Phosphatidylcholine Synthesis in Castor Bean Endosperm : Occurrence of an S-Adenosyl-l-Methionine:Ethanolamine N-Methyltransferase

The methylation steps in the biosynthesis of phosphatidylcholine by castor bean (Ricinus communis L.) endosperm have been studied by pulse-chase labeling. Endosperm halves were incubated with [methyl-¹⁴C]S-adenosyl-l-methionine, [2-¹⁴C]ethanolamine, [¹⁴C]ethanolamine phosphate, or [¹⁴C]serine phosphate. The kinetics of appearance were followed in the free, phospho-, and phosphatidyl-bases. The initial methylation utilized ethanolamine as a substrate to form methylethanolamine, which was then converted to dimethylethanolamine, choline, and phosphomethylethanolamine. Subsequent methylations occurred at the phospho-base and, to a lesser extent, the phosphatidyl-base levels, after which the radioactivity either remained constant or decreased in these compounds and accumulated in phosphatidylcholine. Although the precursors tested did support the synthesis of choline, the kinetics of the labeling make them unlikely to be the major sources of free choline to be utilized for the nucleotide pathway. A model with two pools of choline is proposed, and the implications of these results for the pathways leading to phosphatidylcholine biosynthesis are discussed.     

Role of a specific choline pool in sphingomyelin synthesis in rat heart.

Sphingomyelin synthesis was studied in slices of rat heart by using [Me-14C]choline, [1,2-14C]ethanolamine, S-adenosyl-L-[14C]methionine and [32P]Pi as as precursors. In the presence of both [Me-14C]choline and [32P]Pi the ratio of the specific radioactivities of 14C and 32P in phosphatidylcholine was greater than in sphingomyelin at all the times studied. This suggested that synthesis of phosphatidylcholine and sphingomyelin de novo did not involve the utilization of a common pool of cytidine diphosphate choline. In addition, studies with [1,2-14C]ethanolamine and S-adenosyl-L-[14C]methionine indicated that a quantitatively significant pool of choline, derived from these precursors, was selectively utilized for sphingomyelin formation. This pool was not represented by phosphatidylcholine formed by methylation of phosphatidylethanolamine or by other pathways.     

Sucrose in the free space of translocating maize leaf bundles

Following exposure of portions of mature maize (Zea mays L.) leaf strips to ¹⁴CO₂, xylem exudate from the leaf strips contained [¹⁴C]sucrose. Sucrose was the only sugar in the xylem exudate which was obtained from the cut surface of the leaf strips by reducing the external pressure. The sucrose found in the xylem exudate apparently was obtained from the free space of the vascular bundles, its concentration amounting up to 0.25%. When [¹⁴C]glucose or [¹⁴C]fructose was supplied in the dark to one end of a maize leaf strip, each was taken up by the xylem, and transported to the opposite end. Xylem exudate from such leaf strips contained ¹⁴C-labeled sucrose in addition to the ¹⁴C-labeled hexose. The results of this study support the view that sucrose is loaded into the companion cell-sieve tube complexes from the apoplast of the vascular bundles in the maize leaf.     

Biosynthesis of Choline and Ethanolamine Phospholipids in Crithidia fasciculata*

The incorporation of radioactivity from [1,2-³⁴C]choline, [1,2-³⁴C]ethanolamine, [3-¹⁴C]serine and [methyl-¹⁴C]methionine into lipids was studied in growing cultures of Crithidia fasciculata. Lecithin was formed both from choline and by the methylation of phosphatidylethanolamine. Mono- and dimethylphosphatidylethanolamines were present in no more than trace amounts. Growth of the protozoa in media containing choline (1 mM) did not decrease synthesis by the methylation pathway. Phosphatidylethanolamine was formed from ethanolamine. Radioactivity from serine also was present in both phosphatidylethanolamine and lecithin; however, the presumed intermediate, phosphatidylserine, could not be detected.     

Incorporation of [14C] ethanolamine and [3H] Methionine into phospholipids of rat brain and liver in vivo and in vitro

Comparative studies were undertaken on the in vivo and in vitro incorporation of [¹⁴C] ethanolamine, [³H] methionine and [¹⁴C] S-adenosyl-methionine into phosphatidylethanolamine (PhE) and phosphatidylcholine (PhC) of rat liver and brain. It was observed that brain can synthesize de novo PhC from PhE via the transmethylation pathway, however synthesis rates were (1) markedly lower than those of liver and (2) decreased significantly with age. In the choline-containing lipids more than 95% of the radioactivity was found in PhC. Studies on the localization of the radioactivity in PhC following the intracranial injection of [³H] methionine or [¹⁴C] ethanolamine revealed that both precursors are incorporated almost exclusively into the choline moiety of this phospholipid. There was significant labeling of PhC only when the precursors were administered intracranially and much less incorporation was observed with the systemic routes. Thus following the intravenous administration of [¹⁴C] ethanolamine, the specific radioactivities of liver PhE and PhC were up to 75 times as high as those of brain and 4 to 5 times as high in the organs of the 20-day old as those of the adult. In contrast, when this precursor was administered intracranially the specific radioactivities of both phospholipids in liver were only twice as high as those of brain. Although the short-and long-term time-course studies on the in vivo incorporation of [¹⁴C] ethanolamine and [³H] methionine into PhC of both organs could suggest a precursor-product relationship between the biosynthesis of this phospholipid in liver and brain, this apparent relationship could also be due to the high turnover of PhE in liver, with half-life of 2.87 hr, and its low turnover in brain, with half-life of 10.7 days. The present findings on the low rate of formation of PhC from PhE in brain coupled with the fact that this conversion declines sharply with age, especially when the isotopes are administered systemically, could explain the observation of previous investigators that the brain cannot synthesize its own choline and thus it must derive its choline from exogenous sources such as lipid-choline. It was concluded that the brain can synthesize its own choline; however it remains also dependent on liver and dietary choline which are probably transported into the brain as free choline.     

Radiotracer evidence implicating phosphoryl and phosphatidyl bases as intermediates in betaine synthesis by water-stressed barley leaves

In barley, glycine betaine is a metabolic end product accumulated by wilted leaves; betaine accumulation involves acceleration of de novo synthesis from serine, via ethanolamine, N-methylethanolamines, choline, and betaine aldehyde (Hanson, Scott 1980 Plant Physiol 66: 342-348). Because in animals and microorganisms the N-methylation of ethanolamine involves phosphatide intermediates, and because in barley, wilting markedly increases the rate of methylation of ethanolamine to choline, the labeling of phosphatides was followed after supplying [¹⁴C]ethanolamine to attached leaf blades of turgid and wilted barley plants. The kinetics of labeling of phosphatidylcholine and betaine showed that phosphatidylcholine became labeled 2.5-fold faster in wilted than in turgid leaves, and that after short incubations, phosphatidylcholine was always more heavily labeled than betaine. In pulse-chase experiments with wilted leaves, label from [¹⁴C]ethanolamine continued to accumulate in betaine as it was being lost from phosphatidylcholine. When [¹⁴C]monomethylethanolamine was supplied to wilted leaves, phosphatidylcholine was initially more heavily labeled than betaine. These results are qualitatively consistent with a precursor-to-product relationship between phosphatidylcholine and betaine.     

Synthesis of methylated ethanolamine moieties: regulation by choline in soybean and carrot

The results of experiments in which intact plants of Lemna paucicostata were labeled with either l-[³H₃C]methionine, l-[¹⁴CH₃]methionine, or [1,2-¹⁴C]ethanolamine support the conclusion that growth in concentrations of choline of 3.0 micromolar or above brings about marked decreases in the rate of biosynthesis of methylated forms of ethanolamine (normally present chiefly as phosphatidylcholine, with lesser amounts of choline and phosphocholine). The in vivo locus of the block is at the committing step in the biosynthetic sequence at which phosphoethanolamine is methylated by S-adenosylmethionine to form phosphomethylethanolamine. The block is highly specific: flow of methyl groups originating in methionine continues into S-adenosylmethionine, S-methylmethionine, the methyl moieties of pectin methyl ester, and other methylated metabolites. When choline uptake is less than the total that would be synthesized by control plants, phosphoethanolamine methylation is down-regulated to balance the uptake; total plant content of choline and its derivatives remains essentially constant. At maximum down-regulation, phosphoethanolamine methylation continues at 5 to 10% of normal. A specific decrease in the total available activity of AdoMet: phosphoethanolamine N-methyltransferase, as well as feedback inhibition of this enzyme by phosphocholine, and prevention of accumulation of phosphoethanolamine by down-regulation of ethanolamine synthesis may each contribute to effective control of phosphoethanolamine methylation. This down-regulation may necessitate major changes in S-adenosylmethionine metabolism. Such changes are discussed.     

Transport and phosphorylation of choline in higher plant cells. Phosphorus-31 nuclear magnetic resonance studies

When sycamore cells were suspended in basal medium containing choline, the latter was taken up by the cells very rapidly. A facilitated diffusion system appertained at low concentrations of choline and exhibited Michaelis-Menten kinetics. At higher choline concentrations simple diffusion appeared to be the principal mode of uptake. Addition of choline to the perfusate of compressed sycamore cells monitored by 31P NMR spectroscopy resulted in a dramatic accumulation of P-choline in the cytoplasmic compartment containing choline kinase and not in the vacuole. The total accumulation of P-choline over a 10-h period exhibited Michaelis-Menten kinetics. During this period, in the absence of Pi in the perfusion medium there was a marked depletion of glucose-6-P, and the cytoplasmic Pi resonance disappeared almost completely. When a threshold of cytoplasmic Pi was attained, the phosphorylation of choline was sustained by the continuous release of Pi from the vacuole although at a much lower rate. However, when 100 microM inorganic phosphate was present in the perfusion medium, externally added Pi was preferentially used to sustain P-choline synthesis. It is clear, therefore, that cytosolic choline kinase associated with a carrier-mediated transport system for choline uptake appeared as effective systems for continuously trapping cytoplasmic Pi including vacuolar Pi entering the cytoplasm.     

Modification of glycerolipid metabolism in L-M fibroblasts by an unnatural amino-alcohol, N-isopropylethanolamine.

The unnatural amino-alcohol, N-isopropylethanolamine, is incorporated into a phospholipid by monolayers of L-M fibroblasts. This phospholipid was identified as 1,2-diacyl-$\text{sn}$-glycero-3-phosphoisopropylethanolamine by using chemical and enzymatic procedures combined with thin-layer and gas-liquid chromatography. Since the phospho-N-isopropylethanolamine moiety is removed by phospholipase C, the stereochemistry of the phospholipid analog is identical to naturally occurring phosphoglycerides. Incubation of cells in 10 mM N-isopropylethanolamine inhibited the incorporation of [¹⁴C]choline and [¹⁴C]ethanolamine into phospholipids and stimulated the incorporation of [1-¹⁴C]palmitic acid and [1-¹⁴C]hexadecanol into triacylglycerols and alkyldiacylglycerols. These results indicate that N-isopropylethanolamine affects glycerolipid synthesis at the diradylglycerol branch point.     

Ethanol inhibits phosphatidylcholine and phosphatidylethanolamine biosynthesis in human leukemic monocyte-like U937 cells

The effect of ethanol (ETOH) on the incorporation of [¹⁴C]oleic acid (18:1) into lipid in human monocyte-like U937 cells was investigated. With increasing time of exposure to ETOH, the percentage of the label distributed into neutral lipid (NL) declined from 35 per cent (3 h) to 10 per cent (24 h) accompanied by increased incorporation into phospholipid (PL). [¹⁴C] 18 : 1 was preferentially incorporated into triglyceride (TG) and phosphatidylcholine (PC), comprising over 65 per cent and 50 per cent of the label associated with NL and PL, respectively. Low concentrations of ETOH (⩽ 1·0 per cent; v/v) had no effect. At concentrations greater than 1·5 per cent, there was enhanced incorporation into TG and diacylglycerol (DAG) in a 24-h incubation period, while at 16 h the label in phosphatidylethanolamine (PE) was decreased. The effect of ETOH on the CDP-choline or ethanolamine pathway was examined by monitoring the incorporation of [³H]choline or [¹⁴C]ethanolamine into PC or PE, respectively. At low concentrations ETOH had no effect on either choline uptake or the incorporation into PC. Higher concentrations (≥ 1·5 per cent) for 3 and 6 h resulted in a slightly decreased choline uptake, and the reduction (40–50 per cent) of incorporation into PC suggests that the CDP-choline pathway was inhibited. There was a similar inhibition of the incorporation of [¹⁴C]ethanolamine into PE. When the cells were incubated for 3 h in the presence of 2 per cent ETOH and with labelled 18 : 1 and PL-base, the ratios of incorporation (base/18 : 1) into PC and PE fractions decreased, indicating that the major inhibition lay in blockage of the availability of the base moiety for PL formation. Analysis of the distribution of the label into metabolites revealed that ETOH inhibited the conversion of [¹⁴C] ethanolamine into [¹⁴C]phosphorylethanolamine. The reduction in incorporation was not due to the enhanced breakdown of base-labelled PL. Our results indicate that ETOH has an inhibitory effect on the CDP-choline or ethanolamine pathway.     

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.     

Changes in phospholipid composition during the development of Dictyostelium discoideum

The phospholipid composition of Dictyostelium discoideum cells was determined at various stages of development by two-dimensional, thin-layer chromatography and reaction thin-layer chromatography. Major phospholipids of D. discoideum which were detectable throughout all stages of development were ethanolamine phosphoglyceride and choline phosphoglyceride. Ethanolamine phosphoglyceride and choline phosphoglyceride were found as their plasmalogen forms at 45–58 and 10–24%, respectively. There were no qualitative changes in phospholipid composition during the development, but quantitative changes did occur. The relative content of ethanolamine phosphoglyceride in the total phospholipids gradually decreased from 60% at the vegetative stage to 44% at the 1-day-sorocarp stage. In contrast, choline phosphoglyceride gradually increased from 27% at the vegetative stage to 48% at the preculmination stage, and then gradually decreased to 43% during the culmination. The decrease in ethanolamine phosphoglyceride content during the middle and late development was due mainly to the decreased amount of its plasmalogen form but the increase of choline phosphoglyceride was independent of quantitative changes of its plasmalogen form. Other minor components of phospholipid did not show significant changes in their levels. The causes of these changes in contents of ethanolamine phosphoglyceride and choline phosphoglyceride were examined by label and chase experiments with [³H]ethanolamine and [¹⁴C]choline. It seems that one-third to one-half of the increased amount of choline phosphoglyceride was due to stepwise methylation of ethanolamine phosphoglyceride, and the remaining two-thirds to one-half was caused by de novo synthesis of choline phosphoglyceride from CDP-choline and diglyceride.     

Effects of Hydroxyurea and Deoxyadenosine on the Synthesis of Deoxycytidine Phosphate and on the Uptake of Nucleosides in Excised Root Tips of Vicia faba

The effects of hydroxyurea and deoxyadenosine on the synthesis of deoxycytidine phosphate was studied by measuring the incorporation of [¹⁴C]-cytidine into acid soluble deoxycytidine phosphate in root tips of Vicia faba. Hydroxyurea and deoxyadenosine both markedly depressed the incorporation of [¹⁴C]-cytidine. Deoxyadenosine had the additional effect of inhibiting the uptake of [¹⁴C]-cytidine. Furthermore, millimolar concentrations of deoxyadenosine inhibited the uptake of micromolar concentrations of adenosine, thymidine, and deoxycytidine. The incorporation of [¹⁴C]-cytidine into RNA was only slightly affected by hydroxyurea. Deoxyadenosine inhibited the incorporation into RNA to about the same extent as the uptake of [¹⁴C]-cytidine. It is suggested that hydroxyurea reduced the incorporation of radioactive cytidine into deoxycytidine phosphate mainly by interfering with ribonucleotide reduction. The depression of [¹⁴C]-cytidine incorporation into deoxycytidine phosphate in the presence of deoxyadenosine is believed to be the result of an inhibition of both ribonucleotide reduction and [¹⁴C]-cytidine uptake.     

Accumulation of glycinebetaine and its synthesis from radioactive precursors under salt-stress in the cyanobacterium Aphanothece halophytica

Growth in salt-stressed (2.0 M NaCl) Aphanothece halophytica was initially delayed during the first two days of cultivation and eventually attained the same growth rate as the control (0.5 M NaCl) cells. Glycinebetaine accumulation increased slightly in control cells but a dramatic increase of glycinebetaine occurred in salt-stressed cells during a growth period of six days. There was no apparent increase in the synthesis of [¹⁴C] glycinebetaine in the control cells, in contrast to the marked increase in its synthesis in the salt-stressed cells. Increasing NaCl concentration in the growth medium induced both the accumulation and the synthesis of glycinebetaine. Time course experiments provided evidence that [¹⁴C] choline was first oxidized to [¹⁴C] betaine aldehyde which was further oxidized to [¹⁴C] glycinebetaine in A. halophytica. The supporting data for such a pathway were obtained from the presence of choline and betaine aldehyde dehydrogenase activities found in the membrane and cytoplasmic fractions, respectively. The activities of these two enzymes were also enhanced upon increasing NaCl concentration in the growth medium from 0.5 M to 2.0 M. Under this condition an increaseof approximately 1.5-fold was observed for choline dehydrogenase activity as compared to 2.5-fold for betaine aldehyde dehydrogenase activity, suggesting a preferable induction of the latter enzyme by salt stress. A. halophytica was able to utilize [¹⁴C] ethanolamine and [¹⁴C] glycine for the synthesis of [¹⁴C] glycinebetaine. This revised version was published online in August 2006 with corrections to the Cover Date.     

Phosphatidylcholine synthesis in castor bean endosperm : free bases as intermediates

The methylation steps in the biosynthesis of phosphatidylcholine by castor bean (Ricinus communis L.) endosperm have been studied by pulse-chase labeling. Endosperm halves were incubated with [methyl-(14)C]S-adenosyl-l-methionine, [2-(14)C]ethanolamine, [(14)C]ethanolamine phosphate, or [(14)C]serine phosphate. The kinetics of appearance were followed in the free, phospho-, and phosphatidyl-bases. The initial methylation utilized ethanolamine as a substrate to form methylethanolamine, which was then converted to dimethylethanolamine, choline, and phosphomethylethanolamine. Subsequent methylations occurred at the phospho-base and, to a lesser extent, the phosphatidyl-base levels, after which the radioactivity either remained constant or decreased in these compounds and accumulated in phosphatidylcholine. Although the precursors tested did support the synthesis of choline, the kinetics of the labeling make them unlikely to be the major sources of free choline to be utilized for the nucleotide pathway. A model with two pools of choline is proposed, and the implications of these results for the pathways leading to phosphatidylcholine biosynthesis are discussed.     

Spontaneous Phloem bleeding from cryopunctured fruits of a ureide-producing legume

The vasculature of the dorsal suture of cowpea (Vigna unguiculata [L.] Walp) fruits bled a sugar-rich exudate when punctured with a fine needle previously cooled in liquid N₂. Bleeding continued for many days at rates equivalent to 10% of the estimated current sugar intake of the fruit. A phloem origin for the exudate was suggested from its high levels (0.4-0.8 millimoles per milliliter) of sugar (98% of this as sucrose) and its high K⁺ content and high ratio of Mg²⁺ to Ca²⁺. Fruit cryopuncture sap became labeled with ¹⁴C following feeding of [¹⁴C]urea to leaves or adjacent walls of the fruit, of ¹⁴CO₂ to the pod gas space, and of [¹⁴C] asparagine or [¹⁴C]allantoin to leaflets or cut shoots through the xylem. Rates of translocation of ¹⁴C-assimilates from a fed leaf to the puncture site on a subtended fruit were 21 to 38 centimeters per hour. Analysis of ¹⁴C distribution in phloem sap suggested that [¹⁴C]allantoin was metabolized to a greater extent in its passage to the fruit than was [¹⁴C] asparagine. Amino acid:ureide:nitrate ratios (nitrogen weight basis) of NO₃-fed, non-nodulated plants were 20:2:78 in root bleeding xylem sap versus 90:10:0.1 for fruit phloem sap, suggesting that the shoot utilized NO₃-nitrogen to synthesize amino acids prior to phloem transfer of nitrogen to the fruit. Feeding of ¹⁵NO₃ to roots substantiated this conclusion. The amino acid:ureide ratio (nitrogen weight basis) of root xylem sap of symbiotic plants was 23:77 versus 89:11 for corresponding fruit phloem sap indicating intense metabolic transfer of ureide-nitrogen to amino acids by vegetative parts of the plant.     

(14C) acetylcholine synthesis by cortex slices of rat brain

Abstract—

• 1 A procedure has been developed to measure ACh synthesis from [¹⁴C]-precursors. As little as 10^?9 moles of ACh were detected as the result of de nova synthesis. Following incubation of cortex slices of rat brain with eserine and a tagged metabolite, ACh carrier was added to the incubation medium and to an extract from the slices. ACh was purified by chromatography on Amberlite CG-50, precipitation and recrystallization of ACh chloroaurate.
• 2 [U^?14C]glucose and [2^?14C]pyruvate formed similar amounts of [¹⁴C]ACh. Hydrolysis of ACh with subsequent chromatography of the resultant acetic acid demonstrated that all of the label was located in the acetyl moiety. [¹⁴C]acetate did not serve as a precursor of the acetyl group of ACh. Equivalent incorporation of carbons 1 and 6 of glucose into ACh indicated that glucose metabolism to ACh occurred via the Embden-Meyerhof pathway.
• 3 The amount of ACh detected by bioassay after incubation of cortex slices with [U^?14C]glucose was approximately the same as that calculated as labelled ACh; this demonstrates that all of the acetyl groups of ACh formed during incubation were derived from glucose.
• 4 [¹⁴C]choline, either methyl or chain labelled, formed [¹⁴C]ACh while labelled ethanolamine, serine and methionine did not. Synthesis from labelled choline did not occur in the absence of glucose.
• 5 When both [U^?14C]glucose and [¹⁴C]choline were incubated with brain slices, the acetyl and choline moieties of ACh were equally labelled; this demonstrates that the entire molecule was formed from added precursors. Slices supported a high rate of ACh synthesis without addition of choline. The addition of 10^?4m -hemicholinium-3 inhibited ACh formation by more than 90 per cent from either [U-¹⁴C]glucose or [Me-¹⁴C]choline.
• 6 Study of the time course of ACh synthesis from glucose demonstrated a rapid formation of [¹⁴C]ACh within the slices which reached a maximum during the first hour of incubation. [¹⁴C]ACh in the incubation medium accumulated at a linear rate for 3 hr. Replacement of a portion of the sodium chloride of the incubation medium by potassium chloride to a final concentration of 31 mm -KCI markedly increased the formation of [¹⁴C]ACh found in the incubation medium. Decreased amounts of [¹⁴C]ACh were extracted from the slices by homogenization or by subsequent heating at pH 4 in the high potassium ion medium.

     

Alterations of phospholipid metabolism by phorbol esters and fatty acids occur by different intracellular mechanisms in cultured glioma, neuroblastoma, and hybrid cells

Differences between the influences of phorbol esters (such as 4 beta-12-O-tetradecanoylphorbol 13-acetate) and of fatty acids (such as oleic acid) on the synthesis and turnover of phosphatidylcholine (PtdCho) and other phospholipids have been studied in glioma (C6), neuroblastoma (N1E-115), and hybrid (NG108-15) cells in culture using [methyl-3H]choline, [32P]Pi, [1,2-14C]ethanolamine, or 1-14C-labeled fatty acids as lipid precursors. 100-500 microM oleic acid stimulated PtdCho synthesis 3- to 5-fold in all three cell lines, but had little influence on chase of choline label following a 24-h pulse. Phorbol ester (50-200 nM) stimulated PtdCho synthesis 1.5- to 3-fold in C6 cells, was without effect in N1E-115 cells, and had intermediate effects on NG108-15 cells. Phorbol ester stimulated both uptake of extracellular choline and synthesis of PtdCho, whereas fatty acid stimulated only synthesis. Release of radioactivity from 24-h pulse-labeled PtdCho to the medium was enhanced by phorbol ester in C6 cells. Incorporation of [32P]Pi, primarily into PtdCho, was stimulated, whereas utilization of [1,2-14C]ethanolamine or 1-14C-fatty acid was little altered by phorbol ester. C6 cells "down-regulated" with phorbol ester lost the stimulatory response of subsequent treatment with phorbol esters on PtdCho synthesis, but the response to fatty acid was enhanced. Fatty acid had little influence on the relative binding of phorbol ester or "translocation" of phorbol ester binding sites. Accordingly, metabolism of phospholipids in these cultured cells of neural origin is markedly influenced by cell type, phospholipid class, condition of incubation medium, and nature of stimulator. Phorbol esters and fatty acids appear to enhance phospholipid synthesis and turnover by distinct intracellular mechanisms.     

C Tracer Evidence for Synthesis of Choline and Betaine via Phosphoryl Base Intermediates in Salinized Sugarbeet Leaves

Like other chenopods, sugarbeets (Beta vulgaris L. cv Great Western D-2) accumulate glycine betaine when salinized; this may be an adaptive response to stress. The pathway of betaine synthesis in leaves of salinized (150-200 millimolar NaCl) sugarbeet plants was investigated by supplying [¹⁴C]formate, phosphoryl[¹⁴C]monomethylethanolamine ([¹⁴C][unk] MME) or phosphoryl[¹⁴C]choline ([¹⁴C][unk] choline) to leaf discs and following ¹⁴C incorporation into prospective intermediates. The ¹⁴C kinetic data were used to develop a computer model of the betaine pathway.

When [¹⁴C]formate was fed, [unk] MME, phosphoryldimethylethanolamine ([unk] DME) and [unk] choline were the most prominent methylated products at short labeling times, after which ¹⁴C appeared in free choline and in betaine. Phosphatidylcholine labeled more slowly than [unk] choline, choline, and betaine, and behaved as a minor end product. Very little ¹⁴C entered the free methylethanolamines. When [¹⁴C][unk] MME was supplied, a small amount was hydrolyzed to the free base but the major fate was conversion to [unk] DME, [unk] choline, free choline, and betaine; label also accumulated slowly in phosphatidylcholine. Label from supplied [¹⁴C][unk] choline entered choline and betaine rapidly, while phosphatidylcholine labeled only slowly and to a small extent.

These results are consistent with the pathway [unk] MME →[unk] DME → [unk] choline → choline → → betaine, with a minor side branch leading from [unk] choline into phosphatidylcholine. This contrasts markedly (a) with the pathway of stress-induced choline and betaine synthesis in barley, in which phosphatidylcholine apparently acts as an intermediate (Hitz, Rhodes, Hanson 1981, Plant Physiol 68: 814-822); (b) with choline biogenesis in mammalian liver and microorganisms. Computer modeling of the experimental data pointed strongly to regulation at the [unk] choline → choline step, and also indicated that the rate of [unk] choline synthesis is subject to feedback inhibition by [unk] choline.

     

Glycinebetaine biosynthesis and its control in detached secondary leaves of spinach

In secondary leaves from spinach plants pretreated in vermiculite for 24 h with 300 mM NaCl, glycinebetaine accumulated at a rate of circa 0.16 mol 100 g^-1 Chl d^-1 (2 mol g^-1 FW d^-1), about three times the rate of control plants. The soluble carbohydrate and free amino acid contents did not increase significantly following salinisation until after 4 d when the relative growth rate also decreased. Leaf proline levels remained very low throughout the experimental period. K⁺ on a tissue water basis remained constant at 200 mM while Cl^- and Na⁺ levels increased linearly to reach 175 and 100 mM respectively after 5 d of saline treatment. The osmotic pressure of leaf tissue also increased from 300 to 500 mosmol kg^-1. These experimental conditions were considered suitable to study glycinebetaine biosynthesis and its induction by salinity in the absence of marked growth inhibition or metabolic disturbance. Radioactive labelled [¹⁴C]serine, ethanolamine and choline (all 1 mol, 13.3 MBq in 10 l) were fed to detached secondary leaves via the petiole 24 h after the exposure of plants to salt. The rate of isotope incorporation into water soluble products, lipids and residue was measured over a further 24 h. The major metabolic fate of exogenous [¹⁴C]choline and [¹⁴C]ethanolamine was incorporation into glycinebetaine while less ¹⁴C-label was found in phosphatidyl choline and phosphatidyl ethanolamine. Incorporation rates were identical in control and salinised leaves and were adequate to account for observed values of glycinebetaine accumulation previously reported in spinach. In contrast the labelling of glycinebetaine from [¹⁴C]serine was twice as great in salinated plants as in the controls. These results, together with short term labelling experiment with [¹⁴C]ethanolamine using leaf slices, were consistent with the formation of glycinebetaine via serine, ethanolamine and its methylated derivatives to choline with some control being exerted at the serine level. However a flux through the phosphorylated intermediates is not excluded.From a consideration of these results and the published data on barley subjected to water stress (Hanson and Scott, 1980 Plant Physiol. 66, 342–348) there appear to be significant differences in the biosynthetic pathways in spinach and barley.Abbreviations BHT butylated hydroxytoluerte (2,6-di-tert-butyl-4-methylphenol) - C₁ one-carbon fragment - 1,2DG diglyceride moiety - DW day weight - MCW methanol-chloroform-water (12:5:1, by vol.) - PA phosphatidic acid - PC phosphatidyl choline - PMME phosphatidyl monomethylethanolamine - PDME phosphatidyl dimethylethanolamine - PE phosphatidyl ethanolamine - PPO 2,5-diphenyloxazole - POPOP 1,4-bis(5-phenyloxazoyl) benzene