CDP‐diacylglycerol synthesis in rat liver mitochondria

Mok, Amy; McDougall, G.E.; McMurray, W. C.

doi:10.1016/0014-5793(92)80942-a

Cited by 28 publications

(26 citation statements)

References 36 publications

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“…In rat liver, the majority of CDS activity is found in the microsomal fraction, with some in the mitochondria and nuclei (32). The product of CDS activity (CDP-DAG) can be used to synthesize phosphatidylglycerol as well as PtdIns, and in inositol auxotrophs deprived of inositol there is an increase in phosphatidylglycerol that accompanies the decrease in PtdIns synthesis (5).…”

Section: Discussionmentioning

confidence: 99%

“…Lower panel: Cells were replicately plated in 12-well plastic dishes and allowed to grow for 48 h. The medium was then changed to either serum-free, phosphate-free F-12 medium containing 25 mM HEPES and 50 mCi/ml [ 32 P]orthophosphate for the labeling of PtdIns and PtdCho or serum-free minimal essential medium containing 25 mM HEPES and either 20 mCi/ml radiolabeled arachidonic acid (AA) or 20 mCi/ml radiolabeled oleic acid (OA) plus or minus 50 mM triacsin C (Tc). After another 2 h incubation, lipids were extracted as described in the text and separated by thin-layer chromatography to yield either [ 32 Values shown represent means 6 SD of three separate determinations. Statistical significance for differences seen with triacsin C is as follows: PtdIns, P , 0.0005; PtdCho, no statistical difference; arachidonic acid, P , 0.0025; oleic acid, not determined.…”

Section: Effect Of Pkc Inhibition On the Actions Of Tpamentioning

confidence: 99%

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Regulation of de novo phosphatidylinositol synthesis

Nuwayhid

Vega

Walden

et al. 2006

Journal of Lipid Research

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Mechanisms that function to regulate the rate of de novo phosphatidylinositol (PtdIns) synthesis in mammalian cells have not been elucidated. In this study, we characterize the effect of phorbol ester treatment on de novo PtdIns synthesis in C3A human hepatoma cells. Incubation of cells with 12-O-tetradecanoyl phorbol 13-acetate (TPA) initially (1-6 h) results in a decrease in precursor incorporation into PtdIns; however, at later times (18-24 h), a marked increase is observed. TPA-induced glucose uptake from the medium is not required for observation of the stimulation of PtdIns synthesis, because the effect is apparent in glucose-free medium. Inhibition of the activation of arachidonic acid substantially blocks the synthesis of PtdIns but has no effect on the synthesis of phosphatidylcholine (PtdCho). Increasing the concentration of cellular phosphatidic acid by blocking its conversion to diacylglycerol, on the other hand, enhances the synthesis of PtdIns and inhibits the synthesis of PtdCho. The TPA-induced stimulation of PtdIns synthesis is not the result of the concomitant TPA-induced G1 arrest, because G1 arrest induced by mevastatin has no effect on PtdIns synthesis. Inhibition of protein kinase C activity blocks the stimulatory action of TPA on de novo synthesis of PtdIns but has no effect on TPA-induced inhibition.Potential sites of enzymatic regulation are discussed.-Nuwayhid, S. J., M. Vega, P. D. Walden, and M. E. Monaco. Phosphatidylinositol (PtdIns) is an essential acidic phospholipid found in the membranes of mammalian cells (1) as well as on the chromatin (2). A single molecular species composed of stearate (R9) and arachidonate (R99) accounts for z80% of the total PtdIns. There are two pathways for the synthesis of PtdIns: the de novo pathway and the salvage pathway, which converge at the level of phosphatidic acid (PtdOH). The de novo pathway uses dihydroxyacetone phosphate derived from glucose to make glycerol phosphate, which is subsequently acylated at the sn-1 position with stearate and then at the sn-2 position with arachidonate to yield PtdOH. In the salvage pathway, diacylglycerol (DAG), derived from phospholipase C-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdInsP 2 ), is converted to PtdOH through the action of DAG kinase. In the first instance, there is a net increase in PtdIns, whereas in the second, there is not. The central element of both pathways is the production of PtdOH. For the salvage pathway, it appears that the conversion of DAG to PtdOH by DAG kinase is stimulated as a result of agonist-induced polyphosphoinositide turnover. Whether this results from an increase in DAG substrate and/or an increase in DAG kinase enzyme activity is unclear. This laboratory has previously demonstrated that the two pathways can be differentiated in situ on the basis of the inhibition by triacsin C, a specific inhibitor of arachidonate-specific long-chain fatty acyl-CoA synthetase (ACSL4) in situ (3, 4).Although regulation of the salvage pathway via the activation ...

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Section: Discussionmentioning

confidence: 99%

Section: Effect Of Pkc Inhibition On the Actions Of Tpamentioning

confidence: 99%

Regulation of de novo phosphatidylinositol synthesis

Nuwayhid

Vega

Walden

et al. 2006

Journal of Lipid Research

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“…The average increase in Cds-specific activity was 7.6-fold. The stimulation of Cds activity by GTP was previously proposed to distinguish the microsomal form of the enzyme, which is activated by GTP, from the mitochondrial form (17,40,41). GTP modulation of Cds activity reported by these investigators was potentially an exciting finding at the time since it suggested the possibility of G protein regulation of the enzyme.…”

Section: Isolation and Structure Of The Cds1 And Pis1 Cdnas-thementioning

confidence: 93%

The Role of CDP-Diacylglycerol Synthetase and Phosphatidylinositol Synthase Activity Levels in the Regulation of Cellular Phosphatidylinositol Content

Lykidis¹,

Jackson²,

Rock³

et al. 1997

Journal of Biological Chemistry

118

108

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The regulation of phosphatidylinositol synthesis was examined by cloning and expressing in COS-7 cells the human cDNAs encoding the two enzymes in the biosynthetic pathway. Human CDP-diacylglycerol synthetase (cds1) and phosphatidylinositol synthase (pis1) clones were identified in the human expressed sequencetagged (EST) data base, and full-length cDNAs were obtained by library screening. The cds1 cDNA did not possess a recognizable mitochondrial import signal, and the activity of the expressed Cds1 protein was stimulated by nucleoside triphosphates in vitro, indicating that cds1 did not encode the mitochondrial-specific isozyme. There were two mRNA species (3.9 and 5.6 kilobases) detected on Northern blots hybridized with the cds1 probe that were expressed at distinctly different levels in various human tissues. Consistent with the presence of the two mRNAs, a cDNA predicted to encode a second human CDP-diacylglycerol synthetase (cds2) was also uncovered in the EST data base. In contrast to the two cds mRNAs, a single, 2.1-kilobase pis1 mRNA was uniformly expressed in all human tissues examined. Expression of the pis1 gene led to the overproduction of both phosphatidylinositol synthase and phosphatidylinositol:inositol exchange reactions, indicating that the Pis1 polypeptide catalyzed both of these activities. Phosphatase treatment of cell extracts abolished the CMP-independent phosphatidylinositol:inositol exchange reaction, and exchange activity was completely restored by the addition of CMP. Overexpression of cds1 or pis1 alone or in combination did not enhance the rate of phosphatidylinositol biosynthesis. Also, overexpression did not result in a significant proportional increase in the cellular levels of CDP-diacylglycerol or phosphatidylinositol. These data illustrate that the levels of Cds1 and Pis1 protein expression are not critical determinants of cellular PtdIns content and argue against a determining role for the activity of either of these enzymes in the regulation of PtdIns biosynthesis.

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“…These membrane domains have biochemical properties consistent with the phospholipid-rich, nonraft, ER membrane domains predicted by Shaikh and Edidin ( 35 ). 32 P]CDP-DAG ( 40 ). Fifty microliter samples from each gradient fraction were mixed with an equal volume of assay buffer composed of 20 mM Tris-HCl pH 7.4, 40 mM MgCl 2 , 2 mM EGTA, and 20 Ci/ml [ ␣ 32 P]CTP.…”

Section: Subcellular Fractionation Of A431 Cellsmentioning

confidence: 99%

“…Caveolae were immunoisolated as described previously ( 40 ). For the isolation of calnexin-containing membranes, samples were precleared with 20 µl Protein G Sepharose beads for 1 h at 4°C.…”

Section: Immunoprecipitation Of Caveolae and Calnexin-rich Domainsmentioning

confidence: 99%