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.     

In vivo labeling of myelin lipids and proteolipid protein with [3H]myristate, [14C]linoleate,and [14C]linolenate

To investigate the incorporation of essential fatty acids into myelin components, 24-day-old rabbits were injected intracerebrally with [¹⁴C]linoleate, [¹⁴C]linolenate, or [³H]Myristate for comparison. Animals were killed 22 hr later and myelin was isolated. [³H]myristate labeled all myelin lipids including monogalactosyl diglyceride, with the exception of sulfatides. With¹⁴C-essential fatty acids, only glycerophospholipids were efficiently labeled and their specific activities were in the following decreasing orders: PC>PI>PE>PS with [¹⁴C]linoleate, and PE>PC>PI=PS with [¹⁴C]linolenate. Among myelin proteins, PLP and DM-20 were labeled with all 3 precursors. PLP was purified from myelin labeled with¹⁴C-essential fatty acids. The label was then cleaved from the protein by alkaline methanolysis and was identified as a dienoic ([¹⁴C]linoleate) or a tetraenoic ([¹⁴C]linolenate) fatty acid. MBP was not labeled with [³H]myristate, but was slightly labeled with both¹⁴C-essential fatty acids. The signification of the latter result is discussed.Abbreviations FA fatty acid(s) - HPTLC high-performance thin-layer chromatography - MBP myelin basic protein - PLP proteolipid protein - PC phosphatidylcholine - PE phosphatidylethanolamine and ethanolamine plasmalogens - PI phosphatidylinositol - PS phosphatidylserine - SDS sodium dodecylsulfate     

Myristoylation of neutrophil proteins and their biological characteristics

We have studied protein acylation in neutrophils of guinea pigs using [3H]myristate. A large number of neutrophil proteins were acylated with exogenously added myristic acid. The myristoylation was detected on 110, 77, 56, 54, 52, 42, and 37 kDa proteins. These myristoylations were stronger in peripheral blood than in peritoneal cells. Myristic acid was found to be covalently linked by an amid bond to these proteins since the proteins were resistant to boiling, chloroform/methanol and hydroxylamine treatment. Most myristoylated proteins appeared to be associated with the membrane fraction, while some of the proteins such as 77 kDa one was distributed also in the cytoplasm and translocated from the cytoplasm to the plasma membrane by stimulation. Lysozyme was myristoylated in vitro by the N-hydroxysuccinimide ester of myristic acid. The myristoylated lysozyme had an ability to be associated with phospholipid liposomes, and the membrane-associated lysozyme became a substrate of the rat brain Ca2+- and phospholipid dependent protein kinase (protein kinase C). These results indicate that myristoylation in neutrophil proteins may have an important role in metabolic regulation through their membrane association.     

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 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.     

Palmitoylation of membrane proteins inSpiroplasma floricola

Covalent modification ofSpiroplasma floricola membrane proteins by fatty acids was determined by in vivo labeling of the cells with radioactive fatty acids followed by separation on one-dimensional SDS-polyacrylamide gels and visualization by autoradiography. Approximately 25 different proteins were found to be labeled with [³H]-palmitate, whereas almost none were labeled with [³H]-oleate. The radioactivity could not be removed from the palmitoylated membrane proteins by boiling in SDS or by exhaustive extraction with chloroform-methanol (21). Nevertheless, treating the palmitoylated proteins with a 0.1N KOH solution removed approximately 70% of the bound [³H]-palmitate. The major protein-bound fatty acid species were identified, following their release from the protein by chemical cleavage, as palmitic acid and stearic acid (83% and 7.5%, respectively).     

Posttranslational processing of p21 ras proteins involves palmitylation of the C-terminal tetrapeptide containing cysteine-186.

The p21 proteins of ras oncogenes are synthesized as precursors in the cytosol. After processing, which involves acylation, the products are associated with the plasma membrane in eucaryotic cells. The p21 overproduced in Escherichia coli, however, is not processed by acylation. A synthetic tetrapeptide of the p21 C terminus is used to identify the acylation site in eucaryotic p21 as cysteine-186. The same peptide of bacterial p21 is not acylated. Although p21 of Harvey murine sarcoma virus-transformed NRK cells can be metabolically labeled with either [3H]palmitate or [3H]myristate, the lipid moiety of the hydrophobic peptide is identified as palmitic acid. We suggest that the enzymatic mechanism for p21 palmitylation may be different from N-terminal myristylation of many other membrane proteins.     

Further characterization of membrane proteins involved in the transport of organic anions in hepatocytes Comparison of two different affinity labels: 4,4′-diisothiocyano-1,2-diphenylethane-2,2′-disulfonic acid and brominated taurodehydrocholic acid

4,4′-Diisothiocyano-1,2-diphenylethane-2,2′-disulfonic acid (H₂DIDS) known as an irreversible inhibitor of the anion transport in red blood cells (Cabantchik, Z.I. and Rothstein, A. (1972) J. Membrane Biol. 10, 311–330) blocks also the uptake of bile acids and of some foreign substrates in isolated hepatocytes (Petzinger, E. and Frimmer, M. (1980) Arch. Toxicol. 44, 127–135). [³H]H₂DIDS was used for labeling of membrane proteins probably involved in anion transport of rat liver cells. The membrane proteins modified in vitro by [³H]H₂DIDS were compared with those labeled by brominated taurodehydrocholic acid. The latter is one of a series of suitable taurocholate derivatives, all able to bind to defined membrane proteins of hepatocytes and also known to block the uptake of bile acids as well as of phallotoxins and of cholecystographic agents (Ziegler, K., Frimmer, M., Möller, W. and Fasold, H. (1982) Naunyn-Schmiedeberg's Arch. Pharmacol. 319, 254–261). The radiolabeled proteins were compared after SDS-electrophoresis with and without reducing agent present, solubilization by detergents, two-dimensional electrophoresis and after separation of integral and peripheral proteins. Our results suggest that the anion transport system of liver cells cannot distinguish between bile acids and the anionic stilbene derivative (DIDS). The labeling pattern for both kinds of affinity labels was very similar. Various combinations of separation techniques gave evidence that the radiolabeled membrane proteins are not subunits of a single native channel protein.     

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.     

Acylation of proteins by myristic acid in isolated mitochondria

Isolated and highly purified mitochondria from rat liver were incubated with [1-14C]myristate, solubilized in boiling sodium dodecyl sulfate, and analyzed by polyacrylamide gel electrophoresis and autoradiography. Six to eight protein bands were found to be radioactively labeled. If the mitochondria were heated for 5 min at 95 degrees C prior to incubation with this fatty acid, no labeling was observed. By preexposing the mitochondria to unlabeled fatty acids of varying chain lengths, the extent of labeling by [1-14C]myristate was reduced in a chain length-dependent manner, exhibiting maximal inhibition at lauric acid. Reversibility of the labeling was demonstrated by chasing the incorporated radioactivity with unlabeled fatty acids of varying chain length, resulting in a maximal displacement of the tracer again by lauric acid. Fractionation of the labeled mitochondria into mitochondrial matrix and inner mitochondrial membrane components before or after labeling showed that the modified proteins are located inside the inner mitochondrial membrane. In both cases, the pattern of labeling was different from the one observed with intact mitochondria. The labeled bands in the gel were sensitive to alkaline methanol or hydroxylamine treatment. The radioactivity recovered after this treatment co-migrated with myristic acid on thin layer chromatography plates. The chain length specificity and the rapid reversibility of the observed acylation argue for a new type of reaction, different from the acylation observed in whole cells. The possible involvement of the acylated proteins in the regulation of oxidative phosphorylation is discussed.     

Myristoylation, phosphorylation, and subcellular distribution of the 80-kDa protein kinase C substrate in BC3H1 myocytes

Numerous reports have described a phosphoprotein with an apparent molecular mass of 68-87 kDa, often referred to as the 80K protein, which serves as a major specific substrate for protein kinase C in a wide variety of cell types. This protein has been shown to be myristoylated in macrophages, apparently in a stimulus-dependent manner. In the present study, we have defined the kinetics for myristoylation of the 80K protein in BC3H1 myocytes and have examined the subcellular distribution of the [3H]myristate and 32P-labeled forms of the protein before and after activation of protein kinase C by phorbol dibutyrate (PDBu). The 80K protein was identified in BC3H1 myocytes by apparent molecular mass of 68 kDa (consistent with the previously reported size of the murine homologue), isoelectric point of 4.6-4.8, PDBu-inducible phosphorylation, peptide mapping, and labeling with [3H]myristate. Incorporation of [3H]myristate by this protein occurred through an amide linkage and was abolished completely by cycloheximide. Pulse labeling of quiescent cells with [3H]myristate revealed no alteration in myristoylation of the 80K protein in either the crude membrane or soluble fractions after PDBu-induced phosphorylation. The subcellular distribution of this protein (approximately 80% membrane, approximately 20% cytosol) also was the same in control and PDBu-stimulated cells. Phosphorylation of both the membrane-bound and soluble forms was increased approximately 6-fold upon stimulation of cultures with PDBu; the soluble form was phosphorylated to a 4-fold higher stoichiometry than its membrane-bound counterpart. Together, these data demonstrate that the 80K protein is myristoylated cotranslationally in BC3H1 cells and that protein kinase C-dependent phosphorylation of the 80K protein does not alter its subcellular distribution or degree of myristoylation. The fact that 20% of total myristoylated 80K protein resides in the cytosol also indicates that myristoylation alone is not sufficient to target this protein to the plasma membrane.     

Rapid Plasma Membrane Anchoring of Newly Synthesized p59 fyn: Selective Requirement for NH2-Terminal Myristoylation and Palmitoylation at Cysteine-3

The trafficking of Src family proteins after biosynthesis is poorly defined. Here we studied the role of dual fatty acylation with myristate and palmitate in biosynthetic transport of p59^fyn. Metabolic labeling of transfected COS or NIH 3T3 cells with [³⁵S]methionine followed by analysis of cytosolic and total membrane fractions showed that Fyn became membrane bound within 5 min after biosynthesis. Newly synthesized Src, however, accumulated in the membranes between 20– 60 min. Northern blotting detected Fyn mRNA specifically in soluble polyribosomes and soluble Fyn protein was only detected shortly (1–2 min) after radiolabeling. Use of chimeric Fyn and Src constructs showed that rapid membrane targeting was mediated by the myristoylated NH₂-terminal sequence of Fyn and that a cysteine at position 3, but not 6, was essential. Examination of Gα_o-, Gα_s-, or GAP43-Fyn fusion constructs indicated that rapid membrane anchoring is exclusively conferred by the combination of N-myristoylation plus palmitoylation of cysteine-3. Density gradient analysis colocalized newly synthesized Fyn with plasma membranes. Interestingly, a 10–20-min lag phase was observed between plasma membrane binding and the acquisition of non-ionic detergent insolubility. We propose a model in which synthesis and myristoylation of Fyn occurs on soluble ribosomes, followed by rapid palmitoylation and plasma membrane anchoring, and a slower partitioning into detergent-insoluble membrane subdomains. These results serve to define a novel trafficking pathway for Src family proteins that are regulated by dual fatty acylation.     

Dissociation of Phosphorylation and Translocation of a Myristoylated Protein Kinase C Substrate (MARCKS Protein) in C6 Glioma and N1E-115 Neuroblastoma Cells

Abstract: An 80-kDa protein labeled with [³H]myristic acid in C6 glioma and N1E-115 neuroblastoma cells has been identified as the myristoylated alanine-rich C kinase substrate (MARCKS protein) on the basis of its calmodulin-binding, acidic nature, heat stability, and immunochemical properties. When C6 cells preincubated with [³H]myristate were treated with 200 n M 4β-12- O -tetradecanoylphorbol 13-acetate (β-TPA), labeled MARCKS was rapidly increased in the soluble digitonin fraction (maximal, fivefold at 10 min) with a concomitant decrease in the Triton X-100–soluble membrane fraction. However, phosphorylation of this protein was increased in the presence of β-TPA to a similar extent in both fractions (maximal, fourfold at 30 min). In contrast, β-TPA–stimulated phosphorylation of MARCKS in N1E-115 cells was confined to the membrane fraction only and no change in the distribution of the myristoylated protein was noted relative to α-TPA controls. These results indicate that although phosphorylation of MARCKS by protein kinase C occurs in both cell lines, it is not directly associated with translocation from membrane to cytosol, which occurs in C6 cells only. The cell-specific translocation of MARCKS appears to correlate with previously demonstrated differential effects of phorbol esters on stimulation of phosphatidylcholine turnover in these two cell lines.     

Acetylcholine receptor-associated 43K protein contains covalently bound myristate

Torpedo electroplaque and vertebrate neuromuscular junctions contain high levels of a nonactin, 43,000-Mr peripheral membrane protein referred to as the 43K protein. 43K protein is associated with the cytoplasmic face of postsynaptic membranes at areas of high acetylcholine receptor density and has been implicated in the establishment and/or maintenance of these receptor clusters. Cloning of cDNAs encoding Torpedo 43K protein revealed that its amino terminus contains a consensus sequence sufficient for the covalent attachment of the rare fatty acid myristate. To examine whether 43K protein is, in fact, myristoylated, mouse muscle BC3H1 cells were metabolically labeled with either [35S]cysteine or [3H]myristate and immunoprecipitated with a monospecific antiserum raised against isolated Torpedo 43K protein. In cells incubated with either precursor, a single labeled species was specifically recovered that comigrated on SDS-PAGE with 43K protein purified from Torpedo electric organ. Approximately 95% of the 3H labeled material released from [3H]myristate-43K protein by acid methanolysis was extractable in organic solvents and eluted from a C18 reverse-phase HPLC column exclusively at the position of the methyl myristate internal standard. Thus, 43K protein contains authentic myristic acid rather than an amino or fatty acid metabolite of [3H]myristate. Myristate appears to be added to 43K protein cotranslationally and cannot be released from it by prolonged incubation in SDS, 2-mercaptoethanol, or hydroxylamine (pH 7.0 or 10.0), characteristics consistent with amino terminal myristoylation. Covalently linked myristate may be responsible for the high affinity of purified 43K protein for lipid bilayers despite the absence of a notably hydrophobic amino acid sequence.     

Binding of sulfosuccinimidyl fatty acids to adipocyte membrane proteins: Isolation and ammo-terminal sequence of an 88-kD protein implicated in transport of long-chain fatty acids

Summary We recently reported (Harmon et al., J. Membrane Biol. 124:261–268, 1991) that sulfo-N-succinimidyl derivatives of long-chain fatty acids (SS-FA) specifically inhibited transport of oleate by rat adipocytes. These compounds bound to an 85–90 kD membrane protein which was also labeled by another inhibitor of FA transport [³H]DIDS (4,4-diisothiocyanostilbene-2-2-sulfonate). These results indicated that the protein was a strong candidate as the transporter for long-chain fatty acids. In this report we determined that the apparent size of the protein is 88 kD and its isoelectric point is 6.9. We used [³H]SS-oleate (SSO), which specifically labels the 88-kD protein, to isolate it from rat adipocyte plasma membranes. Identification of 15 amino acids at the N-terminus region revealed strong sequence homology with two previously described membrane glycoproteins: CD36, a ubiquitous protein originally identified in platelets and PAS IV, a protein that is enriched in the apical membranes of lipidsecreting mammary cells during lactation. Antibody against PAS IV cross-reacted with the adipocyte protein. This, together with the N-terminal sequence homology, suggested that the adipocyte protein belongs to a family of related intrinsic membrane proteins which include CD36 and PAS IV.     

Reduction of opioid binding in neuroblastoma x glioma cells grown in medium containing unsaturated fatty acids

Neuroblastoma x glioma cells NG108-15 were cultured in lipid-free medium supplemented with fatty acids of various chain length and unsaturation. Binding of ³H-labelled [DAla²]-[Dleu⁵]-enkephalin by membranes of cells grown in saturation fatty acids of different chain length was not significantly different from that of the control. On the other hand, a proportional decrease of binding capacity with no change in residual receptor affinity was noticed when cells were cultured in medium containing fatty acids of increasing unsaturation. This decrease was time dependent and reached a maximum at about 48 h. Binding of [³H]dihydromorphine and [³H]naloxone was similarly affected. In contrast, when membranes of cells grown in normal medium were preincubated up to 3 h with unsaturated fatty acid and tested for opioid binding, no significant reduction was observed. Examination of the fatty acid composition of phospholipid from cells grown in linolenate indicated that a significant alteration of the acyl composition has occurred. To wval;uate the underlying cause of this type of inhibition, the effect of linolenic acid on cell growth and protein synthesis was examined. When cells were cultured in 100 μM of this fatty acid, both growth and protein synthesis were retarded by 28% and 19%, respectively. Since opiate receptors are proteineous in nature, a reduction of protein synthesis may partially account for the loss of opioid binding activity. On the other hand, an increase of membrane fluidity is known to affect a number of cellular functions, including ligan-receptor recognition. Whether this can offer a satisfactory explanation for our obervations remains to be established.     

Geranylgeraniol Overcomes the Block of Cell Proliferation by Lovastatin in C6 Glioma Cells

Abstract: It is well documented that 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors prevent cultured mammalian cells from progressing through the cell cycle, suggesting a critical role for a mevalonate-derived product. Recently, it has been shown that free geranylgeraniol (GG-OH) and farnesol (F-OH) can be utilized by C6 glioma cells for protein isoprenylation. The ability of GG-OH and F-OH to restore protein geranylgeranylation or farnesylation selectively has enabled us to examine the possibility that mevalonate is essential for cell proliferation because it is a precursor of farnesyl pyrophosphate or geranylgeranyl pyrophosphate, the isoprenyl donors involved in the post-translational modification of key regulatory proteins. In this study we report that GG-OH, as well as mevalonate, overcomes the arrest of cell proliferation of C6 glioma cells treated with lovastatin, as assessed by increased cell numbers and a stimulation in [³H]thymidine incorporation. The increase in cell number and [³H]thymidine incorporation were significantly lower when F-OH was added. Under these conditions [³H]mevalonate and [³H]GG-OH are actively incorporated into a set of isoprenylated proteins in the size range of small, GTP-binding proteins (19–27 kDa) and a polypeptide with the molecular size (46 kDa) of the smaller isoform of 2′,3′-cyclic nucleotide 3′-phosphodiesterase. Analysis of the proteins metabolically labeled by [³H]mevalonate and [³H]GG-OH reveals the presence of labeled proteins containing geranylgeranylated cysteinyl residues. Consistent with geranylgeranylated proteins playing a critical role in the entry of C6 cells into the cell cycle, a (phosphonoacetamido)oxy derivative of GG-OH, a drug previously shown to interfere with protein geranylgeranylation, prevented the increase in cell number when mevalonate or GG-OH was added to lovastatin-treated cells. These results strongly suggest that geranylgeranylated proteins are essential for progression of C6 cells into the S phase of the cell cycle and provide the first evidence that the “salvage” pathway for the utilization of the free isoprenols is physiologically significant in the CNS.     

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.     

Fatty Acid Acylated Proteins of the Halotolerant Alga Dunaliella salina

The unicellular, wall-less alga Dunaliella salina has been shown to contain an array of proteins modified by the covalent attachment of fatty acids. Myristic acid (14:0) comprised approximately 80% by weight of the protein-linked acyl groups in samples derived from cells cultured in medium containing 1.7 molar NaCl and 93% in samples from cells grown in medium containing 3.0 molar NaCl. Palmitic and stearic acids accounted for most of the remaining protein-bound acyl chains. Approximately 0.2% of the incorporated radioactivity was estimated to be in linkage with protein. The bulk of acyl chains (about 99%) were resistant to cleavage by alkali, indicating a preponderance of amide bonding. The sodium dodecyl sulfate-polyacrylamide electrophoresis labeling pattern of proteins from [³H]myristic-labeled cells was significantly different from that of proteins from cells exposed to [³H]palmitate. The appearance of radioactivity in certain proteins was also influenced by the salinity of the culture medium. Thus growth in moderate (1.7 molar) salt favored the acylation of a 48-kilodalton polypeptide whereas in high (3.0 molar) salt, a 17-kilodalton polypeptide was more heavily labeled.     

Inhibition of astroglial glutamate transport by polyunsaturated fatty acids: Evidence for a signalling role of docosahexaenoic acid

Brain cells are especially rich in polyunsaturated fatty acids (PUFA), mainly the n-3 PUFA docosahexaenoic acid (DHA) and the n-6 PUFA arachidonic acid (AA). They are released from membranes by PLA2 during neurotransmission, and may regulate glutamate uptake by astroglia, involved in controlling glutamatergic transmission. AA has been shown to inhibit glutamate transport in several model systems, but the contribution of DHA is less clear and has not been evaluated in astrocytes. Because the high DHA content of brain membranes is essential for brain function, we investigated the role of DHA in the regulation of astroglial glutamate transport.We evaluated the actions of DHA and AA using cultured rat astrocytes and suspensions of rat brain membranes (P1 fractions). DHA reduced d-[³H]aspartate uptake by cultured astrocytes and cortical membrane suspensions, while AA did not. This also occurred in astrocytes enriched with α-tocopherol, indicating that it was not due to peroxidation products. The reduction of d-[³H]aspartate uptake by DHA did not involve any change in the concentrations of membrane-associated astroglial glutamate transporters (GLAST and GLT-1), suggesting that DHA reduced the activity of the transporters. In contrast with the inhibition induced by free-DHA, we found no effect of membrane-bound DHA on d-[³H]aspartate uptake. Indeed, the uptake was similar in astrocytes with varying amount of DHA in their membrane (induced by long-term supplementation with DHA or AA). Therefore, DHA reduces glutamate uptake through a signal-like effect but not through changes in the PUFA composition of the astrocyte membranes. Also, reactive astrocytes, induced by a medium supplement (G5), were insensitive to DHA. This suggests that DHA regulates synaptic glutamate under basal condition but does not impair glutamate scavenging under reactive conditions.These results indicate that DHA slows astroglial glutamate transport via a specific signal-like effect, and may thus be a physiological synaptic regulator.