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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↗
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↗
PubMed1988

Phorbol myristate acetate (PMA) augments chemoattractant-induced diglyceride generation in human neutrophils but inhibits phosphoinositide hydrolysis. Implications for the mechanism of PMA priming of the respiratory burst.

Pretreatment ("priming") of neutrophils with a non-activating concentration (2 nM) of phorbol myristate acetate (PMA) augments superoxide (O2-) production in response to the chemoattractant formylmethionylleucylphenylalanine (fMLP). We initially examined the effect of sphinganine, an inhibitor of protein kinase C (Ca2+/phospholipid-dependent enzyme), on activation of primed neutrophils. In both primed and unprimed cells activation by fMLP was blocked, and inhibition occurred at identical concentrations, supporting a common inhibited site. PMA also augmented (about 2-fold) fMLP-induced generation of sn-1,2-diglyceride (DG), the level of which correlated with O2- generation. In contrast to its effects on DG, PMA diminished by about 50% the magnitude of the fMLP-stimulated rise in cytosolic Ca2+. Thus, PMA priming dissociates the fMLP-stimulated Ca2+ increase from DG and O2- generation. The effect of PMA on Ca2+ levels appeared to be due in part to lowered levels of inositol trisphosphate. Lowering of inositol phosphate levels correlated with inhibition of fMLP-induced hydrolysis of inositol-containing phospholipids, particularly phosphatidylinositol 4,5-bisphosphate. PMA did not inhibit (and in fact augmented at early time points) formation of [32P] phosphatidic acid in response to fMLP, indicating that the increase in DG was not due to inhibition of cellular diglyceride kinase. Thus, the data suggest that PMA enhances fMLP-stimulated DG generation concomitant with switching the source of DG from phosphatidylinositol 4,5-bisphosphate to an alternative lipid(s). Increased DG and inhibition of activation by sphinganine are consistent with a role for protein kinase C in activation of the respiratory burst in PMA-primed neutrophils.

Diglycerides↗
PubMedDate not supplied

Simple method for derivatization of monoglycerides and diglycerides.

A method has been developed for derivatization of monoglycerides and diglycerides to their propyl esters by using propionic anhydride in the presence of pyridine. The derivatives are volatile and thus can be separated and identified by gas chromatography (GC). Completion of the reaction is monitored by thin-layer chromatography. Monoglyceride propyl esters were positively identified by gas chromatography and by gas chromatography/mass spectrometry. No side products were observed in the derivatization reaction which requires 35 min at 75 degrees C. GC analysis of the mono- and diglyceride propyl esters can be completed in approximately 30 min.

Chemical Phenomena↗
PubMed1981

Gas-liquid chromatographic detection an determination of diacetyl tartaric acid ester of diglyceride in dairy and nondairy coffee cream powders.

Diacetyl tartaric acid ester of diglyceride was directly extracted from dairy or nondairy coffee cream powder under acidic conditions with ethyl acetate; then the extract was saponified with methanolic potash. After acidification with HCl, free fatty acid was removed with ether and the reaction mixture was absorbed on an anion exchange column. Tartaric acid was eluted with 2N HCl-acetone (1+1). An aliquot of the trimethylsilyl (TMS) derivative of the eluate was injected into a gas chromatograph with flame ionization detection, and a 1.5% SE-30 column. Recoveries of diacetyl tartaric acid ester of diglyceride at 50, 200, and 2000 ppm were 85.6-99.5%.

Chromatography, Gas↗
PubMed1990

Synthetic sialyl glycolipids (sialo-cholesterol and sialo-diglyceride) induce granulocytic differentiation of human myelogenous leukemia cell line HL-60.

When HL-60 cells were cultivated with synthetic sialyl glycolipids, sialo-cholesterol and sialo-diglyceride, the cells were found to be differentiated into mature granulocytes on morphological and functional criteria. The differentiation of cells was accompanied by inhibition of cell proliferation. The differentiation-inducing activity of sialo-cholesterol was greater than that of sialo-diglyceride on a molar basis, and the alpha-anomer of each compound was more potent than the beta-anomer, suggesting that the stereospecific structure of the compounds is important for the differentiation-inducing activity.

Cell Transformation, Neoplastic↗
PubMed1972

1-(O- -glucosaminyl)-2,3-diglyceride in Bacillus megaterium.

1. A lipid that contains glucosamine but not phosphorus has been isolated from Bacillus megaterium. It constitutes about 5% of the total lipid glucosaminide in this organism and can be distinguished chromatographically from 2'-(O-beta-glucosaminyl)phosphatidylglycerol and 3'-(O-beta-glucosaminyl)phosphatidylglycerol, which are also present. 2. The lipid contains glycerol, fatty acids and glucosamine in the molar proportion 1:2:1. The fatty acids are bound by an ester linkage and are similar to those found in other lipids of this organism. Partial acid hydrolysis or alkaline hydrolysis of the lipid yields 1-(O-beta-glucosaminyl)glycerol and degradation with nitrite yields 2,5-anhydromannose and diglyceride. 3. The lipid has been identified as 1-(O-beta-glucosaminyl)-2,3-diglyceride.

Bacillus megaterium↗
PubMed1970

Metabolism of the glycosyl diglycerides and phosphatidylglucose of Staphylococcus aureus.

A glucose containing lipid, phosphatidylglucose (probably 3-sn-phosphatidyl-1'-glucose) and a lipid tentatively identified as phosphatidylethanolamine have been characterized in the lipids of Staphylococcus aureus. These lipids together comprise less than 2% of the total phospholipids of exponentially growing S. aureus and accumulate to 14% of the total phospholipid in stationary-phase cells. These lipids lost no (32)P when cells grown with H(3) (32)PO(4) were transferred to nonradioactive medium during the exponential growth phase. This was in marked contrast to the other phospholipids which lost (32)P rapidly. The loss of (32)P from phosphatidic acid and cardiolipin in exponentially growing cells was biphasic, suggesting heterogeneity of phospholipid phosphate metabolism. The mono- and diglucosyl diglycerides showed a rapid loss of (14)C-glucose during growth in nonradioactive medium but no loss of (14)C from the fatty acids of these lipids. The (14)C in the glucose and fatty acids of the glucosyl diglycerides was derived from glucose.

Autoradiography↗
PubMed1970

Biosynthesis of cytidine diphosphate diglyceride by human platelets.

Homogenates of human platelets contain an enzyme which catalyzes the formation of cytidine diphosphate diglyceride from cytidine triphosphate and phosphatidic acid. The enzymatic activity could not be dissociated from platelet particles and the greatest specific activity was found in the membrane fraction. The K(m) for cytidine triphosphate was 0.16 mmole/liter and the apparent K(m) for phosphatidic acid was 6.2 mmoles/liter. The pH optimum was 7.0 and the most effective buffers were triethanolamine-HCl and Tris-HCl. The reaction was dependent on the presence of divalent cations, magnesium being the most effective of those investigated. Monovalent cations did not alter the reaction rate. Evidence is presented that the cytidine diphosphate diglyceride produced can serve as a precursor for the synthesis of phosphatidylinositol. No difference was found in the enzymatic activity in platelets from normal subjects and from patients with diseases known to interfere with platelet thromboplastic function.

Blood Platelet Disorders↗
PubMed1992

In vitro and in vivo evaluation of macrofilaricidal activity of GABA and 1,3-dipalmitoyl-2-(4-aminobutyryl)glycerol HCl: a diglyceride prodrug.

A new therapeutic target has been identified from the filaria Molinema dessetae: the gabaergic system. gamma-aminobutyric acid (GABA) itself showed antifilarial effect in vitro and a macrofilaricidal action in vivo at high dose by intraperitoneal route (10(-2) M). Nevertheless, no action was observed by oral route. The study we report here consists to obtain an antifilarial effect by oral route using a diglyceride prodrug. Such a strategy is based on the triglycerides metabolism. A diglyceride prodrug of gamma-aminobutyric acid has been synthesized and its filaricidal activity compared with that of GABA, in vitro on adults of Molinema dessetae and in vivo on Molinema dessetae infected Proechimys oris. In vitro, GABA at 2.5 x 10(-3) M induced a temporary paralysis and the ester drug at the same concentration was fully active on adults. In vivo, no significant activity was observed by oral administration of a daily dose of GABA (10(-2) M). A five day course of GABA at 10(-2) M via the intraperitoneal route induced a significant reduction of male and female worms. We did not find any activity of the prodrug in vivo, either by the oral route (10(-2) M) or after an intraperitoneal administration (10(-3) M). The interest of GABA and GABA derivatives as potential filaricidal drugs was discussed.

Animals↗