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PubMed1988

Large decreases in membrane phosphatidylethanolamine and diphosphatidylglycerol upon mutation to duramycin resistance do not change the protonophore resistance of Bacillus subtilis.

Duramycin-resistant mutant strains were selected from wild-type Bacillus subtilis (BD99) and its protonophore-resistant mutant derivative, strain AG1A3. Analyses of the membranes of the duramycin-resistant mutants showed that they had little or no phosphatidylethanolamine and diphosphatidylglycerol as determined by chemical detection after thin-layer chromatography. Small amounts of these phospholipids must remain in the mutant strains, however, because during studies of incorporation of exogenous, radioactive fatty acids, label associated with palmitoleic acid was found in chromatographic positions that corresponded to the expected positions of phosphatidylethanolamine and diphosphatidylglycerol. The duramycin-resistant strains both showed elevated levels of phosphatidylglycerol and aminoacyl(lysyl)phosphatidylglycerol. The duramycin-resistant derivative of protonophore-resistant AG1A3 (AG1A3-DR4), but not that of the wild type, also showed a decreased content of neutral relative to polar lipid in the membrane. The composition of neutral lipid in that strain was higher in free fatty acids and lower in 1,2-diacylglycerol than its parent strain. AG1A3-DR4 also contained appreciable levels of lysophosphatidylethanolamine and somewhat elevated diglycosyldiacylglycerol relative to the other strains in the study. The protonophore resistance of AG1A3 was unaltered by mutation to duramycin resistance. Nor was there any change in the efficacy of exogenous palmitoleic acid in diminishing the protonophore resistance of AG1A3-DR4. This phenomenon persists upon dramatic reduction in the content of phosphatidylethanolamine and diphosphatidylglycerol even though those phospholipids are normally the preferred sites of incorporation of the exogenous unsaturated fatty acids that mediate the effect.

Anti-Bacterial Agents↗
PubMed1992

Isoprenoid modification of proteins distinct from membrane acyl proteins in the prokaryote Acholeplasma laidlawii.

Isoprenylation is an important posttranslational modification that affects the activity, subunit interactions and membrane anchoring of different eukaryotic proteins. The small, cell-wall-less prokaryote Acholeplasma laidlawii has more than 20 membrane acyl-proteins enriched in myristoyl and palmitoyl chains. Radioactive mevalonate, a precursor to isoprenoids, was incorporated into several specific membrane proteins of 20 to 45 kDa and two soluble proteins of 23-25 kDa, respectively. No acyl proteins and none of the polar acyl lipids became labelled but these are all labelled by radioactive fatty acids. Mevalonate was incorporated mainly into a minor neutral, non-saponifiable lipid which migrated just above a C30-isoprenoid (squalene) on TLC-plates. The isoprenoid chains could not be released by mild alkaline hydrolysis from most of the isoprenylated proteins, although this procedure releases acyl chains from lipids and all acylated proteins. Isoprenylated proteins were enriched in the detergent phase upon partition with the non-ionic detergent Triton X-114. This behaviour is similar to the acyl proteins of this organism and indicates that the isoprenoid chains give the proteins a hydrophobic character.

Acholeplasma laidlawii↗
PubMed1987

Acyl-chain specificity and properties of cholesterol esterases from normal and Wolman lymphoid cell lines.

Cholesteryl esters with various chain lengths of fatty acid, radioactive (C2-C18:1) and fluorescent (pyrene butanoic and decanoic acid, P4 and P10, respectively) were synthesized and their hydrolysis was investigated in lymphoid cell lines from normal subjects and from Wolman's disease patients. The comparison of their hydrolysis showed that three cholesterol esterases were present in normal lymphoid cell lines: the first, active at pH 4.0, hydrolysed preferentially cholesteryl esters of acyl chain length more than 8 carbons, and P10-cholesteryl ester. This acid cholesterol esterase, strongly inhibited by SH-blocking agents and resistant to E600, was severely deficient in Wolman lymphoid cell lines and corresponded to acid lysosomal lipase. The second and the third cholesterol esterases, active at pH 6.0 and 8.0, respectively, hydrolysed shorter-chain derivatives: the pH 8.0 enzyme was specific for short-chain derivatives (cholesteryl acetate, butyrate and P4), whereas the pH 6.0 activity showed a broader specificity, since it hydrolysed all the cholesteryl esters, with a maximum of activity on cholesteryl acetate and butyrate. The pH 6.0 and 8.0 enzymes were heat-labile, inhibited by E600, resistant to SH-blocking agents and not deficient in Wolman lymphoid cell lines. The hypothetical physiological role of these enzymes is discussed.

Carboxylic Ester Hydrolases↗
PubMed1987

A novel inositol glycophospholipid (IGPL) and the serum dependence of its metabolism in bovine adrenocortical cells.

Adrenocortical cells in primary culture actively incorporated [3H]-inositol into phosphatidylinositol (PI) and its mono (PIP) and bisphosphate (PIP2) derivatives. In addition to these well known phosphoinositides, a inositol-containing component was detected in the cell lipid extract when analyzed by proper chromatographic systems. This component was also labeled when the cells were provided with 32P or radioactive fatty acids and a distinctive character was its ability to incorporate [3H]-glucosamine. This novel phospholipid was thus characterized as an inositol glycophospholipid (IGPL). Study of IGPL metabolism in adrenocortical cells disclosed that the presence of serum in the culture medium strikingly increased glucosamine as well as inositol incorporation by a factor of about 10 and 5, respectively, within 36 hours. These observations suggest that IGPL turnover rate, especially at the level of its inositol-glycan moiety may be regulated by extracellular signals. A possible role of IGPL in membrane signalling systems and cell regulation remains to be clarified.

Adrenal Cortex↗
PubMed1995

Synthesis of fluorescent and radioactive analogues of two lactosylceramides and glucosylceramide containing beta-thioglycosidic bonds that are resistant to enzymatic degradation.

Condensation of 2-S-(2,3,4,6-tetra-O-acetyl-beta-D-galactopyranosyl)-2- thiopseudourea hydrobromide with 2,3,6-tri-O-benzoyl-4-O-trifluoromethylsulfonyl-beta-D-galactopyra nosyl- (1-->1)-(2S,3R,4E)-3-O-benzoyl-2-dichloroacetamido-4-octa decen-1,3-diol afforded S-(2,3,4,6-tetra-O-acetyl-beta-D-galactopyranosyl)-(1-->4)-2,3,6-tri-O- benzoyl-4-thio-beta-D-glucopyranosyl-(1-->1)-(2S,3R,4E)-3-O-benzoy l-2- dichloroacetamido-4-octadecen-1,3-diol in good yield. Removal of the protecting groups, followed by selective N-acylation of the sphingosine amino group with either a fluorescent or a radioactive fatty acid, gave labeled lactosylceramide analogues in good yield. Since these products contained a beta-thioglycosidic bond between the two sugar moieties, they were totally resistant to the action of acid lysosomal glycosidases. Likewise, condensation of 2-S-(2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosyl)-2- thiopseudourea hydrobromide and 2,3,4,6-tetra-O-acetyl-beta-D-galactopyranosyl-(1-->4)-2,3,6- tri-O-acetyl-1-S-acetyl-1-thio-beta-D-glucopyranose with (2R,3R,4E)-3-O-benzoyl-2-dichloroacetamido-1-iodo-4-octad ecen-3-ol in methanolic sodium acetate afforded the corresponding beta-thioglycosides 14 and 16, respectively, in good yield. These beta-thioglycosides were converted into glucosylceramide and lactosylceramide analogues following removal of the protecting groups and by subsequent selective N-acylation using either a fluorescent or adioactive fatty acid N-succinimidyl ester. Whereas the glucosylthioceramides thus obtained proved to be completely undegradable by lysosomal glucocerebrosidase, the lactosylceramides containing the beta-thioglycosidic bond between the lactose and the ceramide residues could be degraded by lysosomal GM1-beta-galactosidase to give the corresponding glucosylthioceramides. These compound did not yield to any further enzymatic degradation.

Carbohydrate Sequence↗
PubMed2003

The N-terminal SH4 region of the Src family kinase Fyn is modified by methylation and heterogeneous fatty acylation: role in membrane targeting, cell adhesion, and spreading.

The N-terminal SH4 domain of Src family kinases is responsible for promoting membrane binding and plasma membrane targeting. Most Src family kinases contain an N-terminal Met-Gly-Cys consensus sequence that undergoes dual acylation with myristate and palmitate after removal of methionine. Previous studies of Src family kinase fatty acylation have relied on radiolabeling of cells with radioactive fatty acids. Although this method is useful for verifying that a given fatty acid is attached to a protein, it does not reveal whether other fatty acids or other modifying groups are attached to the protein. Here we use matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry to identify fatty acylated species of the Src family kinase Fyn. Our results reveal that Fyn is efficiently myristoylated and that some of the myristoylated proteins are also heterogeneously S-acylated with palmitate, palmitoleate, stearate, or oleate. Furthermore, we show for the first time that Fyn is trimethylated at lysine residues 7 and/or 9 within its N-terminal region. Both myristoylation and palmitoylation were required for methylation of Fyn. However, a general methylation inhibitor had no inhibitory effect on myristoylation and palmitoylation of Fyn, suggesting that methylation occurs after myristoylation and palmitoylation. Lysine mutants of Fyn that could not be methylated failed to promote cell adhesion and spreading, suggesting that methylation is important for Fyn function.

Acylation↗
PubMed2004

Palmitoylation of inducible nitric-oxide synthase at Cys-3 is required for proper intracellular traffic and nitric oxide synthesis.

A number of cell types express inducible nitric-oxide synthase (NOS2) in response to exogenous insults such as bacterial lipopolysaccharide or proinflammatory cytokines. Although it has been known for some time that the N-terminal end of NOS2 suffers a post-translational modification, its exact identification has remained elusive. Using radioactive fatty acids, we show herein that NOS2 becomes thioacylated at Cys-3 with palmitic acid. Site-directed mutagenesis of this single residue results in the absence of the radiolabel incorporation. Acylation of NOS2 is completely indispensable for intracellular sorting and .NO synthesis. In fact, a C3S mutant of NOS2 is completely inactive and accumulates to intracellular membranes that almost totally co-localize with the Golgi marker beta-cop. Likewise, low concentrations of the palmitoylation blocking agents 2-Br-palmitate or 8-Br-palmitate severely affected the .NO synthesis of both NOS2 induced in muscular myotubes and transfected NOS2. However, unlike endothelial NOS, palmitoylation of inducible NOS is not involved in its targeting to caveolae. We have created 16 NOS2-GFP chimeras to inspect the effect of the neighboring residues of Cys-3 on the degree of palmitoylation. In this regard, the hydrophobic residue Pro-4 and the basic residue Lys-6 seem to be indispensable for palmitoylation. In addition, agents that block the endoplasmic reticulum to Golgi transit such as brefeldin A and monensin drastically reduced NOS2 activity leading to its accumulation in perinuclear areas. In summary, palmitoylation of NOS2 at Cys-3 is required for both its activity and proper intracellular localization.

Amino Acid Sequence↗
PubMed1976

Topological asymmetry of phospholipid metabolism in rat erythrocyte membranes. Evidence for flip-flop of lecithin.

1. The distribution of phospholipids between inside and outside of rat erythrocyte membranes was studied by incubating the cells with phospholipase A2 from Naja naja venom and sphingomyelinase from Staphylococcus aureus. 2. Choline-containing phospholipids were found to comprise the majority of the outer layer of the membrane. 3. The incorporation of radioactive fatty acids into phospholipids occurred predominantly at the inside of the membrane. 4. Exchange of phospholipids between red cell membranes and plasma lipoproteins occurred at the outside of the membrane. 5. Indications were found for a rather slow flip-flop of lecithin across the membrane.

Animals↗
PubMed1975

Studies on the asymmetric arrangement of membrane-lipid-enveloped virions as a model system.

Lipids of BHK 21 cells (baby hamster kidney) grown in tissue culture were labelled with radioactive fatty acids. The enveloped vesicular stomatitis virus was propagated in this host cell type. The virions were purified by density gradient centrifugation. Neuraminidase treatment of the intact virions led to a complete transformation of hematoside [N-acetylneuraminosyl(alpha2-3)lactosyl(beta1-1)ceramide] into lactosylceramide, with identical labelling of the ceramide portion in hematoside of the untreated virions and the lactosylceramide of the neuraminidase-treated particles. The morphology of the virions appeared unchanged in electron micrographs, but the neuraminic-acid-free virions had a strong tendency to aggregate. The results of these studies are evidence that gangliosides are integrated exclusively into the outer lamella of the lipid bilayer in the viral envelope. It is also evident that the viral envelope is a suitable model for studies on membrane asymmetry.

Animals↗
PubMed1992

Substrate specificity of neutral phospholipase D from rat brain studied by selective labeling of endogenous synaptic membrane phospholipids in vitro.

We have designed a novel approach for studying the specificity of neutral phospholipase D from rat brain synaptic plasma membranes for endogenous phospholipid substrates in native membranes. A procedure was established that provides synaptic membranes labeled in selected phospholipids. This labeling procedure exploits the presence of endogenous acyl-coenzyme A synthetase and acyl-coenzyme A:lysophospholipid acyltransferase in synaptosomes for acylating various lysophospholipid acceptors with radioactive fatty acid. With [3H]arachidonate for acylation and optimal concentrations of the respective lysophospholipids, membranes were labeled in either of the following phospholipids: phosphatidylcholine (93% of total label in phospholipids), 1-O-alkyl-phosphatidylcholine (87%), phosphatidylinositol (90%), phosphatidylethanolamine (85%), phosphatidylethanolamine-plasmalogen (81%) or phosphatidylserine (59%). These membranes were employed to study the substrate specificity of the neutral, oleate-activated rat brain phospholipase D. This phospholipase exhibited almost absolute specificity for the choline-phospholipids phosphatidylcholine and 1-O-alkyl-phosphatidylcholine: 0.34% of the former labeled substrate were transphosphatidylated to phosphatidylpropanol during the assay and 0.28% of the latter. Activity toward other phospholipids was barely detectable and could largely be accounted for by utilization of residual labeled phosphatidylcholine present in those preparations. The phospholipase D exhibited some preference for fatty acids in the C-2 position of phosphatidylcholine in the following order: 2-oleoyl-phosphatidylcholine (0.67% of this labeled phosphatidylcholine were converted to phosphatidylpropanol), 2-myristoyl-phosphatidylcholine (0.60%), 2-palmitoyl-phosphatidylcholine (0.46%) and 2-arachidonoyl-phosphatidylcholine (0.34%). The present approach of labeling membrane phospholipids in vitro could be useful in studies of phospholipase specificity as an alternative to the use of sonicated vesicles or mixed detergent-phospholipid micellar systems.

1-Acylglycerophosphocholine O-Acyltransferase↗
PubMed1979

Interaction of membranous enzymes with membranous lipid substrates. Hydrolysis of diacylglycerol by lipase in rat brain microsomes.

The phospholipids in rat brain microsomes were labeled with tritium by intracerebral administration of radioactive fatty acids and converted to diacylglycerol with phospholipase C. The latter lipid was hydrolyzed in situ at pH 4.8, to monoacylglycerol and fatty acid by the endogenous microsomal lipase. This paper provides an experimental approach to determine whether the lipid was degraded by enzyme molecules residing in its own membrane (intramembrane interaction) or an adjacent membrane (intermembrane interaction). Direct interaction between separate membranes containing enzyme or substrate showed the existence of the inter-membrane route while dilution experiments provided evidence for the presence of the intramembrane interaction as well. A probable difference in the mechanisms of these two interactions is suggested by different shapes of the curves that describe the reaction rate as a function of the endogenous substrate. The curve resulting from the intermembrane interaction was hyperbolic while that representing the intramembrane route was of a parabola-like shape. Competition experiments suggested that when given a choice between the two, the enzyme utilized preferentially the substrate molecules in its own membrane.

Animals↗
PubMed1980

Studies on the disappearance of palmitate-1-14C from the plasma of pregnant rats.

Intravenously injected radioactive fatty acids disappear from the circulation at an ever-decreasing rate; about 1% of the injected label remains in plasma for hours. This slow elimination of label can be explained by an extensive recycling of label or by a slows turnover of a portion of the injected label. The results show that a recycling of label can not be responsible for the slow elimination of label, because 1. the specific activities of the tissue fatty acids were much lower than the specific activity of the plasma free fatty acids, and 2. reinjection of the remaining label of 1% into control animals showed the same slow elimination of label. An interpretation of the experiments could be that some fatty acids (about 1%) have a dissociation rate from albumin that is much lower than others, and thus they might be responsible for the late portion of the disappearance curve.

Adipose Tissue↗
PubMed1993

Mobilization of acyl chains from endogenous cellular phospholipids into cholesteryl esters during foam-cell formation in mouse peritoneal macrophages.

We previously showed that phosphatidylserine-containing liposomes (phosphatidylserine liposomes) are effectively metabolized and induce foam-cell formation in cultured macrophages (Nishikawa, K., Arai, H. and Inoue, K. (1990) J. Biol. Chem. 265, 5226-5231). When cultured mouse peritoneal macrophages were incubated with phosphatidylserine liposomes of different cholesterol content, cholesteryl esters were formed in the cells in proportion to the cholesterol content of the liposomes added to the culture. Cholesteryl esters were formed only when the cells took up more than a certain amount of cholesterol (approx. 160% above the basal cell level). Fatty acyl moiety derived from the exogenous phospholipids were utilized preferentially for cholesteryl ester formation. Once cholesterol as an acceptor is exhausted within cells, the acyl chain may be utilized for triacylglycerol formation. Not only exogenous lipid acyl chains but also endogenous phospholipid acyl chains were also utilized for cholesteryl ester and triacylglycerol formation under the present conditions, since the radioactivity in phospholipids was incorporated into both cholesteryl esters and triacylglycerols in macrophages whose endogenous phospholipids had been prelabeled with radioactive fatty acids. Among phospholipid classes, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol served as the acyl donor, whereas sphingomyelin and cardiolipin did not. Mobilization of acyl chains from endogenous phospholipids to the neutral lipid fraction was also observed during foam-cell formation induced by acetyl low-density lipoprotein. The substance(s) produced within cell in the process of foam-cell formation may trigger the acyl chain mobilization from endogenous phospholipids.

Animals↗
PubMed1994

Activation and desensitization of phospholipase D in intact rat hepatocytes.

Activation of phospholipase D (PLD) by receptor-coupled stimuli (vasopressin, ATP), phorbol esters, and Ca2+ ionophores was studied in isolated rat hepatocytes, double labeled with [3H]arachidonate and [14C]stearate. Phosphatidylethanol (Peth) was formed when cells were stimulated in the presence of ethanol. The effect of combinations of agonists was not additive, indicating that the same PLD isozyme(s) were activated. With all agonists, the 3H- and 14C-specific radioactivity in Peth was higher than in any of the main phospholipid classes. The 3H/14C ratios of Peth and phosphatidylcholine (PC) were identical and differed from other phospholipid classes, indicating that the predominant PLD substrate was a PC pool labeled preferentially with radioactive fatty acids. Ethanol (50-300 mM) decreased the initial rate of phosphatidic acid (PA) formation, but did not affect total PLD activity. Agonist-induced changes in steady state accumulation of PA or 1,2-diacylglycerol were also unaffected. A slow degradation of Peth (apparent t1/2 > 60 min) occurred after ethanol removal from cells prestimulated with vasopressin. The rate of degradation was unaffected by agonists that stimulate PLD. Thus, Peth formation is a suitable cumulative indicator for PLD activation in intact hepatocytes. Peth accumulation declined over a period of 5-20 min, depending on the agonist. The decline was not due to increased Peth degradation, or limitations in substrate supply to PLD, or enzyme inhibition by accumulated Peth. Instead, a homologous desensitization of PLD occurs with all agonists. This desensitization may involve the action of selective protein kinase C isozymes.

Adenosine Triphosphate↗
PubMed1977

Lysophospholipase--transacylase from rat lung: isolation and partial purification.

Incubation of rat lung supernatant with 1-[1(-14)C] palmitoyl-sn-glycero-3-phosphocholine in the absence of any cofactors resulted in the release of radioactive fatty acid and the formation of phosphatidylcholine. The production of fatty acids (lysophospholipase activity) exceeded phosphatidylcholine formation (transacylase activity) about thereefold, although the relative extent of phosphatidylcholine formation was considerably greater than previously reported by Abe et al. (Biochim. Biophys. Acta, 369: 361-370, 1974). In agreement with these authors, evidence is presented suggesting that a single enzyme is responsible for both catalytic activities. The enzyme, provisionally denoted lysophospholipase-transacylase, was found primarily in the soluble fraction of rat lung and was purified approximately 250-fold. The enzyme had an estimated mol wt of 50,000. The ratio of lysophospholipase to transacylase activity in the purified enzyme could be varied depending upon the concentration and character of the lysophosphatidylcholine and the ration of substrate to products. The degree of esterification of 1-acyl lysophosphatidylcholine was altered with mixtures of different molecular species of substrate, indicating acyl chain selectivity in the transfer process. This enzyme was capable of synthesizing disaturated phosphatidylcholine, an important component of the pulmonary surfactant. Three lysophospholipases purified from other sources did not possess this transacylase activity.

Acyltransferases↗
PubMed1985

Metabolism of linoleic acid and other essential fatty acids in the epidermis of the rat.

Essential fatty acids are absolutely necessary for maintaining the proper condition of the water barrier (stratum compactum) in the skin. Even direct topical application of linoleic acid or any other Z,Z-(n-6, n-9)-fatty acid to the skin restores the barrier in essential fatty acid-deficient animals. In order to investigate the mechanism by which these polyunsaturated fatty acids exert their activity, radioactively labelled fatty acids were applied to the skin of the live animal and the epidermal lipids were analysed after 1-4 days. Much radioactivity was incorporated into two peculiar lipids, viz. acyl ceramide and acyl acid, which are characteristic of the barrier, in which linoleate was esterified to the end-position of very-long-chain (C30-34) unsaturated omega-hydroxy fatty acids. Strong evidence was obtained which showed that these lipids carry linoleate into the barrier layer where it is converted, probably by lipoxygenase(s), into a series of peroxidated lipids. The lipoxygenase inhibitor, eicosatetraynoic acid, prevents both oxygenation of the polyunsaturated fatty acid and the formation of a healthy skin. This peroxidation may supply the mediators which induce the proper differentiation of the epidermal cells into an effective stratum compactum and a horny layer.

Animals↗
PubMed1980

Digestion and absorption of milk triacylglycerols in 14-day-old suckling rats.

The fatty acid compositions of the major classes of lipid recovered from the contents of the stomach and small intestine, as well as, the chylomicron fraction of the serum of 14-day-old rats have been compared to that of the milk triacylglycerols. The results indicate that at least two lipolytic activities are involved. As well as pancreatic lipase in the small intestine, a second lipase is present in the stomach and removes approximately half of the medium-chain acids (8:0, 10:0, 12:0) which together comprise between 30 and 50% of the total milk fatty acids. Tracer amounts of radioactively labeled fatty acids were then fed with carrier milk lipid and the kinetics of the appearance of the label in the blood determined. The very rapid appearance (less than 5 minutes) of 8:0 and 10:0 suggested that these acids are absorbed directly across the stomach wall and transported as the free acid. The longer times required for the other acids fed indicate that they are abosrbed from the small intestine. While both 16:0 and 18:1 were predominantly esterified into triacylglycerols and secreted in the chylomicrons, 12:0 was transported mainly as the free acid via the portal vein. Unlabeled 9:0, 11:0 and 13:0 were each absorbed and transported as the next higher homolog.

Animals↗