Acylated proteins in Acholeplasma laidlawii.

The covalent modification of membrane proteins by long-chain fatty acids was determined in two strains of Acholeplasma laidlawii by one-dimensional gel electrophoresis of radiolabeled membranes. Of the more than 50 membrane polypeptides detected, approximately 30 were labeled with [3H]palmitate, whereas covalent binding of [3H]oleate to membrane proteins could not be demonstrated. We suggest that in these wall-less bacteria, membrane protein acylation with saturated fatty acids may serve to ensure the structural integrity of the membrane.     

The Rh polypeptide is a major fatty acid-acylated erythrocyte membrane protein

The erythrocyte Rh antigens contain an Mr = 32,000 integral protein which is thought to contribute in some way to the organization of surrounding phospholipid. To search for possible fatty acid acylation of the Rh polypeptide, intact human erythrocytes were incubated with [3H]palmitic acid prior to preparation of membranes and sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. Several membrane proteins were labeled, but none corresponded to the glycophorins or membrane proteins 1-8. An Mr = 32,000 band was prominently labeled on Rh (D)-negative and -positive erythrocytes and could be precipitated from the latter with anti-D. No similar protein was labeled on membranes from Rhmod erythrocytes, a rare phenotype lacking Rh antigens. Labeling of the Rh polypeptide most likely represents palmitic acid acylation through thioester linkages. The 3H label was not extracted with chloroform/methanol, but was quantitatively eluted with hydroxylamine and co-chromatographed with palmitohydroxamate and free palmitate by thin layer chromatography. The fatty acid acylations occurred independent of protein synthesis and were completely reversed by chase with unlabeled palmitate. It is concluded that the Rh polypeptide is fatty acid-acylated, being a major substrate of an acylation-deacylation mechanism associated with the erythrocyte membrane.     

Fatty acid-acylated proteins in secretory mutants of Saccharomyces cerevisiae.

Yeast secretory (sec) mutants that are blocked in the transport of secretory proteins and accumulate membrane organelles were used to study the biosynthesis of fatty acid-acylated proteins. Four proteins were labeled with [3H]palmitate in sec mutants accumulating endoplasmic reticulum membranes. Three of these (molecular weights approximately equal to 20,000, 50,000, and 120,000) were N-linked glycoproteins, based on their ability to be labeled with [3H]mannose and their sensitivity to endoglycosidase H. The fourth protein (molecular weight approximately equal to 30,000) also was labeled with [3H]mannose but was insensitive to endoglycosidase H; it appeared to contain O-linked sugars. In sec mutants accumulating Golgi membranes or post-Golgi vesicles, a 35-kilodalton protein was labeled with [3H]palmitate. Analysis of Staphylococcus aureus protease V8 digests and pulse-chase experiments indicated that the 30-kilodalton protein was a precursor of 35 kilodaltons. None of these proteins was labeled with [3H]palmitate in a sec mutant that blocked the penetration of nascent polypeptides into endoplasmic reticulum; thus, acylation occurred in endoplasmic reticulum. All four proteins could be recovered from fractions enriched for yeast membranes. Fatty acids were not released from proteins by boiling in sodium dodecyl sulfate or extraction with organic solvents but were recovered as methyl esters after proteins were treated with KOH-methanol, a reaction characteristic of an acyl ester linkage.     

Fatty acids of Treponema pallidum and Borrelia burgdorferi lipoproteins.

A fundamental ultrastructural feature shared by the spirochetal pathogens Treponema pallidum subsp. pallidum (T. pallidum) and Borrelia burgdorferi, the etiological agents of venereal syphilis and Lyme disease, respectively, is that their most abundant membrane proteins contain covalently attached fatty acids. In this study, we identified the fatty acids covalently bound to lipoproteins of B. burgdorferi and T. pallidum and examined potential acyl donors to these molecules. Palmitate was the predominant fatty acid of both B. burgdorferi and T. pallidum lipoproteins. T. pallidum lipoproteins also contained substantial amounts of stearate, a fatty acid not typically prevalent in prokaryotic lipoproteins. In both spirochetes, the fatty acids of cellular lipids differed from those of their respective lipoproteins. To characterize phospholipids in these organisms, spirochetes were metabolically labeled with [3H]palmitate or [3H]oleate; B. burgdorferi contained only phosphatidylglycerol and phosphatidylcholine, while T. pallidum contained phosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, and cardiolipin. Although palmitate predominated in the lipoproteins, there were no apparent differences in the incorporation of these two fatty acids into phospholipids (putative acyl donors). Phospholipase A1 and A2 digestion of phosphatidylcholine from B. burgdorferi and T. pallidum labeled with either [3H]palmitate or [3H]oleate also revealed that neither fatty acid was incorporated preferentially into the 1 and 2 positions (potential acyl donor sites) of the glycerol backbone. The combined findings suggest that fatty acid utilization during lipoprotein synthesis is determined largely by the fatty acid specificities of the lipoprotein acyl transferases. These findings also provide the basis for ongoing efforts to elucidate the relationship between lipoprotein acylation and the physiological functions and inflammatory activities of these molecules.     

Mechanism for binding of fatty acids to hepatocyte plasma membranes

The purpose of this study was to examine the interaction between fatty acids and plasma membranes from liver cells. We were unable to reproduce the reported effect of heating on the capacity of these membranes to bind [3H]oleate (Stremmel et al. 1985 Proc. Natl. Acad. Sci. USA. 82: 4-8). In fact, the distribution of [3H]oleate between plasma membranes and unilamellar vesicles of lipids extracted from these membranes was in favor of the lipids, indicating the absence of a detectable amount of binding to a putative fatty acid binding protein in plasma membranes. Radius of curvature of vesicles (125 A vs 475 A) had no effect on the partitioning of fatty acid. In addition, the distribution of [3H]oleate between plasma membranes and other phases had the properties of a partition coefficient over a 200-fold range of [3H]oleate. There was no evidence in this experiment for a binding isotherm, i.e., binding of [3H]oleate at a specific site, superimposed on the nonspecific partitioning of [3H]oleate into the lipids of the plasma membrane. There was no competition between [14C]oleate and [3H]palmitate for entry into plasma membranes. Finally, rates of uptake of [14C]oleate and [3H]palmitate by perfused rat liver were not affected by the presence of the other fatty acid in perfusates. These data indicate that the avidity of hepatocyte plasma membranes for [3H]oleate is a simple consequence of the physical chemical properties of oleate, lipids, and water. The data exclude the idea that the uptake of fatty acids into cells is the result of binding proteins and/or catalyzed reactions at the water-membrane interface of the cell or within the plane of the plasma membrane.     

Specificity of fatty acid acylation of cellular proteins

Labeling of the BC3H1 muscle cell line with [3H] palmitate and [3H]myristate results in the incorporation of these fatty acids into a broad spectrum of different proteins. The patterns of proteins which are labeled with palmitate and myristate are distinct, indicating a high degree of specificity of fatty acylation with respect to acyl chain length. The protein-linked [3H]palmitate is released by treatment with neutral hydroxylamine or by alkaline methanolysis consistent with a thioester linkage or a very reactive ester linkage. In contrast, only a small fraction of the [3H]myristate which is attached to proteins is released by treatment with hydroxylamine or alkaline methanolysis, suggesting that myristate is linked to proteins primarily through amide bonds. The specificity of fatty acid acylation has also been examined in 3T3 mouse fibroblasts and in PC12 cells, a rat pheochromacytoma cell line. In both cells, palmitate is primarily linked to proteins by a hydroxylamine-labile linkage while the major fraction of the myristic acid (60-70%) is linked to protein via amide linkage and the remainder via an ester linkage. Major differences were noted in the rate of fatty acid metabolism in these cells; in particular in 3T3 cells only 33% of the radioactivity incorporated from myristic acid into proteins is in the form of fatty acids. The remainder is presumably the result of conversion of label to amino acids. In BC3H1 cells, palmitate- and myristate-containing proteins also exhibit differences in subcellular localization. [3H]Palmitate-labeled proteins are found almost exclusively in membranes, whereas [3H]myristate-labeled proteins are distributed in both the soluble and membrane fractions. These results demonstrate that fatty acid acylation is a covalent modification common to a wide range of cellular proteins and is not restricted solely to membrane-associated proteins. The major acylated proteins in the various cell lines examined appear to be different, suggesting that the acylated proteins are concerned with specialized cell functions. The linkages through which fatty acids are attached to proteins also appear to be highly specific with respect to the fatty acid chain length.     

Incorporation of myristate and palmitate into the sheep reticulocyte transferrin receptor: evidence for identical sites of labeling

The ability of sheep reticulocytes and plasma membranes isolated from them to incorporate fatty acids into the transferrin receptor has been examined using both [3H]palmitate and [3H]myristate. Both fatty acids, when incorporated into the transferrin receptor, can be released by treating the protein with 1 M hydroxylamine at pH 7.0. After treatment of the 3H-acylated receptor with borohydride, an 3H-labeled alcohol is released, suggesting that the receptor-bound fatty acid is in thioester linkage. With both [3H]myristate and [3H]palmitate, Cleveland maps from immunoprecipitates of the transferrin receptor labeled in intact cells and isolated membranes show that identical peptides are labeled. No evidence was obtained for qualitatively different labeling with the two fatty acids. In intact reticulocytes, incorporation of [3H]palmitate into the transferrin receptor is approximately 3.5 times greater than the incorporation of [3H]myristate from equivalent concentrations of the labeled fatty acids. However, in isolated reticulocyte plasma membranes, there is much less difference between palmitate and myristate incorporation (with ATP) or between their acyl-CoA derivatives. The reason for the discrepancy between cells and membranes is unknown but may be due to the presence in intact cells of more than one enzyme for activating the fatty acids. Acylation of the receptor in isolated plasma membranes is fourfold greater with the CoA derivatives than with the free fatty acids. The fatty acid activating enzyme(s) as well as the acyltransferase(s) appear to be membrane bound in reticulocytes.     

Acylation of cell-associated IL-1 by palmitic acid

To determine whether membrane-associated IL-1 is palmitylated, we labeled LPS-activated human monocytes with [3H]palmitic acid. The plasma membranes were isolated, and the membrane proteins extracted and analyzed simultaneously by SDS-PAGE-autoradiography and Western blot analysis from the same gel. When the monocytes were labeled with [3H]palmitate, 23- and 31-kDa bands were visualized, for membrane-associated IL-1 and its precursor, respectively. The 31- and 23-kDa bands were excised from several gels and rehydrated and analyzed again by SDS-PAGE, autoradiography, and Western blot analysis. The 23- and 31-kDa bands appeared again by both methods. To further investigate membrane-associated IL-1 acylation, human monocytes were labeled with [3H]palmitate, the plasma membranes isolated, and the membrane proteins extracted by CHAPS detergent. Immunoprecipitation of isolated membrane proteins using anti-IL-1 antibodies revealed two bands of 23 and 31 kDa after autoradiography. The studies demonstrate that both membrane-associated IL-1 and the IL-1 precursor are acylated with palmitic acid.     

The majority of cellular fatty acid acylated proteins are localized to the cytoplasmic surface of the plasma membrane

The BC3Hl muscle cell line was previously reported to contain a broad array of fatty acid acylated proteins [Olson, E. N., Towler, D. A., & Glaser, L. (1985) J. Biol. Chem. 260, 3784-3790]. Palmitate was shown to be attached to membrane proteins posttranslationally through thiol ester linkages, whereas myristate was attached cotranslationally, or within seconds thereafter, to soluble and membrane-bound proteins through amide linkages [Olson, E. N., & Spizz, G. (1986) J. Biol. Chem. 261, 2458-2466]. The temporal and subcellular differences between palmitate and myristate acylation suggested that these two classes of acyl proteins might follow different intracellular pathways to distinct subcellular membrane systems or organelles. In this study, we examined the subcellular localization of the major fatty acylated proteins in BC3Hl cells. Palmitate-containing proteins were localized to the plasma membrane, but only a subset of myristate-containing proteins was localized to this membrane fraction. The majority of acyl proteins were nonglycosylated and resistant to digestion with extracellular proteases, suggesting that they were not exposed to the external surface of the plasma membrane. Many proteins were, however, digested during incubation of isolated membranes with proteases, which indicates that these proteins face the cytoplasm. Two-dimensional gel electrophoresis of proteins labeled with [3H]palmitate and [3H]myristate revealed that individual proteins were modified by only one of the two fatty acids and did not undergo both N-linked myristylation and ester-linked palmitylation. Together, these results suggest that the majority of cellular acyl proteins are routed to the cytoplasmic surface of the plasma membrane, and they raise the possibility that fatty acid acylation may play a role in intracellular sorting of nontransmembranous, nonglycosylated membrane proteins.     

Cell-specific fatty acylation of proteins in cultured cells of neuronal and glial origin

Distinct sets of cellular proteins were labeled with [³H]myristic and [³H]palmitic acids in primary (rat neurons and astroglia) and continuous (murine N1E-115 neuroblastoma and rat C6 glioma) cell cultures derived from the nervous system. Both soluble and membrane proteins were modified by myristate in a hydroxylamine-stable (amide) linkage, while palmitoylated proteins were esterlinked and almost exclusively membrane bound. Chain elongation of both labeled fatty acids prior to acylation was observed, but no protein amide-liked [³H]myristate originating from [³H]palmitate was detected. Fatty acylation profiles differed considerably among most of the cell lines, except for rat astroglial and glioma cells in which myristoylated proteins appeared to be almost identical based on SDS gel electrophoresis. An unidentified 47 kDa myristoylated protein was labeled to a significantly greater extent in astroglial than in glioma cells; the expression of this protein could be related to transformation or development in cells of glial origin.     

Acylation of keratinocyte transglutaminase by palmitic and myristic acids in the membrane Anchorage region

The membrane-bound form of keratinocyte transglutaminase was found to be labeled by addition of [3H] acetic, [3H]myristic, or [3H]palmitic acids to the culture medium of human epidermal cells. Acid methanolysis and high performance liquid chromatography analysis of palmitate-labeled transglutaminase yielded only methyl palmitate. In contrast, analysis of the myristate-labeled protein yielded approximately 40% methyl myristate and 60% methyl palmitate. Incorporation of neither label was significantly affected by cycloheximide inhibition of protein synthesis. The importance of the fatty acid moiety for membrane anchorage was demonstrated in three ways. First, the enzyme was solubilized from the particulate fraction of cell extracts by treatment with neutral 1 M hydroxylamine, which was sufficient to release the fatty acid label. Second, solubilization of active enzyme from the particulate fraction upon mild trypsin treatment resulted in a reduction in size by approximately 10 kDa and removal of the fatty acid radiolabels. Third, the small fraction of soluble transglutaminase in cell extracts was found almost completely to lack fatty acid labeling. Keratinocyte transglutaminase translated from poly(A+) RNA in a reticulocyte cell-free system was indistinguishable in size from the native enzyme, suggesting anchorage requires only minor post-translational processing. Thus, the data are highly compatible with membrane anchorage by means of fatty acid acylation within 10 kDa of the NH2 or COOH terminus.     

Proteolipid protein and DM-20 are synthesized by Schwann cells,present in myelin membrane,but they are not fatty acylated

Proteolipid protein (PLP) and DM-20 were intensely labeled after immunoprecipitation of total cellular proteins and myelin proteins labeled with [³⁵S]methionine in nerve slices. These results provided evidence that PLP and DM-20 are incorporated into the myelin membrane following their synthesis in Schwann cells. In contrast, PLP and DM-20 were not fatty acylated after incubation of the nerve slices with [³H]palmitic acid, however, PO glycoprotein and 24kDa protein were heavily fatty acylated. The lack of fatty acylation of PLP and DM-20 in the peripheral nervous system suggests that fatty acyltransferase responsible for their acylation is absent or non-functional in the peripheral nervous system.     

The transfer of myristic and other fatty acids on lipid and viral protein acceptors in cultured cells infected with Semliki Forest and influenza virus.

[3H]Myristic and [3H]palmitic acid were compared as tracers for the fatty acylation of cellular lipids and viral glycoproteins in chicken embryo cells infected with fowl plague and Semliki Forest virus (SFV). Both of these substrates are incorporated into glycerolipids to a similar extent, whereas sphingolipids show much higher levels of palmitate than myristate after a 20 h labeling period. Both fatty acid species were found to be subject to metabolic conversions into longer chain fatty acids yielding 11.7% C16:0 from [3H]myristic and 11.8% C18:0 from [3H]palmitic acid. The reverse, a metabolic shortening of the exogenous acyl-chains yielding, for instance, significant levels of myristic acid from palmitic acid was not observed. Out of the various [3H]fatty acids present after in vivo labeling with [3H]myristic acid (C14:0) the elongated acyl-species arising from metabolic conversion (e.g., C16:0; C18:0) are preferred over myristic acid in the acylation of SFV E1 and E2 and of the influenza viral hemagglutinin (HA2). During acylation of exogenous E1 from SFV in vitro incorporation of palmitic acid from palmitoyl CoA exceeds that of myristic acid from myristoyl CoA by a factor of 37. This indicates that specificity for the incorporation of fatty acids into viral membrane proteins occurs at the level of the polypeptide acyltransferase(s).     

Acylation of the 176R (19-kilodalton) early region 1B protein of human adenovirus type 5.

Antipeptide sera were prepared in rabbits against synthetic peptides corresponding to the predicted amino and carboxy termini of the early region 1B 176R (19-kilodalton [kDa]) protein of human adenovirus type 5. Both antisera specifically immunoprecipitated the 19- and 18.5-kDa forms of the 176R protein observed previously with antitumor sera. These data suggested that both species are full-length molecules of 176 residues. To identify posttranslational modifications that could explain the formation of these multiple species and possibly their known association with membranes, studies were carried out to determine whether they are glycosylated or acylated. Neither the 19- nor the 18.5-kDa species appeared to be a glycoprotein, however, they were labeled with [3H]palmitate and [3H]myristate, indicating that both species are acylated. Thus, whereas acylation does not appear to be the cause of the multiple species, it could play a role in the membrane association of these viral proteins. The acylation of 176R was found to be unusual. The fatty acid linkage was resistant to treatment with hydroxylamine or methanol-KOH, suggesting that acylation was through an amide bond. In addition, both palmitate and myristate were present in 176R, suggesting either a lack of specificity in the acylation reaction or the existence of more than one acylation site.     

In vitro acylation of the transferrin receptor

In vitro fatty acylation of the transferrin receptor with [3H]tetradecanoate or [3H]tetradecanoyl-CoA has been demonstrated for isolated sheep reticulocyte plasma membranes. Although less than 5% of the receptor was labeled in vitro, the acylated protein could be readily observed after sodium dodecyl sulfate-gel electrophoresis. The acylated transferrin receptor in the reticulocyte membrane was specifically precipitated with a monoclonal antibody and was absent from mature red cell membranes. Incorporation of fatty acid was dependent on ATP, and fatty acid was 5-10 times less effective as an acyl donor than the acyl-CoA derivative, pointing out the strong potential of this reagent for in vitro acylation of membrane proteins. During in vitro maturation of reticulocytes, the receptor is released in vesicles into the incubation medium. Using reticulocytes labeled with [3H]tetradecanoate, it can be shown that the 3H-labeled receptor is transferred from the cells to the vesicles without loss of acyl groups, suggesting that the vesiculation process does not involve deacylation.     

Fatty acid acylation of vaccinia virus proteins.

Labeling of vaccinia virus-infected cells with [3H]myristic acid resulted in the incorporation of label into two viral proteins with apparent molecular weights of 35,000 and 25,000 (designated M35 and M25, respectively). M35 and M25 were expressed in infected cells after the onset of viral DNA replication, and both proteins were present in purified intracellular virus particles. Virion localization experiments determined M25 to be a constituent of the virion envelope, while M35 appeared to be peripherally associated with the virion core. M35 and M25 labeled by [3H]myristic acid were stable to treatment with neutral hydroxylamine, suggesting an amide-linked acylation of the proteins. Chromatographic identification of the protein-bound fatty acid moieties liberated after acid methanolysis of M25, isolated from infected cells labeled during a 4-h pulse, resulted in the recovery of 25% of the protein-bound fatty acid as myristate-associated label and 75% as palmitate, indicating that interconversion of myristate to palmitate had occurred during the labeling period. Similar analyses of M25 and M35, isolated from infected cells labeled during a 0.5-h pulse, determined that 46 and 43%, respectively, of the protein-bound label had been elongated to palmitate even during this brief labeling period. In contrast, M25 and M35 isolated from purified intracellular virions labeled continuously during 24 h of growth contained 75 and 70%, respectively, myristate-associated label, suggesting greater stability of these proteins or a favored interaction of the proteins containing myristate with the maturing or intracellular virion.     

Photochemical labeling of membrane hydrophobic core of human erythrocytes using a new photoactivable reagent 2-[3H]diazofluorene

Photoactivable reagents have been useful for studying the structural aspects of membrane hydrophobic core. We have reported earlier (Anjaneyulu, P.S.R., and Lala, A. K. (1982) FEBS Lett. 146, 165-167) the use of diazofluorene as a probe for fluorescent photochemical labeling of hydrophobic core in artificial membranes. To quantitate and enhance the monitoring ability of this probe, we have synthesized 2-[3H]diazofluorene of high specific activity. This reagent rapidly partitions into phosphatidylcholine vesicles and selectively labels the fatty acyl chains of phosphatidylcholine. The insertion yield (13%) is not affected by the presence of scavengers like reduced glutathione. 2-[3H]Diazofluorene also readily partitions into erythrocyte membranes and on photolysis labels the membrane. The overall insertion was 48% with 9.7% in protein fraction and the rest in lipids. The distribution of radioactivity in labeled protein fraction was restricted to integral membrane proteins with Band 3 being the major protein labeled. There is little or no labeling associated with extrinsic proteins like spectrin. Further analysis of labeled Band 3 by treatment with chymotrypsin indicated that the labeling was restricted to the membrane spanning CH-17 and CH-35 fragments. No labeling of the cytoplasmic fragment of Band 3 could be observed. 2-[3H]Diazofluorene should prove useful for studying integral membrane proteins and their membrane-spanning regions.     

Phospholipid metabolism of stimulated lymphocytes. Preferential incorporation of polyunsaturated fatty acids into plasma membrane phospholipid upon stimulation with concanavalin A

Rabbit thymocytes were isolated and incubated for various lengths of time with concanavalin A. The cultures were pulsed for the last 12.5 min of incubation with equimolar mixtures of radioactively labelled fatty acids, either [3H]arachidonate and [14C]oleate or [3H]arachidonate and [14C]palmitate, and the uptake of each fatty acid into phospholipid of plasma membrane was determined. Upon binding of the mitogen, the fatty acids were incorporated at an increased rate with a new steady state being reached between 12.5 and 42.5 min after stimulation. Initially after 12.5 min, when the two fatty acids were added together, no preferential incorporation of the polyunsaturated fatty acid arachidonate was seen compared to the saturated or monounsaturated ones, palmitate or oleate. However shortly thereafter arachidonate, when compared to palmitate or oleate, started to be preferentially incorporated into plasma membrane phospholipid so that by 4 h after activation, only arachidonate was incorporated at an increased rate: the uptake of palmitate and oleate had reverted to that of unstimulated cells. In contrast, when palmitate or oleate were added alone, after 4 h of activation incorporation was increased similar to that of arachidonate, suggesting that all long chain fatty acids compete for the same activated enzyme(s). A detailed analysis of incorporation into phospholipid species showed that all fatty acids were taken up with the highest rate into phosphatidylcholine. After activation, fatty acid incorporation was increased by approx. 50% for phosphatidylcholine: the highest stimulation rates were observed with phosphatidylinositol (3-7-fold) and phosphatidylethanolamine (2-3-fold). The data suggest that shortly after stimulation with mitogens, the membrane phospholipids start to change by replacing saturated and monounsaturated fatty acids by polyunsaturated ones, thus creating a new membrane.     

The role of fatty acid unsaturation in minimizing biophysical changes on the structure and local effects of bilayer membranes

Studying the effects of saturated and unsaturated fatty acids on biological and model (liposomes) membranes could provide insight into the contribution of biophysical effects on the cytotoxicity observed with saturated fatty acids. In vitro experiments suggest that unsaturated fatty acids, such as oleate and linoleate, are less toxic, and have less impact on the membrane fluidity. To understand and assess the biophysical changes in the presence of the different fatty acids, we performed computational analyses of model liposomes with palmitate, oleate, and linoleate. The computational results indicate that the unsaturated fatty acid chain serves as a membrane stabilizer by preventing changes to the membrane fluidity. Based on a Voronoi tessellation analysis, unsaturated fatty acids have structural properties that can reduce the lipid ordering within the model membranes. In addition, hydrogen bond analysis indicates a more uniform level of membrane hydration in the presence of oleate and linoleate as compared to palmitate. Altogether, these observations from the computational studies provide a possible mechanism by which unsaturated fatty acids minimize biophysical changes and protect the cellular membrane and structure. To corroborate our findings, we also performed a liposomal leakage study to assess how the different fatty acids alter the membrane integrity of liposomes. This showed that palmitate, a saturated fatty acid, caused greater destabilization of liposomes (more “leaky”) than oleate, an unsaturated fatty acid.     

Selective Transacylation of 1-O-Alkylglycerophosphoethanolamine by Docosahexaenoate and Arachidonate in Rat Brain Microsomes

The mechanism involved in the enzymic acylation of 1-[3H]alkylglycero-3-phosphoethanolamine (1-[3H]alkyl-GPE) in brain microsomes was investigated in comparison with the acylation of 1-[3H]alkylglycero-3-phosphocholine (1-[3H]alkyl-GPC). Both the alkyllsophospholipids were acylated without exogenously added cofactors to similar extents. The [14C]arachidonoyl moiety of exogenously added 1-stearoyl-2-[14C]arachidonoyl-GPC was transferred to the alkyllysophospholipids and the transfer was not inhibited by exogenously added free arachidonate. These results indicated that the transferase activity was due to a transacylase that catalyzes the transfer of fatty acids between intact phospholipids. The addition of CoA increased the acylation of 1-[3H]alkyl-GPC two or three times with a high acceptor concentration, and the highest rate of acylation of 1-[3H]alkyl-GPC was observed in the presence of CoA, ATP, and Mg2+. On the other hand, the addition of such cofactors only slightly increased the acylation of 1-[3H]alkyl-GPE. HPLC analysis revealed that docosahexaenoate and arachidonate were transferred to the second position of both [3H]alkyllysophospholipids without cofactors and that other fatty acids were transferred to much lower extents. With the addition of cofactors, the acylation of 1-[3H]alkyl-GPC by both docosahexaenoate and arachidonate increased 1.5-2 times, and high amounts of palmitate, oleate, and linoleate were newly transferred. High amounts of oleate were also transferred to 1-[3H]alkyl-GPE in the presence of cofactors but the acylation by both docosahexaenoate and arachidonate scarcely increased on the addition of these cofactors.(ABSTRACT TRUNCATED AT 250 WORDS)