scholarly journals Phosphatidylcholine biosynthesis and choline transport in the anaerobic protozoon Entodinium caudatum

1976 ◽  
Vol 160 (3) ◽  
pp. 481-490 ◽  
Author(s):  
F L Bygrave ◽  
R M C Dawson
Keyword(s):  
Cell Envelope ◽  
Choline Kinase ◽  
Choline Uptake ◽  
Choline Transport ◽  
Intact Cells ◽  
Phosphatidylcholine Biosynthesis ◽  
Free Choline ◽  
Choline Phosphate ◽  
Rate Limiting ◽  
Phosphatidylcholine Synthesis

Choline accumulation and phosphatidylcholine biosynthesis were investigated in the choline-requiring anaerobic protozoon Entodinium caudatum by incubating whole cells or subcellular fractions with [14C] choline, phosphoryl [14C] choline and CDP-[14C] choline. 2. All membrane fractions contained choline kinase (EC 2.7.1.32) and CDP-choline-1,2-diacylglycerol cholinephosphotransferase (EC 2.7.8.2), although the specific activities were less in the cell-envelope fraction. Choline phosphate cytidylyltransferase (EC 2.7.7.15) was limited to the supernatant, and this enzyme was rate-limiting for phosphatidylcholine synthesis in the whole cell. 3. Synthesis of phosphatidylcholine from free choline by membranes was only possible in the presence of supernatant. Such reconstituted systems required ATP (2.5 mM), CTP (1 mM) and Mg2+ (5 mM) for maximum synthesis of the phospholipid. CTP and Mg2+ were absolute requirements. 4. Hemicholinium-3 prevented choline uptake by the cells and was strongly inhibitory towards choline kinase; the other enzymes involved in phosphatidylcholine synthesis were minimally affected. 5. Ca2+ ions (0.5 mM) substantially inhibited CDP-choline-1,2-diacylglycerol cholinephosphotransferase in the presence of 15 mM-Mg2+, but choline phosphate cytidylyltransferase and choline kinase were less affected. 6. No free choline could be detected intact cells even after short (10-180s) incubations or at temperatures down to 10° C. The [14C] choline entering was mainly present as phosphorylcholine and to a lesser extent as phosphatidylcholine. 7. It is suggested that choline kinase effectively traps any choline within the cell, thus ensuring a supply of the base for future growth. At low choline concentrations the activity of choline kinase is rate-limiting for choline uptake, and the enzyme might possibly play an active role in the transport phenomenon. Thus the choline uptake by intact cells and choline kinase have similar Km values and show similar responses to temperature and hemicholinium-3.

Effect of amino acids on choline uptake and phosphatidylcholine biosynthesis in the isolated hamster heart

1988 ◽  
Vol 66 (5) ◽  
pp. 418-424 ◽  
Author(s):  
Grant M. Hatch ◽  
Willem K. Stevens ◽  
Patrick C. Choy
Keyword(s):  
Amino Acids ◽  
Direct Interaction ◽  
Choline Uptake ◽  
Choline Transport ◽  
Neutral Amino Acids ◽  
Acidic Amino Acids ◽  
Phosphatidylcholine Biosynthesis ◽  
Concentration Threshold ◽  
Hamster Heart ◽  
Dose Dependent

Choline uptake by the hamster heart has been shown to be enhanced by exogenous glycine. In this study, the effect of neutral, basic, and acidic amino acids on choline uptake was assessed. Hamster hearts were perfused with labelled choline, and in the presence of L-alanine, L-serine, or L-phenylalanine (≥0.1 mM), choline uptake was enhanced 20–38%. L-Arginine, L-lysine, L-aspartate, and L-glutamate did not influence choline uptake. The rate of phosphatidylcholine biosynthesis was unaffected by all amino acids tested. Enhancement of choline uptake by neutral amino acids was not additive or dose dependent but required a concentration threshold. The enhancement of choline uptake by neutral amino acids was not influenced by preperfusion with the same amino acid. Exogenous choline had no effect on the uptake of amino acids. We postulate that choline and the neutral amino acids are not cotransported and modulation of choline uptake is facilitated by direct interaction of the neutral amino acids with the choline transport system.

scholarly journals The Phospholipid N-Methyltransferase and Phosphatidylcholine Synthase Pathways and the ChoXWV Choline Uptake System Involved in Phosphatidylcholine Synthesis Are Widely Conserved in Most, but Not All Brucella Species

2021 ◽  
Vol 12 ◽  
Author(s):  
Beatriz Aragón-Aranda ◽  
Leyre Palacios-Chaves ◽  
Miriam Salvador-Bescós ◽  
María Jesús de Miguel ◽  
Pilar M. Muñoz ◽  
...  
Keyword(s):  
Cell Envelope ◽  
Innate Immune ◽  
Intracellular Bacteria ◽  
Choline Uptake ◽  
Uptake System ◽  
Innate Immune Responses ◽  
A Cell ◽  
Direct Linkage ◽  
Phosphatidylcholine Synthesis

The brucellae are facultative intracellular bacteria with a cell envelope rich in phosphatidylcholine (PC). PC is abundant in eukaryotes but rare in prokaryotes, and it has been proposed that Brucella uses PC to mimic eukaryotic-like features and avoid innate immune responses in the host. Two PC synthesis pathways are known in prokaryotes: the PmtA-catalyzed trimethylation of phosphatidylethanolamine and the direct linkage of choline to CDP-diacylglycerol catalyzed by the PC synthase Pcs. Previous studies have reported that B. abortus and B. melitensis possess non-functional PmtAs and that PC is synthesized exclusively via Pcs in these strains. A putative choline transporter ChoXWV has also been linked to PC synthesis in B. abortus. Here, we report that Pcs and Pmt pathways are active in B. suis biovar 2 and that a bioinformatics analysis of Brucella genomes suggests that PmtA is only inactivated in B. abortus and B. melitensis strains. We also show that ChoXWV is active in B. suis biovar 2 and conserved in all brucellae except B. canis and B. inopinata. Unexpectedly, the experimentally verified ChoXWV dysfunction in B. canis did not abrogate PC synthesis in a PmtA-deficient mutant, which suggests the presence of an unknown mechanism for obtaining choline for the Pcs pathway in Brucella. We also found that ChoXWV dysfunction did not cause attenuation in B. suis biovar 2. The results of these studies are discussed with respect to the proposed role of PC in Brucella virulence and how differential use of the Pmt and Pcs pathways may influence the interactions of these bacteria with their mammalian hosts.

scholarly journals The regulation of phosphatidylcholine biosynthesis in rye (Secale cereale) roots. Stimulation of the nucleotide pathway by low temperature

1987 ◽  
Vol 242 (3) ◽  
pp. 755-759 ◽  
Author(s):  
A J Kinney ◽  
D T Clarkson ◽  
B C Loughman
Keyword(s):  
Secale Cereale ◽  
Choline Chloride ◽  
Rate Limiting Step ◽  
Phosphatidylcholine Biosynthesis ◽  
Secale Cereale L ◽  
Choline Phosphate ◽  
Relative Differences ◽  
Rate Limiting

The incorporation of [14C]choline chloride and [14C]glycerol into segments taken from rye (Secale cereale L., cv. Rheidal) roots was greater in segments from roots grown at 5 degrees C than in segments taken from roots growing at 20 degrees C. The incorporation was measured at the temperature at which the root had been growing. Measurements in vitro of the enzymes of the nucleotide pathway showed activity of choline kinase (EC 2.7.1.32), choline-phosphate cytidylyltransferase (EC 2.7.7.15) and cholinephosphotransferase (EC 2.7.8.2) to be higher in homogenates from the cooler roots when assayed at 5 degrees C than the activities assayed at 20 degrees C in the 20 degrees C-root homogenates. Changes in vivo in the pool sizes of the CDP-base intermediates with temperature, relative differences in nucleotide-pathway-enzyme activities and a pulse-chase experiment with [14C]choline indicated that the rate-limiting step for phosphatidylcholine biosynthesis in this tissue, at both temperatures, was the reaction catalysed by cytidylyltransferase.

Na(+)-dependent and Na(+)-independent systems of choline transport by plasma membrane vesicles of A549 cell line

1994 ◽  
Vol 267 (5) ◽  
pp. C1279-C1287 ◽  
Author(s):  
A. Kleinzeller ◽  
C. Dodia ◽  
A. Chander ◽  
A. B. Fisher
Keyword(s):  
A549 Cells ◽  
Membrane Vesicles ◽  
Choline Uptake ◽  
Plasma Membrane Vesicles ◽  
Choline Transport ◽  
High Affinity ◽  
A Value ◽  
Carbonyl Cyanide ◽  
Rate Limiting ◽  
A549 Lung

Membrane vesicles of A549 lung cells accumulate choline by two pathways: the Na(+)-independent uphill uptake of choline [Michaelis-Menten constant (Km) approximately 44 microM; steady-state gradient approximately 45 at 5 microM external choline] is dependent on a transmembrane H+ gradient, is relatively insensitive to hemicholinium-3, is amiloride sensitive, and is abolished by valinomycin plus carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). The Na(+)-dependent active choline uptake (Km approximately 4 microM, inhibitor constant for hemicholinium-3 approximately 0.1 microM), is specific for Na+, is amiloride and FCCP sensitive, and is electrogenic: the overshoot using K(+)-loaded vesicles and NaCl gradient was increased by valinomycin. The time of the overshoot peak, T was approximately 90 s in a NaSCN medium (or in presence of other lipid-soluble anions), a value close to that for alpha-aminoisobutyrate as substrate (T = approximately 1.5 min). T was lengthened in NaCl medium to approximately 10 min, and the overshoot was abolished by impermeant anions. External Cl- is not required for the choline uptake: valinomycin produced an overshoot in the presence of only impermeant anions, with T approximately 90 s. Most of the above properties are shared by the high-affinity Na(+)-dependent choline transport in synaptosomes. The characteristics of the Na(+)-dependent choline uptake by membrane vesicles of A549 cells are consistent with an electrogenic choline(+)-Na+ cotransport, with the rate-limiting anion (e.g., Cl-) influx balancing the positive charges transferred into the vesicles. The data are also consistent with an involvement of an amiloride-sensitive choline+/H+ antiport (or choline(+)-OH- symport) in the low- and high-affinity choline uptake pathways.

Transport of choline by plasma membrane vesicles from lung-derived epithelial cells

1992 ◽  
Vol 263 (6) ◽  
pp. C1250-C1257 ◽  
Author(s):  
A. B. Fisher ◽  
C. Dodia ◽  
A. Chander ◽  
A. Kleinzeller
Keyword(s):  
Plasma Membrane ◽  
Steady State ◽  
Epithelial Cells ◽  
Concentration Gradient ◽  
A549 Cells ◽  
Membrane Vesicles ◽  
Choline Uptake ◽  
Plasma Membrane Vesicles ◽  
Choline Transport ◽  
Intact Cells

A549 cells, a lung epithelium-derived cell line, were used as a model system to study choline transport by granular pneumocytes. Intact cells accumulated free choline against a concentration gradient by a low-affinity transport system with kinetic characteristics similar to that previously described for granular pneumocytes (Am. J. Respir. Cell Mol. Biol. 1: 455, 1989). Membrane vesicles prepared from these cells showed a 10-fold enrichment in plasma membrane marker enzymes with a vesicular H2O space of 5.7 +/- 0.05 (SE) microliters/mg protein. Vesicles showed a time- and concentration-dependent uptake of free [3H]choline in Na(+)-free medium. With 5 microM choline, choline uptake reached an apparent steady-state concentration gradient (inside/outside) of 50. 3H that was membrane associated ("bound" choline) represented approximately 5% of total uptake. In the presence of an initial gradient of NaCl, choline uptake showed an overshoot with a plateau value similar to Na(+)-free conditions; a similar effect was observed for plasma membrane vesicles from rat lung type 2 epithelial cells. The steady-state uptake of choline was inhibited at low pH (6.5) and by the presence of valinomycin or carbonyl cyanide p-tri-fluoromethoxyphenylhydrazone and was abolished when both were present. These results show that plasma membrane vesicles from A549 cells accumulate choline by binding to the membranes and by Na(+)-dependent and -independent transport mechanisms, the latter apparently reflecting a transmembrane proton gradient.

scholarly journals Tight connection between choline transport and phosphatidylcholine synthesis in MDCK cells

1996 ◽  
Vol 315 (3) ◽  
pp. 983-987 ◽  
Author(s):  
Philippe ZLATKINE ◽  
Christine LEROY ◽  
Gert MOLL ◽  
Christian LE GRIMELLEC
Keyword(s):  
Mdck Cells ◽  
Water Soluble ◽  
Choline Uptake ◽  
Choline Transport ◽  
Apical Side ◽  
Preferential Localization ◽  
Basolateral Side ◽  
Tight Connection ◽  
Uptake Sites ◽  
Phosphatidylcholine Synthesis

In MDCK cells, choline uptake, the first step in the CDP-choline pathway for the biosynthesis of choline-containing phospholipids and osmolytes, occurs via both a transport system highly specific for choline and a non-specific pathway. The specific choline carrier is present at the apical domain of cells grown on dishes and is sodium-independent. Growing the cells on a permeant support results in the preferential localization of the specific choline carrier at the basolateral domain. To characterize the relationships between the choline uptake sites and the synthesis of phosphatidylcholine, MDCK cells were incubated with [Me-3H]choline and/or [Me-14C]choline for various times (up to 36 h) and the incorporation of label into phospholipids and water-soluble molecules was determined. For cells grown on dishes, addition of [Me-3H]choline at the apical side was followed by rapid incorporation of the label into the successive intermediates of the CDP-choline pathway. A comparable situation was found when growing the cells on a permeant support and adding the labelled choline at the basolateral side of the culture. On the other hand, radioactive choline added to the apical bath entered the CDP pathway to only a very low extent. Efflux experiments on cells loaded with choline from either the apical or the basolateral side demonstrate the existence of intracellular pools of choline. Addition of hemicholinium-3, an inhibitor of the specific choline carrier, markedly reduced the metabolism of choline taken up by the cells on the basolateral side but had no effect on that transported at the apical side. These results strongly suggest the existence of a tight connection between the entry of choline through the specific choline carrier and phosphatidylcholine synthesis in MDCK cells.

Effect of cAMP on choline uptake and phosphatidylcholine biosynthesis in cultured chick heart cells

1988 ◽  
Vol 66 (3) ◽  
pp. 231-237 ◽  
Author(s):  
Paul S. Sunga ◽  
P. Haydn Pritchard ◽  
Simon W. Rabkin
Keyword(s):  
Phosphodiesterase Inhibitor ◽  
Cardiac Cells ◽  
Heart Cells ◽  
Choline Uptake ◽  
Ctp:Phosphocholine Cytidylyltransferase ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Phosphatidylcholine Biosynthesis ◽  
Chick Heart ◽  
Rate Limiting

The effect of an analogue of cAMP on the uptake and metabolism of choline in the heart was studied in isolated cardiac cells. The cells were obtained from 7-day-old chick embryos and maintained in culture. The effects of cAMP were studied using the dibutyryl cAMP analogue and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine. After a 2-h incubation with [3H]choline, about 85% of the label was recovered in phosphocholine, with most of the rest in phospholipid. During a subsequent chase incubation, [3H]phosphocholine was transferred to phosphatidylcholine with little accumulation in CDP-choline. This suggests the rate-limiting step for the conversion of phosphocholine to phosphatidylcholine in these cells is the synthesis of CDP-choline. cAMP decreased the incorporation of choline into phosphatidylcholine, but did not change the flux of metabolites through the step catalyzed by CTP:phosphocholine cytidylyltransferase. cAMP had little effect on choline uptake at low (1–25 μM) extracellular choline concentrations, but significantly (p < 0.05) decreased choline uptake at higher (37.5–50 μM) extracellular choline concentrations. Thus, cardiac cells take up and metabolize choline to phosphocholine, with CTP:phosphocholine cytidylyltransferase being the rate-limiting step in phosphatidylcholine biosynthesis. cAMP decreases [3H]choline uptake and its subsequent incorporation into phosphocholine and phospholipid. However, the metabolism of choline within the cell is unaffected.

scholarly journals Pathological ATX3 Expression Induces Cell Perturbations in E. coli as Revealed by Biochemical and Biophysical Investigations

2021 ◽  
Vol 22 (2) ◽  
pp. 943
Author(s):  
Diletta Ami ◽  
Barbara Sciandrone ◽  
Paolo Mereghetti ◽  
Jacopo Falvo ◽  
Tiziano Catelani ◽  
...  
Keyword(s):  
Cell Envelope ◽  
Spinocerebellar Ataxia Type ◽  
Spinocerebellar Ataxia Type 3 ◽  
Intact Cells ◽  
E Coli ◽  
Fourier Transform Infrared Microspectroscopy ◽  
Electron Microscopy Analysis ◽  
Microscopy Analysis ◽  
The Fourier Transform ◽  
Type 3

Amyloid aggregation of human ataxin-3 (ATX3) is responsible for spinocerebellar ataxia type 3, which belongs to the class of polyglutamine neurodegenerative disorders. It is widely accepted that the formation of toxic oligomeric species is primarily involved in the onset of the disease. For this reason, to understand the mechanisms underlying toxicity, we expressed both a physiological (ATX3-Q24) and a pathological ATX3 variant (ATX3-Q55) in a simplified cellular model, Escherichia coli. It has been observed that ATX3-Q55 expression induces a higher reduction of the cell growth compared to ATX3-Q24, due to the bacteriostatic effect of the toxic oligomeric species. Furthermore, the Fourier transform infrared microspectroscopy investigation, supported by multivariate analysis, made it possible to monitor protein aggregation and the induced cell perturbations in intact cells. In particular, it has been found that the toxic oligomeric species associated with the expression of ATX3-Q55 are responsible for the main spectral changes, ascribable mainly to the cell envelope modifications. A structural alteration of the membrane detected through electron microscopy analysis in the strain expressing the pathological form supports the spectroscopic results.

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