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At least 19 recordsLinked to original sources
PubMed1974

Degradation of monogalactosyl diglyceride and digalactosyl diglyceride by sheep pancreatic enzymes.

1. Saline extract of sheep pancreas acetone-dried powder was shown to catalyse acyl ester hydrolysis of spinach leaf galactosyl diglycerides and also galactosylglucosyl diglyceride of Lactobacillus casei. 2. Sodium deoxycholate stimulated the enzyme activity. Ca(2+) had no effect on the hydrolysis of monogalactosyl diglyceride, but it enhanced that of digalactosyl diglyceride. When added together, there was considerably less activity with both the substrates. 3. Optimal hydrolysis was observed at pH7.2. 4. The initial point of hydrolysis was at position-1, leading to the formation of monogalactosyl monoglyceride and digalactosyl monoglyceride. Further hydrolysis to the corresponding galactosylglycerols and later to galactose and glycerol was also observed, indicating the presence of alpha- and beta-galactosidases in the enzyme preparation. 5. Formation of monogalactosyl diglyceride from digalactosyl diglyceride by the action of alpha-galactosidase was noted. 6. Monogalactosyl diglyceride was also hydrolysed by beta-galactosidase to a limited extent, giving rise to diacylglycerol and galactose. 7. Attempts at purification of monogalactosyl diglyceride acyl hydrolase by using protamine sulphate treatment, Sephadex G-100 filtration and DEAE-cellulose chromatography gave a partially purified enzyme which showed 9- and 81-fold higher specific activity towards monogalactosyl diglyceride and digalactosyl diglyceride respectively. This still showed acyl ester hydrolysis activity towards methyl oleate, phosphatidylcholine and triacylglycerol. 8. When sheep, rat and guinea-pig tissues were compared, guinea-pig tissues showed the highest activity towards both monogalactosyl diglyceride and digalactosyl diglyceride. In all the species pancreas showed higher activity than intestine.

Animals↗
PubMed1979

Diglyceride kinase mutants of Escherichia coli: inner membrane association of 1,2-diglyceride and its relation to synthesis of membrane-derived oligosaccharides.

Mutants of Escherichia coli defective in diglyceride kinase contain 10 to 20 times more sn-1,2-diglyceride than normal cells. This material constitutes about 8% of the total lipid in such strains. We now report that this excess diglyceride is recovered in the particulate fraction, primarily in association with the inner, cytoplasmic membrane. The diglyceride kinase of wild-type cells was recovered in the same inner membrane fractions. The conditions employed for the preparation of the membranes did not appear to cause significant redistribution of lipids and proteins. The biochemical reactions leading to the formation of diglyceride in E. coli are not known. To determine whether diglyceride formation requires concurrent synthesis of the membrane-derived oligosaccharides (H. Schulman and E. P. Kennedy, J. Biol. Chem. 252:4250-4255, 1977), we have constructed a double mutant defective in both the kinase (dgk) and phosphoglucose isomerase (pgi). When oligosaccharide synthesis was inhibited in this organism by growing the cells on amino acids as the sole carbon source, the diglyceride was no longer present in large amounts. When glucose was also added to the medium, the pgi mutation was bypassed, oligosaccharide synthesis resumed, and diglyceride again accumulated. These findings suggest that diglyceride may arise during the transfer of the sn-glycero-1-P moiety from phosphatidylglycerol (and possibly cardiolipin) to the oligosaccharides. In wild-type cells the kinase permits the cyclical reutilization of diglyceride molecules for phospholipid biosynthesis.

Diglycerides↗
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↗
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↗
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↗