reaction takes place on the external leaflet which corresponds to the cytoplasmic one in situ. The transfer of these molecules from the outer leaflet to the inner one is a slow process and the mechanisms that control the transbilayer movement of the phosphatidylcholine seem to be independent of those that control their biosynthesis.