[30] Ethanolamine-phosphate cytidylyltransferase

Lb, Tijburg; Ps, Vermeulen; Golde, van

doi:10.1016/0076-6879(92)09032-x

Cited by 33 publications

(31 citation statements)

References 6 publications

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“…After centrifugation at 13,000 g and 4jC to pellet cell debris, the supernatants were assayed for mPcyt2 activity as described (23), except that commercially available [ 14 C]phosphoethanolamine (American Radiolabeled Chemicals) was used at a final specific activity of 2 mCi/mmol. The final concentrations of phosphoethanolamine and CTP were 1 and 2 mM, respectively.…”

Section: Enzyme Activity Assaysmentioning

confidence: 99%

“…In addition, the molar concentration of mPcyt2a was determined using spectrophotometry, and Western blotting with the anti-myc antibody followed by densitometry (data not shown) was used to equalize the molar amounts of each enzyme used in the experiments. Enzyme activity assays were carried out as described (23), with CTP at a fixed concentration of 2 mM and unlabeled phosphoethanolamine varying from 31.25 mM to 1.0 mM. [ 14 C]phosphoethanolamine was used at a final specific activity of 0.2 mCi/mmol.…”

Section: Radiolabeling With [mentioning

confidence: 99%

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Alternative splicing of CTP:phosphoethanolamine cytidylyltransferase produces two isoforms that differ in catalytic properties

Tie

Bakovic

2007

Journal of Lipid Research

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CTP:phosphoethanolamine cytidylyltransferase (Pcyt2) catalyzes the rate-controlling reaction of the CDPethanolamine (Kennedy) pathway. We have previously established that Pcyt2 is encoded by a single gene that can be alternatively spliced from an internal exon into two transcripts, designated Pcyt2a and Pcyt2b. Little is currently known about the regulation of Pcyt2. Here, we functionally express both murine Pcyt2 (mPcyt2) transcripts and investigate the roles of the two proteins in the regulation of mPcyt2 activity. We demonstrate that the tagged and purified a and b proteins differ significantly in their kinetic properties. The K m of mPcyt2a for phosphoethanolamine was 318.4 mM, compared with 140.3 mM for mPcyt2b. The maximal velocities of the a and b isoforms at saturating conditions for both substrates were 138.0 and 114.4 nmol/ min/mmol enzyme, respectively. When phosphoethanolamine was used at a fixed concentration of 1 mM, the K m of mPcyt2a for CTP was 102.0 mM and that of mPcyt2b was 84.09 mM. Using a combination of nondenaturing PAGE, gel filtration chromatography, and immunoprecipitation, we provide evidence that mPcyt2a and mPcyt2b proteins can form both homodimeric and heterodimeric complexes. We show that alternative splicing of the mPcyt2 transcript is ubiquitous but could also be regulated in a tissue-specific manner, producing a variable ratio of mPcyt2a/mPcyt2b mRNAs. The expression of two distinct protein isoforms maybe an important mechanism by which Pcyt2 activity is regulated.-Tie, A., and M. Bakovic. Alternative splicing of CTP:phosphoethanolamine cytidylyltransferase produces two isoforms that differ in catalytic properties. J. Lipid Res. 2007Res. . 48: 2172Res. -2181

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Section: Enzyme Activity Assaysmentioning

confidence: 99%

Section: Radiolabeling With [mentioning

confidence: 99%

Alternative splicing of CTP:phosphoethanolamine cytidylyltransferase produces two isoforms that differ in catalytic properties

Tie

Bakovic

2007

Journal of Lipid Research

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“…The activity of ECT was measured in 40 µl of homogenate (approx. 100 µg of protein), as described by Tijburg et al [22]. Radioactive CDP[1,2-14 C]ethanolamine and phospho[1,2-14 C]ethanolamine were separated and quantified as described for the water-soluble ethanolamine-containing metabolites.…”

Section: Transfection Of Cho Cells With Ect Cdna and Assay Of Ect Actmentioning

confidence: 99%

“…Our results strongly suggest that, at physiological ethanolamine concentrations, the supply of CDPethanolamine, via the expression level of ECT, together with the amount of cellular DAG (diacylglycerol) available to EPT are major factors regulating the flux through the CDPethanolamine pathway. [1,[2][3][4][5][6][7][8][9][10][11][12][13][14] C]ethanolamine with partially purified ethanolamine kinase as described previously [22]. sn-1,2-DAG kinase was from Calbiochem (La Jolla, CA, U.S.A.).…”

Section: Introductionmentioning

confidence: 99%

Control of the CDPethanolamine pathway in mammalian cells: effect of CTP:phosphoethanolamine cytidylyltransferase overexpression and the amount of intracellular diacylglycerol

Bleijerveld

Klein

Vaandrager

et al. 2004

Biochemical Journal

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For an insight regarding the control of PtdEtn (phosphatidylethanolamine) synthesis via the CDPethanolamine pathway, rat liver cDNA encoding ECT (CTP:phosphoethanolamine cytidylyltransferase) was transiently or stably transfected in Chinesehamster ovary cells and a rat liver-derived cell line (McA-RH7777), resulting in a maximum of 26-and 4-fold increase in specific activity of ECT respectively. However, no effect of ECT overexpression on the rate of [ 3 H]ethanolamine incorporation into PtdEtn was detected in both cell lines. This was explored further in cells overexpressing four times ECT activity (McA-ECT1). The rate of PtdEtn breakdown and PtdEtn mass were not changed in McA-ECT1 cells in comparison with controltransfected cells. Instead, an accumulation of CDPethanolamine (label and mass) was observed, suggesting that in McA-ECT1 cells the ethanolaminephosphotransferase-catalysed reaction became rate-limiting. However, overexpression of the human choline/ethanolaminephosphotransferase in McA-ECT1 and control-transfected cells had no effect on PtdEtn synthesis. To investigate whether the availability of DAG (diacylglycerol) limited PtdEtn synthesis in these cells, intracellular DAG levels were increased using PMA or phospholipase C. Exposure of cells to PMA or phospholipase C stimulated PtdEtn synthesis and this effect was much more pronounced in McA-ECT1 than in controltransfected cells. In line with this, the DAG produced after PMA exposure was consumed more rapidly in McA-ECT1 cells and the CDPethanolamine level decreased accordingly. In conclusion, our results suggest that the supply of CDPethanolamine, via the expression level of ECT, is an important factor governing the rate of PtdEtn biosynthesis in mammalian cells, under the condition that the amount of DAG is not limiting.

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“…ECT has been regarded as the rate-limiting enzyme for PE synthesis by the nucleotide pathway in mammalian cells (Tijburg et al, 1989;Vermeulen et al, 1993). It is soluble in both mammals (Sundler, 1975;Tijburg et al, 1992;Vermeulen et al, 1993) and yeast (Carman and Henry, 1989). The intracellular distribution reported for castor bean suggests a more complex situation and also contrasts with the distribution of the cytidylyltransferase for CDPcholine synthesis in castor bean, which is found almost entirely in the ER (Kinney et al, 1987); no activity for CCT has been reported as mitochondrial.…”

mentioning

confidence: 77%

Enzymes of the Primary Phosphatidylethanolamine Biosynthetic Pathway in Postgermination Castor Bean Endosperm (Developmental Profiles and Partial Purification of the Mitochondrial CTP:Ethanolaminephosphate Cytidylyltransferase)

Tang

Moore

1997

Plant Physiology

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Ethanolamine kinase, CTP:ethanolaminephosphate cytidylyltransferase (ECT), and ethanolaminephosphotransferase, which sequentially catalyze the primary pathway for phosphatidylethanolamine synthesis, were measured in castor bean (Ricinus communis L. var Hale) endosperm for 6 d after the onset of imbibition. Ethanolamine kinase (EC 2.7.1 3 2 ) activity was cytosolic, increasing slowly during the first 5 d and then declining. Total ECT (EC 2.7.7.14) activity increased until the 4th d, but the endoplasmic reticulum fraction of the activity peaked at d 3, and the mitochondrial activity peaked at d 4. Diacylglycero1:CDPethanolamine ethanolaminephosphotransferase (EC 2.7.8.1) increased during the first 2 d after imbibition began, after which it declined. The lowest activity of ethanolamine kinase during postgermination was more than 5-fold higher than the maximum activity of ECT, and the total activity of diacylglycero1:CDPethanolamine ethanolaminephosphotransferase at d 2 was at least triple that of ECT of the endoplasmic reticulum. We have partially purified ECT from mitochondrial fractions of postgermination castor bean endosperm starting with mitochondria purified by sucrose (SUC) density gradient centrifugation and broken by osmotic shock and homogenization. The membranebound ECT was solubilized with 1.5% 3-[(3-cholamidopropyI) dimethylammonio]-2-hydroxy-l -propanesulfonate and purified approximately 1 18-fold by polyethylene glycol precipitation, chromatography on Sephacryl S-200, and then SUC gradient centrifugation. The continuous presence of both salt (0.5 M NaCI) andhydroxy-1 -propanesulfonate) was necessary to prevent aggregation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the final activity peak resulted in a prominent protein band at 35 kD, which correlated with bands from peak ECT activity fractions after both SUC gradient centrifugation and gel filtration on Sephacryl S-200. The activity of this enzyme was enhanced by the addition of several phospholipids.

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[30] Ethanolamine-phosphate cytidylyltransferase

Cited by 33 publications

References 6 publications

Alternative splicing of CTP:phosphoethanolamine cytidylyltransferase produces two isoforms that differ in catalytic properties

Alternative splicing of CTP:phosphoethanolamine cytidylyltransferase produces two isoforms that differ in catalytic properties

Control of the CDPethanolamine pathway in mammalian cells: effect of CTP:phosphoethanolamine cytidylyltransferase overexpression and the amount of intracellular diacylglycerol

Enzymes of the Primary Phosphatidylethanolamine Biosynthetic Pathway in Postgermination Castor Bean Endosperm (Developmental Profiles and Partial Purification of the Mitochondrial CTP:Ethanolaminephosphate Cytidylyltransferase)

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