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PubMed1994

Epidermal growth factor induces the production of biologically distinguishable diglyceride species from phosphatidylinositol and phosphatidylcholine via the independent activation of type C and type D phospholipases.

An early response to epidermal growth factor in A431 cells is the generation of diglyceride, a physiological activator of protein kinase C. By differentially prelabeling cellular phospholipids with [3H]arachidonate and [3H]myristate, which are incorporated primarily into phosphatidylinositol and phosphatidylcholine, respectively, we have found that epidermal growth factor induces an increase in diglyceride levels from both phosphatidylinositol and phosphatidylcholine via distinct mechanisms and kinetics. The epidermal growth factor-induced increase in phosphatidylinositol-derived diglyceride was transient and peaked at 5 min. As diglyceride levels dropped, there was a corresponding increase in phosphatidic acid, suggesting that the diglyceride is efficiently converted to phosphatidic acid by a diglyceride kinase. In contrast, epidermal growth factor-induced increases in phosphatidylcholine-derived diglyceride peaked at 30 min and remained elevated for greater than 2 h. The epidermal growth factor-induced increases in phosphatidic acid detected in [3H]myristate-prelabeled cells paralleled the increase in diglyceride, suggesting that the phosphatidylcholine-derived diglyceride is produced from phosphatidic acid via a phosphatidic acid phosphatase. Consistent with this hypothesis, epidermal growth factor also induced a protein kinase C-independent phospholipase D activity that was specific for phosphatidylcholine. These data suggest that epidermal growth factor induces diglyceride production from phosphatidylinositol and phosphatidylcholine via two distinct mechanisms: a rapid and transient induction of diglyceride that likely involves phospholipase c-gamma-mediated hydrolysis of phosphatidylinositol-4,5-bisphosphate and a slower, more sustained induction of diglyceride via a phospholipase D-mediated hydrolysis of phosphatidylcholine to produce phosphatidic acid, which is then converted to diglyceride by a phosphatidic acid phosphatase.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaloids↗
PubMed1992

Complement receptor-mediated phagocytosis is associated with accumulation of phosphatidylcholine-derived diglyceride in human neutrophils. Involvement of phospholipase D and direct evidence for a positive feedback signal of protein kinase.

Complement receptor (CR)-mediated phagocytosis is associated with an increased accumulation of diglyceride (sn-1,2-diacylglycerol and/or 1-O-alkyl-2-acyl-glycerol) in human neutrophils. The C3bi-mediated increase in diglyceride (5-20 min) was only partially impaired when phosphoinositide-specific phospholipase C (PLC) activity was abolished by reduction of cytosolic free Ca2+. At an early time point (1 min), however, diglyceride production was barely detectable in control cells, whereas production was considerable in cells with a reduced cytosolic free Ca2+ concentration. C3bi stimulation of 32P-labeled neutrophils caused a rapid and significant breakdown of [32P]phosphatidylcholine (PC) which was not affected by inhibition of Ca(2+)-dependent phosphoinositide-specific PLC. Thus, PC hydrolysis could be involved in C3bi-induced diglyceride formation. Stimulation of cells labeled with [3H]1-O-alkyl-lyso-PC ([3H]alkyl-lyso-PC), resulted in an increased formation of [3H]1-O-alkyl-phosphatidic acid ([3H]alkyl-PA) and a later and slower formation of [3H]1-O-alkyl-diglyceride ([3H]alkyl-diglyceride); this suggests activation of phospholipase D (PLD). When these labeled cells were stimulated in the presence of 0.5% ethanol a marked accumulation of [3H]1-O-alkyl-phosphatidylethanol ([3H]alkyl-PEt) was observed in both controls and calcium-reduced cells, further strengthening the suggested involvement of PLD activity. In parallel with the sustained increase in diglyceride formation, CR-mediated phagocytosis was also associated with phosphorylation of a cellular protein kinase C substrate (MARCKS). Therefore it seems reasonable to suggest a causal relationship between C3bi-induced PLD activation, which results in diglyceride formation, and activation of protein kinase C. In electropermeabilized cells which were incapable of ingesting particles, C3bi particles were still able to activate PLD and induce formation of diglyceride. This signaling event must therefore be triggered by binding of particles to the cell and not by the engulfment process. Most importantly, introduction of the protein kinase C inhibitor peptides, PKC(19-36) and PKC(19-31), into these permeabilized cells resulted in a clear reduction of the C3bi-induced production of diglyceride, indicating that CR-mediated activation of protein kinase C directly triggers a positive feedback mechanism for additional diglyceride formation. Taken together, these data further clarify the mechanisms of CR-mediated diglyceride formation and give added support to the concept that protein kinase C plays an important role in the phagocytic process.

Blotting, Western↗
PubMed1976

Partial purification and characterization of cytidine 5'-diphosphate-diglyceride hydrolase from membranes of Escherichia coli.

Cytidine 5'-diphosphate (CDP)-diglyceride is hydrolyzed to phosphatidic acid and cytidine 5'-monophosphate by a specific membrane-bound enzyme in cell-free extracts of Escherichia coli. The hydrolase can be extracted from the particulate fraction with Triton X-100 and purified 1,000-fold in the presence of this detergent. Several nucleoside disphosphate diglycerides were synthesized to determine the substrate specificity of the hydrolase. CDP-diglyceride was hydrolyzed preferentially, although uridine 5'-diphosphate-diglyceride, guanosine 5'-diphosphate-diglyceride, and adenosine 5'-diphosphate (ADP)-diglyceride were also slowly hydrolyzed. Surprisingly, the purified enzyme did not catalyze detectable cleavage of deoxy-CDP (dCDP)-diglyceride. The liponucleotide pool of E. coli contains dCDP-diglyceride and CDP-diglyceride in approximately equal amounts (Raetz and Kennedy, 1973). Water-soluble nucleoside pyrophosphates, such as CDP-choline, nicotinamide adenine dinucleotide, or adenosine 5'-triphosphate are not attacked by this specific hydrolase. Hydrolysis of CDP-diglyceride is strongly inhibited by adenosine 5'-monophosphate and by ADP-diglyceride.

Cell Membrane↗
PubMed1984

Isolation and characterization of Escherichia coli strains defective in CDP-diglyceride hydrolase.

CDP-diglyceride, an obligatory intermediate in the biosynthesis of the glycerophospholipids in Escherichia coli, is cleaved in vitro to phosphatidic acid and CMP by a membrane-bound hydrolase. Previous work from our laboratory (Bulawa, C.E., Hermes, J.D., and Raetz, C. R. H. (1983) J. Biol. Chem. 258, 14974-14980) has demonstrated that this enzyme also catalyzes the transfer of CMP from CDP-diglyceride to phosphate and numerous phosphomonoesters. We now report the isolation of E. coli mutants which are defective in CDP-diglyceride hydrolase. These mutations, designated cdh, map at minute 88 between pfkA and tpi. This information permitted the identification of a ColE1 hybrid plasmid, pLC16-4, which causes the overproduction of hydrolase activity. The isolation of deletion and Tn10 insertion mutants at cdh suggests that the hydrolase is nonessential for cell growth. Hydrolase mutants are defective in both CDP-diglyceride hydrolysis and CDP-diglyceride-dependent cytidylylation, indicating that both activities are encoded by the cdh gene. Although previously described as a ribospecific enzyme, we have found that incubation of the partially purified hydrolase with [alpha-32P]dCDP-diglyceride and phosphate yields two products, [32P]dCMP and [alpha-32P]dCDP. That a single enzyme utilizes both CDP- and dCDP-diglyceride is demonstrated by the following. (i) The hydrolysis of [alpha-32P]CDP-diglyceride is inhibited by nonradioactive dCDP-diglyceride and vice versa. (ii) Utilization of both liponucleotides is inhibited by AMP. (iii) Mutants in the cdh gene are defective in both CDP- and dCDP-diglyceride hydrolysis, while cdh clones overproduce both activities. (iv) Hydrolase mutants accumulate both CDP- and dCDP-diglyceride.

CDPdiacylglycerol-Serine O-Phosphatidyltransferase↗
PubMed1990

Kinetic and molecular species analyses of mitogen-induced increases in diglycerides: evidence for stimulated hydrolysis of phosphoinositides and phosphatidylcholine.

A wide variety of agonist-induced events appear to be mediated through an increase in cellular diglyceride levels. With regard to the ability of diglycerides to mediate these events, three important parameters must be considered: a) the kinetics of diglyceride generation, b) the absolute mass levels, and c) their molecular species. While this increase is often due to a stimulated hydrolysis of phosphoinositides, there is increasing evidence that the stimulated hydrolysis of phosphatidylcholine also contributes to agonist-induced increases in diglyceride levels. The kinetics of mass increases in diglyceride levels stimulated in cultured fibroblasts are agonist-dependent. High concentrations of alpha-thrombin stimulate a biphasic increase in diglyceride levels with the first phase peaking at 15 s and the second phase peaking at 5 min. In contrast, stimulation with epidermal growth factor, or platelet-derived growth factor, results in a monophasic increase in cellular diglyceride levels. Furthermore, the molecular species and phospholipid source of the stimulated diglycerides are also agonist-dependent. While the hydrolysis of phosphoinositides is major source of diglycerides initially generated in response to some agonists (15 s with alpha-thrombin at 500 ng/ml), phosphatidylcholine is hydrolyzed as well. Following longer incubations, or at all times following stimulation by epidermal growth factor or platelet-derived growth factor, phosphatidylcholine hydrolysis is the principal source of the stimulated diglycerides.

Cells, Cultured↗
PubMed1995

Interleukin-1 and endothelin stimulate distinct species of diglycerides that differentially regulate protein kinase C in mesangial cells.

Diglycerides are phospholipid-derived second messengers that serve as cofactors for protein kinase C activation. We have previously shown that, in rat glomerular mesangial cells, the cytokine, interleukin-1 alpha, and the vasoactive peptide, endothelin, generate diglycerides from unique phospholipid precursors. However, neither the molecular species of these diglycerides nor their biological actions were determined. It is now hypothesized that interleukin-1- and endothelin-treated mesangial cells form distinct molecular species of diglycerides which may serve different roles as intracellular signaling molecules. Diglyceride molecular species were resolved and quantified by TLC and high performance liquid chromatography as diglyceride-[14C]acetate derivatives. Endothelin stimulates predominantly ester-linked species (diacylglycerols) in contrast to interleukin-1 which stimulates only ether-linked species (alkyl, acyl- and alkenyl,acylglycerols). In support of these data, interleukin-1-treated mesangial cells hydrolyze ethanolamine plasmalogens, vinyl ether-linked phospholipids. It has been reported that ether-linked, in contrast to ester-linked, diglyceride species do not activate protein kinase C activity. Thus, we next assessed membrane protein kinase C activity in endothelin- or interleukin-1-treated mesangial cells. Even though interleukin-1 has no effect upon basal protein kinase C activity, this cytokine, through the formation of ether-linked diglyceride second messengers, inhibits endothelin, platelet-activating factor, or arginine vasopressin-stimulated protein kinase C activity. We further demonstrate that ester-linked diacylglycerols but not alkyl,acyl- or alkenyl,acylglycerols substitute for phorbol esters in a cell-free protein kinase C assay. In addition, alkenyl,acylglycerols inhibit diacylglycerol-stimulated immunoprecipitated protein kinase C alpha activity in vitro and total protein kinase C activity in permeabilized mesangial cells ex vivo. Taken together, these data suggest that interleukin-1-induced formation of ether-linked diglycerides may physiologically serve to down-regulate receptor-mediated protein kinase C activity and that individual molecular species of diglycerides may serve different roles as intracellular signaling molecules.

Acetates↗
PubMed1976

Cytidine diphosphate diglyceride of bovine brain. Positional distribution of fatty acids and analysis of major molecular species.

A method is described for the isolation of CDP-diglyceride from bovine brain. Yields of the product ranged from 9.2-15.5 mumol per kilogram of tissue, which corresponds to about 1% of the level of phosphatidic acid. Mild alkaline hydrolysis of the product gave three water-soluble phosphate esters which had the same electrophoretic mobilities as CMP, CDP-glycerol and glycerol 3-phosphate. The liponucleotide was quantitatively hydrolysed by CDP-diglyceride hydrolase from Escherichia coli to phosphatidic acid and CMP. No dCMP was recovered in enzymatic or alkaline hydrolysates and it is concluded there can be little or no dCDP-diglyceride in bovine brain. Brain CDP-diglyceride was similar to phosphatidylinositol in that in both lipids stearate was the major saturated fatty acid and arachidonate the most abundant unsaturated fatty acid. This differed significantly from the fatty acid patterns of other metabolically related phospholipids, phosphatidic acid and cardiolipin. Brain CDP-diglyceride was hydrolysed with phospholipase C from Clostridium welchii with the liberation of the diglyceride moiety in high yield. Treatment of the diglyceride with pancreatic lipase showed CDP-diglyceride with the asymmetric distribution of fatty acids characteristic of most mammalian phospholipids, saturated fatty acids being found mostly at position 1 and polyunsaturated fatty acids at position 2. The derived diglyceride acetates were separated into different molecular species by argentation thin-layer chromatography. These analyses showed that 1-stearoyl, 2-arachidonoyl was the major species of brain CDP-diglyceride.

Animals↗
PubMed1976

Biosynthesis of molecular species of CDP-diglyceride from endogenously-labeled phosphatidate in rat liver microsomes.

The biosynthesis of [14C] CDP-diglyceride was studied using rat liver microsomes which were endogenously labeled with [14C] phosphatidic acid by preincubation of unlabeled microsomes with sn-[14C] glycerol-3-phosphate and appropriate cofactors. The formation of CDP-diglyceride from radioactive phosphatidate showed an absolute requirement for CTP and MgCl2. The newly formed [14C] CDP-diglyceride was characterized by thin layer chromatography (TLC), isotopic labeling from radioactive CTP, and its ability to serve as substrate for the microsomal enzyme, CDP-diglyceride: inositol phosphatidyltransferase. The distributions of radioactive glycerol-3-phosphate among the various chemical classes of microsomal [14C] phosphatidate and [14C] CDP-diglyceride were determined following argentation TLC of their 1,2-diglyceride acetate derivatives. Most of the radioactivity among the phosphatidic acids was present in the monoenoic (36%) and dienoic (33%) molecular species, whereas 10, 8, 4, and 8% were associated with the saturates, trienes, tetraenes, and polyenes, respectively. Similar distributions of radioactivity were found among the corresponding classes of newly formed CDP-diglyceride. Only a slight enrichment of radioactivity in the tetraenoic CDP-diglyceride was found relative to the corresponding phosphatidates. Therefore, under the conditions of study, the microsomal CTP: phosphatidate cytidylyltransferase produces mainly monoenoic and dienoic species of CDP-diglyceride and shows little specificity towards different molecular species of phosphatidic acids. The present results suggest also that the arachidonoyl phosphatidate derived from the microsomal acylation of sn-glycerol-3-phosphate is not likely the major source of arachidonic acid in liver phosphatidylinositol.

Animals↗
PubMed1987

Comparison of cytidinediphospho-sn-1,2-diglyceride transfer from microsomal and liposomal to mitochondrial membranes.

Transfer of [3H]CDP-diglycerides from isolated guinea pig liver microsomal and liposomal membranes to guinea pig mitochondrial membranes was studied by incubating microsomal or liposomal membranes carrying [3H]CDP-diglycerides with mitochondrial membranes and determining the CDP-diglyceride-dependent incorporation of sn-3-[14C]glycerolphosphate into mitochondrial [14C]polyglycerophosphatides. A significant difference in the amount of transferred [3H]CDP-diglycerides and the composition of mitochondrial [14C]polyglycerophosphatides was found depending on whether [3H]CDP-diglycerides were transferred from microsomal or liposomal membranes. This amount was around 12% when [3H]CDP-diglycerides were transferred from the microsomal membranes and around 4.6% when they were transferred from the liposomal membranes. Furthermore, about 60% of [14C]phosphatidylglycerol and 35% of [14C]phosphatidylglycerophosphate were found in the microsomes-mitochondria system and about 9% of [14C]phosphatidylglycerol and 79% of [14C]phosphatidylglycerophosphate were found in the liposomes-mitochondria system, establishing an important role for the membrane donor in the transfer of [3H]CDP-diglycerides to mitochondria. Furthermore, if the transfer of [3H]CDP-diglycerides from the microsomal to the mitochondrial membranes was assayed by the determination of [3H]CDP-diglycerides in reisolated mitochondrial membranes without further incorporation into mitochondrial polyglycerophosphates, it amounted to about 38%.

Animals↗
PubMed1979

Production of diglyceride from phosphatidylinositol in activated human platelets.

Human platelets generate diglyceride within 5 s of exposure to thrombin. Production of diglyceride is transient. 15 s after the addition of thrombin, the levels of diglyceride have increased up to 30-fold, but decrease thereafter. Prior incubation of platelets with 2 mM dibutyryl cyclic AMP prevents both the generation of diglyceride and the secretion of serotonin. Acetylsalicylic acid (100 microgram/ml), which completely inhibits prostaglandin endoperoxide synthesis, does not block diglyceride production and serotonin secretion induced by thrombin. Based on studies examining the incorporation of [3H]arachidonic acid into diglyceride of prelabeled platelets exposed to thrombin, it is concluded that neither phosphatidic acid nor triglyceride is the source of the diglyceride. Phosphatidylinositol appears to be the most likely source, both because its loss of radiolabel is sizable and rapid enough to account for the appearance of radiolabel in diglyceride, and because a phosphatidylinositol-specific phosphodiesterase, described in this report, exists in platelets. The phosphatidylinositol-phosphodiesterase, which produces diglyceride and inositol phosphate, requires Ca+2 and shows optimal activity at pH 7. The enzyme does not act upon phosphatidylcholine, phosphatidylethanolamine, or phosphatidylserine.

Arachidonic Acids↗
PubMedDate not supplied

Localization of the tri- and digalactosyl diglyceride in the thylakoid membrane with serological methods.

Trigalactosyl diglyceride was isolated from leaves of Urtica dioica and characterized by thin layer chromatography, infrared spectroscopy and by its fatty acid composition. An antiserum to the trigalactolipid was obtained by immunization of rabbits. By means of inhibition experiments with oligosaccharides and mono- and digalactosyl glycerol it was demonstrated that the antibodies are directed towards the alpha-galactosyl-(1 leads to 6)-alpha-galactosyl-(1 leads to 6)-beta-galactosyl-(1 leads to 1)-glycerol configuration of the trigalactosyl diglyceride. Monogalactosyl diglyceride and sulfoquinovosyl diglyceride do not react with this antiserum. However, a cross reaction was observed with digalactosyl diglyceride. The presence of antibodies to tri- and digalactosyl diglyceride was demonstrated in antisera to different chloroplast preparations of Antirrhinum majus and Spinacia oleracea. The antiserum to the trigalactolipid agglutinates stroma-freed chloroplasts. Membrane fragments obtained by the ultra sonication were precipitated. The antiserum is exhausted by trigalactosyl diglyceride but not by digalactosyl diglyceride or digalactosyl glycerol. The antiserum treated with digalactosyl glycerol and digalactosyl diglyceride also agglutinated stroma-freed chloroplasts. 1 g stroma-freed chloroplasts binds 0.17 g antibodies to trigalactolipid. Membrane fragments bind more antibodies to trigalactolipids than stroma-freed chloroplasts. From the agglutination tests it follows that the antigenic determinants of the trigalactolipid and the digalactolipid are localized in the outer surface as well as in the surface directed towards the inside of the thylakoid membrane.

Animals↗
PubMed1990

Differential metabolism of diradyl glycerol molecular subclasses and molecular species by rabbit brain diglyceride kinase.

Elevations in the mass of ether-linked diglycerides (i.e. 1-O-alk-1'-enyl-2-acyl-sn-glycerol (AAG) and 1-O-alkyl-2-acyl-sn-glycerol (Alkyl AG)) during cellular activation are prolonged in comparison to their 1,2-diacyl-sn-glycerol (DAG) counterparts. Since the metabolic removal of DAG is determined, in large part, by the rate of its phosphorylation by diglyceride kinase, we quantified differences in the activity of diglyceride kinase utilizing individual subclasses of diradyl glycerols as substrate. Rabbit brain microsomal diglyceride kinase activity was over 30-fold greater utilizing DAG as substrate (25.8 nmol.mg-1.min-1) in comparison to AAG (0.8 nmol.mg-1.min-1). No alterations in the affinity of microsomal diglyceride kinase for ATP were present (Km approximately 0.5 mM) utilizing each diradyl glycerol subclass. Similar subclass specificities for diglyceride kinase (i.e. DAG greater than Alkyl AG much greater than AAG) were present in brain and liver cytosol as well as in liver microsomes utilizing multiple assay conditions. In sharp contrast, Escherichia coli diglyceride kinase phosphorylated DAG, Alkyl AG, or AAG diradyl glycerol molecular subclasses at identical rates. Furthermore, although DAG was rapidly hydrolyzed by diglyceride lipase, catabolism of AAG or Alkyl AG by plasmalogenase, alkyl ether hydrolase, or diglyceride/monoglyceride lipase was undetectable. Collectively, these results demonstrate the importance of the differential catabolism of each diradyl glycerol molecular subclass as a primary determinant of their biologic half-lives. Since individual subclasses of diglycerides have distinct physical properties and physiologic functions, these results underscore the importance of lipid subclass specific metabolism in tailoring individual cellular responses during activation.

Animals↗
PubMed1990

Molecular species analysis of mitogen-stimulated 1,2-diglycerides in fibroblasts. Comparison of alpha-thrombin, epidermal growth factor, and platelet-derived growth factor.

Recent studies have implicated the hydrolysis of phosphoinositides and phosphatidylcholine in agonist-stimulated events. The potent mitogen, alpha-thrombin, stimulates the generation of diglycerides in a biphasic and sustained manner in IIC9 fibroblasts (Wright, T. M., Rangan, L. A., Shin, H. S., and Raben, D. M. (1988) J. Biol. Chem. 263, 9374-9380). Using measurements of radiolabeled headgroup release and molecular species analysis, we previously determined that alpha-thrombin generates diglycerides through the hydrolysis of both the phosphoinositides and phosphatidylcholine at early times (15 s), and at later times (greater than or equal to 5 min) through the hydrolysis of primarily, if not exclusively, phosphatidylcholine (Pessin, M. S., and Raben, D. M. (1989) J. Biol. Chem. 264, 8729-8738). In contrast, IIC9 fibroblasts respond to the mitogenic treatments of (a) alpha-thrombin following chymotrypsin pretreatment or (b) epidermal growth factor by increasing their levels of diglycerides in a monophasic and sustained manner (Wright, T. M., Rangan, L. A., Shin, H. S., and Raben, D. M. (1988) J. Biol. Chem. 263, 9374-9380). In this report, we have analyzed the molecular species of the diglycerides generated by these two different treatments and have also examined the lipid response of IIC9 fibroblasts to platelet-derived growth factor. Based on both the molecular species analyses and the release of radiolabeled head-groups, all three of these different mitogenic treatments generate diglycerides primarily through the stimulation of phosphatidylcholine hydrolysis. However, while similar, the molecular species profiles of the diglycerides generated by these three treatments are not identical to the molecular species profile of total cellular phosphatidylcholine. In addition, the molecular species profiles of the diglycerides generated by these three mitogenic treatments greatly resemble each other, with significant differences between any two profiles occurring in at most one molecular species. This finding differs from that seen with alpha-thrombin stimulation alone, where the molecular species profile of the diglycerides generated following 5 min of alpha-thrombin stimulation is nearly identical to the molecular species profile of total cellular phosphatidylcholine. These data support the possibility of hormone-sensitive phosphatidylcholine pools or selective diglyceride metabolism.

Animals↗
PubMed1988

Structural studies on the diglyceride-mediated activation of protein kinase C.

Diglyceride analogs were studied with respect to their abilities to activate protein kinase C (Ca2+- and phospholipid-dependent protein kinase) in the presence of low calcium and phospholipid. Analogs which lacked either a free hydroxyl group at the 3 position or an ester moiety at the 1 position were without activity. It was concluded that the hydrophilic moieties of the active diglycerides are crucial for activity. However, diglyceride analogs containing additional hydrophilic moieties in one of the acyl side chains did not exhibit enhanced activity when compared to diglycerides containing two fatty acyl groups. Diglyceride analogs with a modified glycerol backbone were also studied. Homologous diglycerides with either one or two methylene groups between the 3-methylene group of the diglyceride and the hydroxyl group possessed markedly reduced activities when compared to the appropriate unmodified diglyceride. Isomers of these homologues which contained either a methyl group at the 1 position, or dimethyl groups incorporated at the 1 and 3 positions, were virtually without activity. Where studied, none of the diglyceride analogs prepared possessed antagonist activity. The results of these experiments are discussed with respect to the extreme specificity observed.

Calcium↗
PubMed1969

Biosynthesis of glucosyl diglycerides by Mycoplasma laidlawii strain B.

Monoglucosyl diglyceride is synthesized from 1,2-diglyceride and uridine-5'-diphosphoglucose (UDP); diglucosyl diglyceride from monoglucosyl diglyceride, and uridine-5'-diphosphoglucose by membranes of Mycoplasma laidlawii strain B. All of these enzymatic activities reside in the membrane. Membranes solubilized by detergent action or succinylation and acetone powders of membranes were inactive. Requirements for Mg(2+), UDP, and appropriate lipid acceptor were demonstrated for biosynthesis of both glycolipids. Glucose-1-phosphate plus uridine triphosphate could replace the UDP requirement. A medium of relatively high ionic strength and a critical concentration of sodium lauryl sulfate stimulated biosynthesis of the monoglucosyl diglyceride. The optimal pH for both reactions was 8.0. A specificity for 1,2-diglyceride from the homologous organism was found for optimal synthesis of the monoglucosyl diglyceride, and a specificity for monoglucosyl diglyceride was found in the case of diglucosyl diglyceride synthesis. Both reactions were specific for UDP.

Buffers↗
PubMed2002

Determination of the diglyceride content in greek virgin olive oils and some commercial olive oils by employing (31)P NMR spectroscopy.

In this study, the diglyceride contents of 96 samples of virgin olive oils from the regions of Crete, Lesvos, Messinia, Pilion, Zakynthos, Halkidiki, and Ilia, 15 samples of commercial extra virgin and pure olive oils, and 3 samples each of refined olive oils and pomace oils were determined by a facile method introduced in a previous publication. This method is based on the phosphitylation of the free hydroxyls of the diglycerides with 2-chloro-4,4,5,5-tetramethyldioxaphospholane and the integration of the appropriate peaks in the (31)P NMR spectra. This preliminary study showed interesting trends in the diglyceride content of the virgin olive oils from the various regions of Greece that can be used as simple criteria to assess the olive oil characteristics. Analysis of variance has been carried out for the diglyceride content of each region in an attempt to detect possible differences in the diglyceride levels among the various regions. Finally, the relationship between the ratio of 1,2-diglycerides to the total amount of diglycerides and the total amount of diglycerides has been used to monitor the quality of virgin olive oils, commercial olive oils, refined olive oils, and pomace oils.

Analysis of Variance↗
PubMed1975

Isolation and characterization of cytidine diphosphate diglyceride from beef liver.

Cytidine diphosphate diglyceride was isolated from beef liver by a combination of silicic acid column, DEAE-cellulose column, and this layer chromatography. The product (5.8 to 17.4 mumol/kg of liver) contained cytidine/phosphate/fatty acids in the molar proportions 1.05/2.0/2.05 (theoretical, 1.0/2.0/2.0) (average for three preparations). The liponucleotide was split quantitatively by a partially purified hydrolase from Escherichia coli, specific for CDP-diglyceride, (Raetz, C. R. H., Hirschberg, C. B., Dowhan, W., Wickner, W. T., and Kennedy, E. P. (1972) J. Biol. Chem. 247, 2245-2247) into phosphatidic acid and a water-soluble nucleotide that was chromatographically identical with CMP. No dCMP was located in these hydrolysates. The liver liponucleotide was more effective than a synthetic preparation of CDP-diglyceride in promoting the formation of phosphatidylinositol with guinea pig brain microsomes. The fatty acid composition of CDP-diglyceride was compared with metabolically related phospholipids from beef liver. The liponucleotide had a similar composition to phosphatidylinositol, characterized by a high level of stearate and with arachidonate as the major unsaturated fatty acid. The content of arachidonate in both lipids was significantly higher than that in phosphatidic acid. The profile of fatty acids of cardiolipin was quite unlike that of CDP-diglyceride. These findings suggest several alternatives for the metabolic origins of beef liver CDP-diglyceride: (a) CDP-diglyceride is formed from an atypical pool of phosphatidic acid, (b) the enzyme is selective for arachidonoyl-containing species of phosphatidic acid, (c) the liponucleotide may also be derived from phosphatidylinositol by the back-reaction of CDP-diglyceride: inositol phosphatidyltransferase.

Animals↗
PubMed1989

Molecular species analysis of 1,2-diglycerides stimulated by alpha-thrombin in cultured fibroblasts.

Diglycerides derived from the phospholipase C-mediated hydrolysis of phosphoinositides are implicated as important mediators of agonist-induced responses, including the stimulation of cell division. alpha-Thrombin-stimulated proliferation of fibroblasts is associated with a sustained increase in cellular diglycerides, while the hydrolysis of phosphoinositides is transient (Wright, T. M., Rangan, L. A., Shin, H. S., and Raben, D. M. (1988) J. Biol. Chem. 263, 9374-9380). A rigorous assessment of this apparent discrepancy requires an analysis of the molecular species of the lipids involved. In this report, we have analyzed the molecular species of 1,2-diglycerides present in quiescent and alpha-thrombin-stimulated IIC9 Chinese hamster embryo fibroblasts. The molecular species profiles of the stimulated diglycerides were compared to the profiles of molecular species contained in cellular phospholipids. We demonstrate that 1) stimulation of IIC9 cells by alpha-thrombin results in an increase in the levels of diglyceride molecular species already present in control, quiescent cultures, without the addition of new species or the complete loss of existing species; 2) the diglycerides present in control cultures as well as in cultures stimulated with alpha-thrombin are all ester-linked; and 3) while the phosphoinositides contribute a significant proportion of the diglycerides generated 15 s following alpha-thrombin addition, phosphatidylcholine contributes most of the diglycerides generated after 5 min and 1 h.

Animals↗