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PubMed1978

Identification of an acyl donor in steryl ester biosynthesis by enzyme preparations from spinach leaves.

A pathway for steryl ester biosynthesis in acetone powder preparations from spinach (Spinacia oleracea L.) leaves has been elucidated; free sterol and 1,2-diglyceride were the substrates. Although animals synthesize cholesteryl esters by three distinct biosynthetic pathways, none of these pathways utilizes 1,2-diglyceride as an acyl donor. Phosphatidylcholine, phosphatidic acid, triglyceride, 1,3-diglyceride, 1-monoglyceride, free fatty acid, and fatty acyl-CoA were not acyl donors for spinach leaf steryl ester biosynthesis in our assay system. The unstable 2 isomer of monoglyceride was not tested. It is possible that 1,2-diglyceride and 2-monoglyceride were both acyl donors for spinach leaf steryl ester biosynthesis. Acyl-labeled phosphatidylcholine and acyl-labeled phosphatidylethanolamine were rapidly degraded by acetone powder preparations to 1,2-diglyceride via phosphatidic acid. The 1,2-diglycerides were slowly metabolized to monoglycerides, triglycerides, free fatty acids, and steryl esters. The monoglycerides were rapidly degraded to free fatty acids and glycerol.

Journal Article↗

PubMedDate not supplied

Effect of liposomes on substrate uptake by isolated guinea-pig liver mitochondrial and microsomal membranes.

The effect of adding small unilamellar lecithin liposomes, prepared in the presence of cytidine-diphosphoryl-1,2-diglycerides (CDP-diglycerides) or cytochrome c, on microsomal biosynthesis of phosphatidylinositol and NADPH-cytochrome c reduction and on mitochondrial biosynthesis of polyglycerophosphatides and succinate-cytochrome c reduction was studied in isolated guinea-pig liver subcellular membranes. Both microsomal biosynthesis of phosphatidylinositol and mitochondrial biosynthesis of phosphatidylglycerol were significantly reduced when CDP-diglycerides associated with liposomes were used, suggesting that some CDP-diglycerides were entrapped by liposomal membranes and were not available to subcellular membranes as substrates. The degree of decrease in phospholipid biosynthesis depended on the membrane and the nature of fatty acids in CDP-diglycerides. The composition of mitochondrial polyglycerophosphatides synthesized in the presence of CDP-diglycerides-liposomes was also affected in respect to the amount of phosphatidylglycerol formed. The reduction of cytochrome c in both microsomal and mitochondrial membranes was also decreased when liposomes were present in the assay system, but to a lesser degree than the phospholipid biosynthesis. These results indicate that the cytochrome c liposome association did not provide efficient protection of this substrate from the subcellular reduction. When chlorpromazine was also present with liposomes in the assay system, the NADPH-cytochrome c reduction in microsomes was scarcely affected, while the succinate-cytochrome c reduction in mitochondria was dependent on the concentration of chlorpromazine and could be completely abolished. These results were interpreted in terms of liposomal interaction with substrates in competition with subcellular membranes for the same substrates.

Animals↗

PubMed1991

Epidermal growth factor-induced hydrolysis of phosphatidylcholine by phospholipase D and phospholipase C in human dermal fibroblasts.

The enzymatic pathways for formation of 1,2-diradylglyceride in response to epidermal growth factor in human dermal fibroblasts have been investigated. 1,2-Diradylglyceride mass was elevated 2-fold within one minute of addition of EGF. Maximal accumulation (4-fold) occurred at 5 minutes. Since both diacyl and ether-linked diglyceride species occur naturally and may accumulate following agonist activation, we developed a novel method to determine separately the alterations in diacyl and ether-linked diglycerides following stimulation of fibroblasts with EGF. Utilizing this method, it was found that approximately 80% of the total cellular 1,2-diradylglyceride was diacyl, the remaining 20% being ether-linked. Addition of EGF caused accumulation of 1,2-diacylglyceride without alteration in the level of ether-linked diglyceride. Thus, the observed induction of 1,2-diradylglyceride by EGF was due exclusively to increased formation of 1,2-diacylglyceride. In cells labelled with [3H]choline, the water soluble phosphatidylcholine hydrolysis products, phosphorylcholine and choline, were increased 2-fold within 5 minutes of addition of EGF. No hydrolysis of phosphatidylethanolamine, phosphatidylserine, or phosphatidylinositol was observed. Quantitation by radiolabel and mass revealed equivalent elevations in phosphorylcholine and choline, suggesting stimulation of both phospholipase C and phospholipase D activities. To identify the presence of EGF-induced phospholipase D activity, cells were labelled with exogenous [3H]1-0-hexadecyl, 2-acyl phosphatidylcholine and its conversion to phosphatidic acid in response to EGF determined. Radiolabelled phosphatidic acid was detectable in 15 seconds after addition of EGF and was maximal (3-fold) at 30 seconds. Consistent with the presence of EGF-induced phospholipase D activity, treatment of cells with EGF, in the presence of [14C]ethanol, resulted in the rapid formation of [14C]phosphatidylethanol, the product of phospholipase D-catalyzed transphosphatidylation. The formation of phosphatidylethanol, which competes for the formation of phosphatidic acid by phospholipase D, did not diminish the induction of 1,2-diglyceride by EGF. These data suggest that the phosphatidic acid formed by phospholipase D-catalyzed hydrolysis of phosphatidylcholine is not a major precursor of the observed increased 1,2-diglyceride. Thus, the induction of 1,2-diacylglycerol by EGF may occur primarily via phospholipase C-catalyzed hydrolysis of phosphatidylcholine.

Cells, Cultured↗

PubMed1993

Phospholipase D and exocytosis of the ram sperm acrosome.

We have investigated whether phospholipase D (PLD) is involved in events leading to acrosomal exocytosis. Ram spermatozoa pre-labelled with [3H]alkyl-lysophosphatidylcholine and stimulated with the ionophore A23187 (1 microM) and Ca2+ (3 mM) in the presence of ethanol, showed a slow time-dependent increase in [3H]phosphatidic acid and [3H]phosphatidylethanol (PEt), the latter being clear evidence of PLD activity. Unlabelled cells similarly treated underwent acrosomal exocytosis. However, [3H]PEt formation was inhibited by high Ca2+ concentrations, although such conditions result in maximal acrosomal exocytosis. Treatment with A23187/Ca2+ led to a fast generation of [3H]alkyl-diglyceride and an increase in 1,2-diacylglycerol mass, which preceded [3H]PEt formation. The rises in [3H]alkyl-diglyceride and 1,2-diacylglycerol mass took place regardless of the presence or absence of ethanol. Inclusion of propranolol, a phosphatidic acid phosphohydrolase inhibitor, did not affect the early rise of labelled or unlabelled 1,2-diglycerides either. Stimulation of spermatozoa with A23187/Ca2+ in the presence of either ethanol or propranolol did not affect the occurrence of acrosomal exocytosis. Taken together, these results indicate that although Ca2+ entry triggers a late activation of PLD, this enzyme is not involved in the early generation of diglycerides. Moreover, they suggest that PLD does not make a substantial contribution in events leading to exocytosis of the sperm acrosome. Therefore, generation of diglycerides may take place primarily via phospholipase C.

Acrosome↗

PubMed1991

Source and role of diacylglycerol formed during phagocytosis of opsonized yeast particles and associated respiratory burst in human neutrophils.

The results presented in this paper demonstrate that in human neutrophils phagocytosis of C3b/bi and IgG-opsonized yeast particles is associated with activation of phospholipase D and that this reaction is the main source of diglycerides. The demonstration is based upon the following findings: 1) the challenge of neutrophils with these opsonized particles was followed by a rapid formation of [3H]alkyl-phosphatidic acid [( 3H]alkyl-PA) and [3H]alkyl-diglyceride [( 3H]alkyl-DG) in cells labeled with [3H]alkyl-lyso-phosphatidylcholine; 2) in the presence of ethanol [3H]alkyl-phosphatidylethanol was formed, and accumulation of [3H]alkyl-PA and [3H]alkyl-DG was depressed; 3) propranolol, by inhibiting the dephosphorylation of [3H]alkyl-PA, completely inhibited the accumulation of [3H]alkyl-DG and depressed by about 75% the formation of diglyceride mass. Evidence is also presented that phagocytosis of C3b/bi and IgG-opsonized yeast particles and associated respiratory burst can take place independently of diglyceride formation and of the activity of this second messenger on protein kinase C. In fact: a) propranolol while completely inhibited the formation of diglyceride mass did not modify either the phagocytosis or respiratory burst; b) these two processes were insensitive to staurosporine.

Alkaloids↗

PubMed1990

Modulation by neuropeptides of bradykinin-stimulated second messenger release in dorsal root ganglion neurons.

Fetal rat dorsal root ganglion neurons (7-8 days in culture) were labeled with [3H]arachidonic acid for 24 h. Stimulation with 10 microM bradykinin (BK) for 30 s resulted in nearly 2-fold increases in levels of radioactive diglyceride and arachidonic acid. A similar result was obtained in the absence of receptor stimulation using the Ca2+ channel agonist BAY K 8644 (10 microM, in the presence of 100 mM potassium chloride) or the Ca2+ ionophore, ionomycin (2.5 microM). If Ca2+ influx was inhibited by adding 3 mM Co2+, a blocker of voltage-sensitive calcium channels, or 2.5 mM EDTA, then BK-stimulated accumulation of both arachidonate and diglyceride was inhibited. These data suggest Ca2+ influx is required for ligand-stimulated accumulation of both arachidonate (a product of diglyceride-lipase or phospholipase A2) and diglyceride (a product of phospholipase C). Two distinct populations of channels may be involved in these reactions since pretreatment with 10 microM nifedipine or 50 microM verapamil (agents which block a subset of voltage-sensitive Ca2+ channels) inhibited BK-stimulated accumulation of arachidonic acid, but did not inhibit diglyceride accumulation. Such functional discrimination appears to have physiological importance; the inhibitory effect of nifedipine and verapamil on BK-stimulated arachidonate release was mimicked by pretreatment with peptides which decrease Ca2+ channel conductance in dorsal root ganglion neurons. The three peptides used were 1 microM neuropeptide Y, 10 microM somatostatin, and 10 microM [N-MePhe3,D-Pro4]-morphiceptin. The effect of neuropeptide Y was blocked by pretreatment with pertussis toxin.(ABSTRACT TRUNCATED AT 250 WORDS)

Analgesics↗

PubMed1983

Neutral lipids of frog and rat rod outer segments.

Neutral lipids of rod outer segments (ROS) from frog and rat comprise roughly 10 mol% of the total lipids and consist of free sterol, free fatty acids, and 1,2-diglycerides, but contain no detectable sterol esters or triglycerides. Expressed as nmol per mg ROS protein, the levels of both free fatty acids and free sterol are higher in the rat than in the frog, whereas the diglycerides in frogs are about four times higher than in rats. The level of 22:6 omega 3 in the free fatty acids fraction of the frog is twice that of the rat, although the level of 22:6 omega 3 in both the frog and rat free fatty acids is lower than in any of the glycerolipids. The diglyceride fraction from both animals consists almost entirely of two molecular species: in the frog, the C-38 and C-40 type predominate in a molar distribution of 52 and 42% respectively, whereas in the rat, the C-36 and C-38 types are most abundant in a molar distribution of 28 and 60%, respectively. Comparison of the diglyceride fatty acid and molecular species compositions with those of phosphatidylinositol and phosphatidic acid led to the suggestion that ROS diglycerides are derived from phosphatidylinositol.

Animals↗

PubMed1990

The stereochemical requirement for protein kinase C activation by 3-methyldiglycerides matches that found in naturally occurring tumor promoters aplysiatoxins.

Protein kinase C is stereospecifically activated by sn-1,2-(S)-diglycerides. A second chiral center was introduced into the diglycerides by preparing the 3-methyl derivatives. The activation of protein kinase C was also stereospecific with respect to the new chiral center established at the C3 position of the methylated diglycerides. The stereospecifically of protein kinase C directed towards the C2 and C3 positions of the diglycerides is matched in the analogous C29 and C30 stereocenters of the tumor promoting debromoaplysiatoxins. This finding strengthens the view that the structurally diverse tumor promotors contain the embedded diglyceride-like pharmacophore.

Animals↗

PubMed1976

Ribosomal-associated phosphatidylserine synthetase from Escherichia coli: purification by substrate-specific elution from phosphocellulose using cytidine 5'-diphospho-1,2-diacyl-sn-glycerol.

Cytidine 5'-diphospho-1,2-diacyl-sn-glycerol (CDPdiglyceride):L-serine O-phosphatidyltransferase (EC 2.7.8.8, phosphatidylserine synthetase) is bound tightly to the ribosomes in crude extracts of Escherichia coli. After separation of the enzyme from the ribosomes by the method of Raetz and Kennedy (Raetz, C.R.H., and Kennedy, E.P. (1974), J. Biol. Chem. 249, 5038), we have purified the enzyme to 97% of homogenekty. The major portion of the overall 5500-fold purification was attained by substrate-specific elution from phosphocellulose using CDP-diglyceride in the presence of detergent. The purified enzyme migrated as a single band with an apparent minimum molecular weight of 54 000 when subjected to electrophoresis on polyacrylamide disc gels containing sodium dodecyl sulfate. The purified enzyme catalyzed exchange reactions between cytidine 5'- monophosphate (CMP) and CDP-diglyceride and between serine and phosphatidylserine. The enzyme also catalyzed the hydrolysis of CDP-diglyceride to form CMP and phosphatidic acid. dCDP-diglyceride was equivalent to CDP-diglyceride in all reactions catalyzed by the enzyme. In addition, the purified enzyme catalyzed the formation of phosphatidylglycerol or phosphatidylglycerophosphate at a very slow rate when serine was replaced as substrate by glycerol or sn-glycero-3-phosphate, respectively. These results suggest catalysis occurs via a ping-pong mechanism through the formation of a phosphatidyl-enzyme intermediate.

CDPdiacylglycerol-Serine O-Phosphatidyltransferase↗

PubMed1984

Biosynthesis of triglyceride and other fatty acyl esters by developing rat brain.

The biosynthesis of triglyceride from 1,2-diglyceride and long-chain acyl coenzyme A (CoA) was studied in developing rat brain. Diglyceride acyltransferase activity was highest in a microsomal fraction, had a neutral pH optimum, and was stimulated by MgCl2. Palmitoyl CoA and oleoyl CoA served equally well as acyl donors. The enzyme catalyzed the acylation of both endogenous diglyceride and several naturally occurring and synthetic exogenous diglycerides. In addition, short-chain primary and secondary alcohols were found to be acylated under these conditions. A second acylation system, active at low pH, was found to catalyze esterification of ethanol and cholesterol, but not diglyceride, with free fatty acid. These results demonstrate that brain has the capacity to acylate a wide variety of physiological and nonphysiological hydroxyl compounds.

Acyl Coenzyme A↗

PubMed1985

Inhibition of vasopressin-stimulated water flow in toad bladder by phorbol myristate acetate, dioctanoylglycerol, and RHC-80267. Evidence for modulation of action of vasopressin by protein kinase C.

The action of vasopressin (AVP) in transporting epithelia is mediated by cyclic AMP(cAMP), whereas its effects in hepatocytes are mediated by calcium and phosphoinositides. Based on our recent observation that AVP stimulates phosphoinositide turnover in toad bladder, we examined the role of calcium-phospholipid-dependent kinase (protein kinase C) as a modulator of AVP's hydroosmotic effect. Phorbol myristate acetate (PMA), which can substitute for diglyceride as an activator of protein kinase C, the diglyceride dioctanoylglycerol, and RHC-80267, a glyceride lipase inhibitor that should increase diglyceride levels, inhibited AVP-stimulated water flow, but not water flow stimulated by cAMP, suggesting inhibition of cyclic AMP production. Both the dioctanoylglycerol and RHC-80267, but not PMA, also decreased water flow in response to 8-bromo cAMP indicating a potential inhibition at post-cAMP events as well. PMA increased prostaglandin synthesis; however, inhibition of water flow persisted even when prostaglandin synthesis was completely blocked by incubation with naproxen. Furthermore, water flow was not inhibited by incubation with the inactive diglyceride substitute phorbol didecanoate, supporting the specificity of the PMA inhibition. Consistent with the site of action at adenylate cyclase suggested by the transport experiments, PMA and RHC-80237 decreased both cell cAMP content and the cyclic AMP-dependent kinase ratio (-cAMP/+cAMP), an index of intracellular cyclic AMP effect. Assay for protein kinase C activity in toad bladder epithelial cell supernatant demonstrated that the toad bladder indeed contains a kinase stimulable by phospholipid, calcium, and PMA. As an apparently independent effect, we found that addition of PMA, but not dioctanoylglycerol or RHC-80267, to the mucosal bath increased both water permeability and the frequency of granular cell luminal membrane aggregates in the absence of vasopressin, consistent with stimulation of fusion events at the luminal membrane. Our data suggest that protein kinase C can modulate AVP-stimulated water flow in toad bladder by inhibiting cAMP generation, and perhaps post-cAMP steps as well, and support the hypothesis that AVP-stimulated turnover of membrane phosphoinositides antagonize the effects of AVP via changes in diglyceride, calcium, and protein kinase C.

Animals↗

PubMed1994

Effect of triamcinolone on parathyroid hormone-stimulated second messenger systems and phosphate transport in opossum kidney cells.

Although PTH is known to stimulate both the adenylate cyclase/protein kinase-A system and the phospholipase-C/protein kinase-C second messenger systems, the relative roles of these second messenger pathways remain unclear. The present studies were designed to examine the effect of triamcinolone on PTH-stimulated second messenger systems and phosphate transport in confluent cultures of opossum kidney cells. Triamcinolone was added to these cultures at a concentration of 10 nM for 24-48 h. Neither cell number nor protein content was changed by this treatment. The addition of triamcinolone did not alter PTH receptor binding or competitive displacement radioligand binding assay curves. PTH-stimulated cAMP generation and activation of protein kinase-A were not altered by triamcinolone. The glucocorticoid, however, increased basal phosphate uptake from 1.0 +/- 0.1 to 1.28 +/- 0.1 pmol/5 min.culture (P < 0.01). Phosphate transport was significantly decreased by PTH (0.01 nM) in the triamcinolone-treated cultures, but not in control cultures. Phosphate uptake in the presence of maximal doses of PTH was similar in both control and triamcinolone-treated cultures. Thus, the PTH-responsive component of phosphate transport was preserved, and the threshold dose for the effect of PTH was reduced after treatment with triamcinolone. Studies were then performed to evaluate the alternate second messenger pathway. In control cultures, PTH rapidly increased the level of diglyceride mass, as measured by diglyceride kinase assay, from 0.18 +/- 0.01 to a peak of 0.26 +/- 0.02 mol/100 mol total phospholipid (P < 0.002), 1 min after addition of the hormone. Triamcinolone pretreatment for 48 h, however, elevated the basal diglyceride levels, but the increase after the addition of PTH was totally abolished. The absence of an increase in diglyceride upon stimulation with PTH correlated with elimination of the PTH-stimulated increase in the activity of particulate protein kinase-C. Thus, in triamcinolone-treated cells, the effect of PTH on phosphate transport was preserved, and the threshold dose of PTH-induced alteration in phosphate transport was reduced in the absence of stimulation of this alternate second messenger pathway. These data show that triamcinolone in opossum kidney cells does not alter PTH activation of the cAMP/protein kinase-A system, but eliminates the increase in diglyceride and the activation of protein kinase-C in response to PTH. These studies emphasize the major role of the protein kinase-A system in the regulation of phosphate transport by PTH.

1-Methyl-3-isobutylxanthine↗

PubMed1980

The enzymes of phospholipid synthesis in Clostridium butyricum.

We have examined extracts of Clostridium butyricum for several enzymes of phospholipid synthesis. Membrane particles were shown to catalyze the formation of CDP-diglyceride from [3H]CTP and phosphatidic acid. The reaction was dependent on Mg2+ and stimulated by monovalent cations. CDP-diglyceride formed in vitro was found to be a substrate for both phosphatidylglycerophosphate synthetase and phosphatidylserine synthetase. The formation of phosphatidylglycerophosphate from added CDP-diglyceride and [U-14C]sn-glycerol-3-phosphate was dependent on Mg2+ and Triton X-100. The dephosphorylation of endogenously-generated phosphatidylglycerophosphate to yield phosphatidylglycerol was observed to be pH-dependent. The formation of phosphatidylserine from CDP-diglyceride and L-[3-14C]serine was stimulated by Mg2+ and Triton X-100. dCDP-diglyceride was a suitable substrate for both phosphatidylglycerophosphate synthetase and phosphatidylserine synthetase. Phosphatidylserine decarboxylase activity was barely detectable in membrane particles from C. butyricum. The addition of E. coli membrane particles provided efficient phosphatidylserine decarboxylase activity in this system. Although plasmalogens are the principal lipids of C. butyricum, none of the products of phospholipid synthesis formed in vitro contained measurable amounts of plasmalogens. The subcellular distribution of both phosphatidylglycerophosphate synthetase and phosphatidylserine synthetase in C. butyricum was also studied. Both were found to be membrane-associated.

CDPdiacylglycerol-Serine O-Phosphatidyltransferase↗

PubMed1986

Enzymes of phospholipid metabolism in rat pancreatic islets: subcellular distribution and the effect of glucose and calcium.

The effect of glucose and calcium on the activities of the phosphatidylinositol cycle enzymes, CDP-diglyceride inositol transferase, diacylglycerokinase, and lysophosphatidylcholine 2-acyltransferase in rat pancreatic islets was studied. Calcium inhibited the activity of CDP-diglyceride inositol transferase but had no effect on lysophosphatidylcholine 2-acyltransferase and diacylglycerokinase activities. Upon preincubation of islets in a concentration of glucose known to stimulate insulin release, the activity of lysophosphatidylcholine 2-acyltransferase, but not that of diacylglycerokinase or the CDP-diglyceride inositol transferase, was stimulated. Subcellular fractionation of pancreatic islets showed that secretory granule membranes were enriched in CDP-diglyceride inositol transferase, whereas lysophosphatidylcholine 2-acyltransferase activity was highest in the microsomal membranes. The activation of 2-acyltransferase by incubating islets in insulinotropic glucose, and the calcium sensitivity of CDP-diglyceride inositol transferase, suggest that these enzymes may have roles in regulation of insulin secretion.

Acyltransferases↗

PubMed1985

[Eicosanoids and phospholipases].

Prostaglandins, thromboxanes, and leukotrienes have been implicated to play an important role in physiology as well as in a growing list of pathophysiologic conditions. These oxidation products of 8.11.14-eicosatrienoic-, 5.8.11.14.-eicosatetraenoic-, and 5.8.11.14.17.-pentaenoic acids have been collectively designated eicosanoids. Many clinically important diseases are associated with altered eicosanoid biosynthesis. Furthermore, a series of hormones are known to induce acutely formation of eicosanoids, suggesting a crucial role in a multitude of tissue responses including phenomena such as secretion, platelet aggregation, chemotaxis, and smooth muscle contraction. The major precursor for the eicosanoids seems to be 5.8.11.14.-eicosatetraenoic acid or arachidonic acid. Virtually all of arachidonic acid however is present in esterified form in complex glycerolipids. Since cyclooxygenase and the lipoxygenases utilize arachidonic acid in its free form, a set of acylhydrolases is required to liberate arachidonic acid from membrane lipids before eicosanoid formation can occur. It became only recently apparent that a minor acidic phospholipid, phosphatidylinositol, comprising only 5%-10% of the phospholipid mass in mammalian cells, plays an important role in arachidonic acid metabolism. Phosphatidylinositol--after phosphorylation to phosphatidylinositolphosphate and phosphatidylinositolbisphosphate--appears to be hydrolyzed by specific phospholipases C generating 1-stearoyl-2-arachidonoyl-diglyceride. Diglyceride serves as substrate for diglyceride lipase to form monoglyceride and free fatty acid. Alternatively diglyceride is phosphorylated by diglyceride kinase yielding phosphatidic acid, which is believed to be reincorporated into phosphatidylinositol. In addition to phosphatidylinositol phosphatidylcholine, phosphatidylethanolamine and phosphatidic acid may contribute to arachidonic acid release. These phospholipids are substrates for phospholipases A2 generating free arachidonic acid and the respective lysophospholipid. Understanding of the biochemistry of arachidonic acid liberation may be critical in developing strategies of pharmacological intervention in a variety of pathological conditions.

Animals↗

PubMed1975

Lipids of cultured hepatoma cells: VII. Structural analyses of glycerolipids in minimal deviation hepatoma 7288C.

Phosphatidylcholine. phosphatidylethanolamine, and triglycerides were isolated from minimal deviation hepatoma 7288C cells cultured as monolayers to confluency in roller flasks containing Swim's 77 medium supplemented with 5% fetal calf serum, plus 20%, 10%, or 5% bovine serum. Fatty acid distribution at each position of glycerol was determined for the 3 glycerolipid classes, and carbon number distributions of triglycerides and diglycerides derived from phosphatidylcholine and phosphatidylethanolamine were quantitated by high temperature gas liquid chromatography. Fatty acid composition was only marginally affected by the level of bovine serum in the culture medium. Percentage composition of fatty acids esterified at each position of the 3 glycerolipids was different, indicating a nonrandom distribution of acyl groups in triglycerides and the 2 diacyl phosphatides. The carbon number distribution of diglycerides derived from phosphatidylcholine and phosphatidylethanolamine was different, and neither carbon number distribution agreed with the calculated 1-random, 2-random diacyl distribution, thus indicating pairing of certain acids in the diglycerides derived from these phospholipd classes. The determined triglyceride carbon number distributions did not show complete agreement with those calculated, assuming a 1-random, 2-random, 3-random type of fatty acyl distribution, suggesting preferential pairing of some acids in this lipid class. The 1-, 2-diglycerides derived from phosphatidylcholine, phosphatidylethanolamine, and triglycerides differed, indicating either selectivity in utilization of diglyceride species in biosynthesis of these glycerolipids, or modification of glycerolipids after their initial synthesis.

Carcinoma, Hepatocellular↗

PubMed1995

Analysis of triglycerides using atmospheric pressure chemical ionization mass spectrometry.

Atmospheric pressure chemical ionization (APCI) mass spectrometry was investigated as a new method for analysis of a mixture of triglycerides separated by reverse-phase high-performance liquid chromatography. A mixture of homogeneous (monoacid) triglyceride standards containing fatty acids with zero to three double bonds was analyzed to demonstrate the quality of mass spectra obtained by using the APCI interface. The mass spectra showed that minimal fragmentation occurs, resulting primarily in diglyceride [M-RCOO]+ ions and [M + 1]+ protonated molecular ions. The degree of unsaturation within the acyl chains had a marked effect on the proportion of diglyceride ions vs. the [M + 1]+ ions formed in the APCI source. The mass spectra of triglycerides containing fatty acids with two or three double bonds showed predominantly protonated triglyceride ions, with diglyceride peaks representing 13 to 25% of the base peak. The triglycerides containing singly unsaturated fatty acids gave diglyceride ions as the base peak, and [M + 1]+ ions with an intensity 20 to 28% that of the base peak. Only diglyceride ions were observable in the spectra of triglycerides containing saturated fatty acids.

Atmospheric Pressure↗

PubMed1981

Protein composition and extractability of lipid-modified membranes from Acholeplasma laidlawii.

Membranes from acholeplasma laidlawii have been extracted with neutral detergents, which solubilize the proteins and lipids selectively, or with sodium deoxycholate, which gives an almost total solubilization. The amounts of individual proteins present in the detergent extracts of membranes with induced variations in lipid compositions were determined by crossed immunoelectrophoresis. Extraction with the neutral detergent Tween 20 showed that ionic lipids and the glucolipid diglucosyl diglyceride were enriched in the Tween extracts whereas the glucolipid monoglucosyl diglyceride (which cannot easily by accommodated in micelles for geometrical reasons) was enriched in the membrane residue. The amount of monoglucosyl diglyceride in the Tween 20 extracts increased when the content of this lipid was increased in the membrane, and protein D12 was also more easily extracted from membranes rich in monoglucosyl diglyceride. This was not correlated with an increase in the total amounts of D12 in the membrane (as analyzed by crossed immunoelectrophoresis after sodium deoxycholate solubilization), indicating that monoglucosyl diglyceride is involved in the anchoring of protein D12 in the membrane. The total amount of the flavoprotein T4a in the membrane was found to increase upon enhanced amounts of ionic membrane lipids. Furthermore, protein T4a was found to be increasingly extractable upon an increase in the amounts of unsaturated fatty acyl chains in the lipids. Several other proteins also displayed lipid-dependent extractabilities. These results support the hypothesis that for membrane proteins the extractability with neutral detergents and thus interactions with lipids are partly dependent upon the molecular shapes of the membrane lipid molecules. Thus, by use of these selective extraction procedures, information about protein-lipid interactions in the membrane was gained.

Acholeplasma laidlawii↗