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PubMed1986

A novel mechanism of diglyceride formation. 12-O-tetradecanoylphorbol-13-acetate stimulates the cyclic breakdown and resynthesis of phosphatidylcholine.

12-O-Tetradecanoylphorbol-13-acetate (TPA) treatment of Madin-Darby canine kidney cells resulted in an increased incorporation of 32Pi and [methyl-3H]choline into choline-containing phosphoglycerides (PC). In pulse-chase experiments, TPA treatment caused an increased release of [methyl-3H]choline from the PC fraction of prelabeled cells. When cells were prelabeled with [3H]arachidonic acid and [14C]palmitic acid, TPA treatment resulted in an increased synthesis of 14C, 3H-diglycerides. Further studies were done to determine the relationship between PC breakdown and diglyceride synthesis. Cells were preincubated with ether-linked 1-O-[3H]hexadecyl-2-lyso-sn-glycero-3-phosphocholine which was acylated to form 1-O-[3H]hexadecyl-2-acyl-sn-glycero-3-phosphocholine. Subsequent treatment of these cells with TPA resulted in an increased synthesis of 1-O-[3H]hexadecyl-2-acyl-sn-glycerol compared to cells not stimulated with TPA. These findings demonstrate that TPA stimulates PC turnover in Madin-Darby canine kidney cells and provide evidence for a novel mechanism of diglyceride formation.

Animals↗

PubMed1980

Phosphatidic acid accumulation in the membranes of Escherichia coli mutants defective in CDP-diglyceride synthetase.

CTP-phosphatidic acid cytidylyltransferase (CDP-diglyceride synthetase) is a key enzyme in the biogenesis of membrane phospholipids in Escherichia coli. Using a modification of a previously described autoradiographic screening procedure (Raetz, C. R. H. (1975) Proc. Natl. Acad. Sci. U.S.A. 72, 2274-2278), we have isolated six mutant strains in which the specific activity of the synthetase is 1 to 10% that of the wild type, as judged by in vitro assays. The synthesis of dCDP-diglyceride, as well as CDP-diglyceride, is defective in these organisms. The mutations responsible for the enzyme defects (designated cds) all map in the same location near minute 4 on the chromosome. Although none of the mutants obtained are temperature-sensitive for growth, all of them exhibit significantly elevated levels of phosphatidic acid in vivo. The highest increase is observed in the mutant GL60, in which phosphatidic acid constitutes about 5% of the membrane lipid, in contrast to 0.2% in typical wild type strains. The accumulation of phosphatidic acid occurs primarily at the expense of phosphatidylglycerol and cardiolipin, but the total lipid-to-protein ratio of GL60 is nearly normal. In vivo labeling of GL60 with 32Pi suggests that the increased phosphatidic acid pool is the result of a partial metabolic block early in the phospholipid pathway, but that most of this expanded pool is nonetheless available for de novo synthesis.

Cell Membrane↗

PubMed1982

Massive accumulation of phosphatidic acid in conditionally lethal CDP-diglyceride synthetase mutants and cytidine auxotrophs of Escherichia coli.

Escherichia coli mutants partially defective in CTP: phosphatidic acid cytidylyltransferase (CDP-diglyceride synthetase) are more resistant to the antibiotic erythromycin than are isogenic wild type strains. When 100 micrograms/ml erythromycin is added to nutrient agar plates, it is possible to obtain a 30-fold enrichment for cds mutants from a mutagen-treated stock, as judged by colony autoradiography (Ganong, B. R., Leonard, J. M., and Raetz, C. R. H. (1980) J. Biol. Chem. 255, 1623-1629). Using this approach, we have isolated 38 new cds mutants, nine of which are unable to grow at a culture pH greater than 8. A typical conditionally lethal mutant like GN80 contains a 3 to 5% phosphatidic acid below pH 7. Above pH 8, GN80 accumulates phosphatidic acid to about 30% of the total membrane lipid, while the de novo syntheses of phosphatidylethanolamine and phosphatidylglycerol are abruptly inhibited by over 10-fold. GN80 loses viability after 60 min at pH 8.5, and the liponucleotide pool of GN80 is about one-seventh that of an isogenic wild type, GN85, under these conditions. The pH optimum of the residual CDP-diglyceride synthetase present in extracts of GN80 is 0.5 pH units lower than normal. Twenty-one of 26 spontaneous pH-resistant revertants of GN80 concomitantly regain parental levels of the enzyme. Our results constitute definitive physiological proof that CDP-diglyceride is an obligatory precursor for over 90% of the phosphatidylethanolamine and phosphatidylglycerol in E. coli. Independent evidence for this is provided by the observation that cytidine auxotrophs, which are defective in the conversion of UTP to CTP, also accumulate very high levels of phosphatidic acid after 1 h of cytidine starvation.

Cytidine↗

PubMed1972

The structure of a glycerylphosphoryldiglucosyl diglyceride from the lipids of Acholeplasma laidlawii strain B.

1. The phosphatidylglucose structure proposed previously (Smith & Henrikson, 1965) for the glucose-containing phospholipid from Acholeplasma laidlawii is incorrect. 2. The structure now proposed is 3-(sn-glycerol-3-phosphoryl-6'-[O-alpha-d-glucopyranosyl-(1-->2)-O-alpha-d-glucopyranosyl])- sn-1,2-diglyceride, a new type of bacterial lipid. 3. Deacylation of the lipid gave a single water-soluble phosphate ester which could be distinguished on chromatography from synthetic samples of glucosylphosphorylglycerols. 4. Hydrolysis of the lipid with alkali gave a mixture of fatty acids, glycerol 2-phosphate, sn-glycerol 3-phosphate and O-alpha-d-glucopyranosyl-(1-->2)-O-alpha- d-glucopyranosyl-(1-->1)-d-glycerol. 5. The lipid was unaffected on incubation with phospholipases A, C and D. 6. Diglucosyl diglyceride was isolated after treatment of the lipid with 60% HF, establishing the location of the fatty acid residues. 7. Periodate oxidation studies showed that the sn-glycerol 3-phosphate was esterified to the 6-hydroxyl group of one of the glucose residues in diglucosyl diglyceride.

Acholeplasma laidlawii↗

PubMed1967

Galactosyl diglycerides: their possible function in Euglena chloroplasts.

Illumination of euglenas grown in the dark induces the formation of chloroplasts characterized by the simultaneous appearance of chlorophyll and galactosyl diglycerides in a relatively fixed ratio. The fatty acyl chains of the galactosyl diglycerides are constructed so that they can provide a stable lock-and-key fit with the phytol chains of chlorophyll in such a way as to localize the porphyrin structures of chlorophyll and space them for efficient photoreception. Light-starved photobiotic euglenas show chloroplast shrinkage with a concurrent partial loss of galactosyl diglycerides.

Chlorophyll↗

PubMed1983

pH-sensitive CDP-diglyceride synthetase mutants of Escherichia coli: phenotypic suppression by mutations at a second site.

In Escherichia coli, mutations which lower the level of CDP-diglyceride synthetase are designated cds and map at min 4. The cds-8 mutation resulted in strikingly defective enzyme activity and also rendered cells pH sensitive for growth. Both the inhibition of growth and the massive accumulation of phosphatidic acid which occur in a cds-8 mutant at pH 8 were suppressed by mutations at a second locus, designated cdsS, which mapped between argG and gltB near min 68. The cdsS3 mutation by itself did not affect CDP-diglyceride synthetase activity in wild-type cells, but it caused a twofold stimulation of the residual activity present in strains harboring cds-8. Both the insensitivity to pH and the twofold stimulation of residual activity were lost by introduction of an F' strain carrying cdsS+ into a recA1 cds-8 cdsS3 host. When a culture of a cds-8 cdsS+ strain was shifted to pH 8, the residual specific activity of synthetase dropped by 75% within 100 min. In a cds-8 cdsS3 double mutant under the same conditions, the activity declined appreciably less, about to the level found in the cds-8 cdsS+ strain under permissive conditions (pH 6). Thus, it appears that mutations in the cdsS gene suppress the pH sensitivity of cds mutants by inhibiting the decay of residual CDP-diglyceride synthetase activity at the nonpermissive pH. The cdsS locus appears to be distinct from any known nonsense or missense suppressor.

Chromosome Mapping↗

PubMed1985

Molecular cloning and sequencing of the gene for CDP-diglyceride synthetase of Escherichia coli.

The cds gene of Escherichia coli codes for the enzyme CDP-diglyceride synthetase. We now report the construction of plasmids which carry cds. Using these plasmids, we have sequenced 1274 base pairs of DNA, including a 750-base pair open reading frame which is the coding region of the cds gene. This DNA sequence allows the deduction of the primary peptide sequence for CDP-diglyceride synthetase. The protein is very hydrophobic, and, assuming no processing or modification, has a molecular weight of 27,570. Furthermore, there is a second open reading frame immediately after cds, implying that cds may be part of an operon. We have also constructed a runaway replication cds-plasmid that directs approximately 50-fold overproduction of CDP-diglyceride synthetase. This overproduction has been utilized in the purification of the enzyme to homogeneity, as described in the accompanying paper (Sparrow, C.P., and Raetz, C.R.H., J. Biol. Chem. 260, 12084-12091). Finally, the molecular cloning work reported herein allows the exact placement of the cds gene on the E. coli genetic map.

Amino Acid Sequence↗

PubMed1991

Cytidine diphosphate diglyceride analogs of antiretroviral dideoxynucleosides: evidence for release of dideoxynucleoside-monophosphates by phospholipid biosynthetic enzymes in rat liver subcellular fractions.

We recently synthesized phospholipid analogs with antiviral nucleosides in the polar headgroup and demonstrated their antiretroviral activity in vitro in human immunodeficiency virus-infected cells (Hostetler, K.Y., Stuhmiller, L.M., Lenting, H.B.M., van den Bosch, H. and Richman, D.D. (1990) J. Biol. Chem. 265, 6112-6117). Dideoxynucleoside analogs of cytidine diphosphate diglyceride (CDP-DG) represent one class of such phospholipid prodrugs from which the antiviral active principle may be released through established pathways of cellular phospholipid metabolism. We now demonstrate that the liponucleotides of dideoxycytidine, 3'-deoxythymidine and 3'-azido-3'-deoxythymidine (AZT, Zidovudine) can substitute to varying extents for CDP-DG in the biosynthesis of phosphatidylinositol, phosphatidylglycerol or diphosphatidylglycerol by rat liver subcellular fractions. In all three biosynthetic pathways dideoxycytidine diphosphate diglyceride was the most active donor of the phosphatidyl unit. The nearly stoichiometric formation of dideoxycytidine-5'-monophosphate during phosphatidylinositol biosynthesis supports the rationale that the antiretroviral liponucleotides may provide cells with a depot form from which the antiviral drug can be released in 5'-monophosphorylated form, thus bypassing the initial phosphorylation of free dideoxynucleosides.

Animals↗

PubMed1987

Analysis of phospholipid molecular species in rat lung as dinitrobenzoate diglycerides by electron capture negative chemical ionization mass spectrometry.

The use of electron capture negative ion desorption chemical ionization mass spectrometry was demonstrated in the analysis of phospholipid molecular species at the 1,3-dinitrobenzoate (DNB) diglyceride derivative. Modification of phosphatidylcholine (PC) or phosphatidylethanolamine (PE) by phospholipase C treatment and acylation of the resultant diglyceride with 3,5-dinitrobenzoylchloride afforded separation of the alkylacyl, alkenylacyl, and diacyl dinitrobenzoate subclasses by thin-layer chromatography (TLC). Separation of alkylacyl DNB into individual molecular species by reverse-phase high-performance liquid chromatography (RPHPLC) was demonstrated. Electron capture desorption chemical ionization of individual molecular species (10-25 ng) from a direct probe yielded a mass spectrum characterized by an intense molecular anion. This molecular anion was the base peak of the spectrum accounting for greater than 80% of the total ionization. From this molecular anion the total carbon number and degree of unsaturation of the fatty chains could be determined. Analysis of fatty acid content of the molecular species allowed unequivocal assignment of structure for the alkyl ether phospholipids. Using selected ion monitoring as little as 0.5 pmol of these species could be detected with a signal-to-noise ratio greater than or equal to 3. This technique was useful in the analysis of low picomolar amounts of molecular species of ether phospholipids in the rat lung. Given an appropriate internal standard, analysis of dynamic changes in turnover, metabolism and precursor product relationships could be undertaken.

Animals↗

PubMed1976

Partial purification and properties of diglyceride kinase from Escherichia coli.

Diglyceride kinase (diacylglycerol kinase, E.C. 2.7.1.-), an enzyme localized in the inner membrane of Escherichia coli, has been purified about 600-fold. The purified enzyme exhibits an absolute requirement for magnesium ion; its activity toward both lipid and nucleotide substrates is stimulated by diphosphatidylglycerol or other phospholipids. Adenine nucleotides are much better substrates for the enzyme than are other purine or pyrimidine nucleotides. The purified enzyme preparation catalyzes the phosphorylation of a number of lipids, including ceramide and several ceramide and diacylglycerol-like analogs. The broad lipid substrate specificity of diglyceride kinase suggests that this enzyme may function in vivo for the phosphorylation of an acceptor other than diacylglycerol.

Cell Membrane↗

PubMed1990

Phorbol-12-myristate-13-acetate activation of phospholipase D in human neutrophils leads to the production of phosphatides and diglycerides.

The contribution of phospholipase D (PLD) to the production of phosphatides (PA) and diglycerides (DG) in phorbol-12-myristate-13-acetate (PMA)-stimulated human neutrophils was studied. Neutrophils were double labeled with 1-O-[3H]alkyl-phosphatidylcholine [( 3H]alkyl-PC) and alkyl-[32P]PC. Upon stimulation with PMA, these cells produced 1-O-alkyl-PA (alkyl-PA) and, in the presence of ethanol, 1-O-alkyl-phosphatidylethanol (alkyl-PEt) both containing 3H and 32P. Lagging behind alkyl-PA and alkyl-PEt formation was the production of 1-O-[3H]alkyl-diglyceride [( 3H]alkyl-DG) and [32P]orthophosphate [( 32P]PO4), suggesting dephosphorylation of alkyl-PA by PA phosphohydrolase (PPH). Furthermore, the PPH inhibitor, propranolol, inhibited the formation of both [3H]alkyl-DG and [32P]PO4, while increasing alkyl-PA levels (containing both 3H and 32P). PMA-induced DG mass accumulation was also inhibited by propranolol. The results of this study demonstrate that PMA activates PLD in neutrophils leading to the generation of PA and that the bulk of the DG mass accumulation is derived from the sequential actions of PLD and PPH on PC.

Diglycerides↗

PubMed1990

Interleukin-1 stimulates diglyceride accumulation in the absence of protein kinase C activation.

Despite advances in the knowledge of the intracellular signalling in response to extracellular messengers, the mechanism of action of interleukin-1 (IL-1) has remained an enigma. In the present study, we have employed human dermal fibroblasts (Detroit 532 cells) to investigate IL-1 beta-induced changes in intracellular signals. Both recombinant human IL-1 beta and a native preparation purified from human placental tissue were employed. Cyclic AMP levels in cell monolayers were unaltered by IL-1 beta. Also, IL-1 beta did not influence significantly the levels of phosphatidylinositol, phosphatidylinositol 4-monophosphate, and phosphatidylinositol 4,5-bisphosphate in the membrane, nor the water-soluble inositol phosphates, inositol monophosphate, inositol bisphosphate and inositol trisphosphate, in cells prelabelled with myo-[3H]inositol. In addition, intracellular calcium as measured by Quin2 was unaffected by interleukin-1. However, in cells labelled with [3H]glycerol or [3H]arachidonic acid, IL-1 beta caused an immediate rise in diglyceride (DG) accumulation. As the effects of IL-1 beta have been reported to be mimicked by tumour-promoting phorbol esters, this rise in DG suggested the involvement of protein kinase C (PKC). However, repeated experiments failed to reveal any acute effect of IL-1 beta on the activity of this enzyme. Furthermore, IL-1 beta did not cause the translocation of PKC between the membrane and the cytosol as has been found in response to other extracellular signals. Rather, IL-1 beta appeared to increase the synthesis of PKC in both membrane and cytosol preparations, an effect which could be prevented by coincubation with cycloheximide. These findings suggest that the diglyceride formed in response to IL-1 beta does not activate protein kinase C.

Calcium↗

PubMed1997

In vivo and in vitro studies on the stereoselective hydrolysis of tri- and diglycerides by gastric and pancreatic lipases.

The stereoselectivity of dog gastric and dog pancreatic lipases was investigated both in vitro, under simulated physiological conditions, and in vivo, during the digestion of a liquid test meal. In vitro it was observed that although both lipases had a stereopreference for the sn-3 position in triglycerides, it was about three times higher in the case of the gastric lipase. On the other hand, both lipases clearly showed a comparable enantioselectivity for the sn-1 position when a racemic diolein was used as the substrate. In the case of pancreatic lipase, the enantiomeric excess of 1,2-sn-diolein generated in vitro by the hydrolysis of triolein was found to decrease significantly, and even to be slightly reversed, at high rates of hydrolysis (above 50%) due to the further stereoselective hydrolysis of diglycerides into monoglycerides. This finding may explain the low enantiomeric excess of the diglycerides observed in vivo during the early phase of intraduodenal digestion when pancreatic lipase plays a predominant role and the rate of triolein hydrolysis is already high. On the other hand, a large enantiomeric excess of 1,2-sn-diolein generated from triolein was always the fingerprint of the gastric lipase in vitro even at high hydrolysis rates. This fingerprinting of gastric lipase was observed during both the intragastric phase and the late intestinal phase of lipolysis. This feature was therefore taken as an index to determine the respective roles of gastric and pancreatic lipases during in vivo lipolysis. To the best of our knowledge, this is the first time that stereoselectivity has been used as a tool to discriminate between the activities of two enzymes hydrolyzing the same substrate in vivo.

Animals↗

PubMed1994

Synthesis and in vitro study of a diglyceride prodrug of a peptide.

A diglyceride derivative of a pentapeptide renin inhibitor, the 1,3-dipalmitoyl-[Iva-Phe-Nle-Sta-Ala-Sta-acetyl]-glycerol was synthesized and tested in vitro as a potential prodrug for oral administration. The ability of the diglyceride analog to inhibit the renin activity was equivalent to that of the parent peptide after predigestion with pancreatic lipase. Furthermore, the presence of the palmitoyl groups was found to induce, in vitro, an efficient protection of the peptide from gastric and intestinal hydrolysis. During incubation with intestinal and gastric fluids, and with alpha-chymotrypsin and pancreatic lipase, the glycerolipidic derivative was more stable than the peptide alone. These results support the use of glycerolipidic prodrug for oral administration of peptides.

Amino Acid Sequence↗

PubMed1989

Effects of mezerein and diglycerides on the PRL stimulation of cell replication in Nb2 node lymphoma cells.

These studies provide further support for the thesis that the activation of protein kinase C is likely involved in the prolactin (PRL) stimulation of mitogenesis in the Nb2 node lymphoma cell line. The diterpene mezerein is shown to potentiate the mitogenic effect of PRL at a hormone concentration which elicits a less than maximum response. A similar response was observed with two diglycerides, diolein and dicaprin. Neither mezerein nor the diglycerides affected the magnitude of response to a maximum stimulatory concentration of PRL.

Carcinogens↗

PubMed1975

Biosynthesis of digalactosyl diglyceride in Vicia faba leaves.

Developing and mature leaf tissue from Vicia faba plants were pulse-fed 14-CO2. The lipids were extracted at intervals after exposure to light, and the galactolipids monogalactosyl diglyceride (MGDG) and digalactosyl diglyceride (DGDG) were separated. After methylation and methanolysis, gas-liquid chromatography was used to separate the two galactose units of DGDG and the galactose of MGDG. The specific activities of the galactoses and the changes over the time period of the experiment were determined. The results support the view that DGDG is formed by galactosylation of MGDG. This does not take place by a rapid two-enzyme system reaction but more slowly in two phases: galactosylation of a pool of newly formed MGDG and a more random galactosylation of MGDG. There is no evidence of a high turnover of galactose in these lipids.

Carbon Dioxide↗

PubMed1977

CDP-diglyceride:inositol transferase from rat liver. Purification and properties.

CDP-diglyceride:inositol transferase, which catalyzes the final step of the de novo synthesis of phosphatidylinositol, was solubilized by sodium cholate from microsomes prepared from rat liver and purified by ammonium sulfate fractionation, sucrose density gradient centrifugation, and DEAE-cellulose column chromatography. Addition of phospholipid during the purification and the assay procedures prevented irreversible loss of the enzyme activity to some extent. The resulting preparation was nearly homogeneous as judged by polyacrylamide gel electrophoresis. The recovery of the purified enzyme from the microsomal fraction was 3 to 3.3% with respect to activity and 0.12% with respect to amount of protein. The molecular weight of the enzyme was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 60,000. The purified enzyme required exogenous phospholipds for its activity. Various phospholipid classes activated the enzyme rather nonspecifically. The Km for myo-inositol was 2.5 X 10(-3) M and that for CDP-diglyceride was 1.7 X 10(-4) M. The pH optimum was 8.6. The enzyme required Mm2+ or Mg2+ for activity. The optimal concentration of Mn2+ for activation was 0.5 mM, while the activity in the presence of Mg2+ increased up to 20 mM. The enzyme was inhibited by thiol-reactive reagents. There was a competition for inositol by inosose-2 but not by scyllitol.

Animals↗

PubMed1990

Complement C5a activation of phospholipase D in human neutrophils. A major route to the production of phosphatidates and diglycerides.

The contribution of phospholipase D (PLD) to the production of phosphatidic acid (PA) and diglyceride (DG) by C5a-stimulated human neutrophils has been studied. Membrane-associated 1-O-alkyl-phosphatidylcholine (alkyl-PC) was double labeled with 3H and 32P by incubating neutrophils with [3H]alkyl-lysoPC and alkyl-[32P]lysoPC. Upon stimulation with recombinant C5a, these labeled neutrophils produce 1-O-alkyl-phosphatidic acid (alkyl-PA) and, in the presence of ethanol, 1-O-alkyl-phosphatidyl-ethanol (alkyl-PEt), containing both 3H and 32P. Formation of radiolabeled alkyl-PEt parallels that of radiolabeled alkyl-PA and requires both extracellular Ca2+ and cytochalasin B. Furthermore, the 3H/32P ratios of alkyl-PA and alkyl-PEt formed during stimulation are very similar to that of th substrate alkyl-PC. These results demonstrate that, in C5a-stimulated neutrophils, alkyl-PA and alkyl-PEt are formed from alkyl-PC almost exclusively by PLD-catalyzed hydrolysis and transphosphatidylation, respectively. Upon C5a stimulation, neutrophils labeled with 3H and 32P also produce 1-O-[3H]alkyl-diglyceride [( 3H]alkyl-DG) and [32P]orthophosphate [( 32P]PO4), but not [32P]phosphocholine. [3H]Alkyl-DG and [32P]PO4 are formed in parallel, although temporally lagging behind alkyl-PA. Propranolol, a PA phosphohydrolase (PPH) inhibitor, decreases the formation of both [3H]alkyl-DG and [32P]PO4, although increasing alkyl-PA accumulation. These data support the conclusion that alkyl-DG is formed from alkyl-PC by the combined activities of PLD and PPH and not by phospholipase C (PLC). Furthermore, by using [3H]acyl-PC-labeled neutrophils, it is demonstrated that, like alkyl-PC, 1-acyl-PC is also degraded sequentially by PLD and PPH to 1-acyl-DG. Propranolol does not inhibit phosphoinositide-specific PLC and yet it causes almost complete inhibition of the total DG mass accumulation in C5a-stimulated neutrophils. We conclude that, in cytochalasin B-treated neutrophils stimulated with C5a, PLD-catalyzed hydrolysis of PC determines the levels of both PA and DG with potentially important ramifications for neutrophil-mediated defense functions.

Alkylation↗