Endogenous fatty acids are covalently and noncovalently bound to interphotoreceptor retinoid-binding protein in the monkey retina

Interphotoreceptor retinoid-binding protein (IRBP) purified from monkey interphotoreceptor matrix contains relatively high concentrations of endogenous fatty acids, 6.51 mol/mol of protein. Sixty-five percent of the total fatty acid bound to IRBP was found to be noncovalently attached, with the remainder covalently bound. The fatty acids are not residual components of phospholipids or neutral lipids, as judged by microchemical methods. The major fatty acids bound to IRBP are: palmitic (35%), stearic (21%), palmitoleic (7%), oleic (29%), linoleic (6%) and docosahexaenoic acids (2%). These fatty acids account for about 90% of the total fatty acid bound to interphotoreceptor matrix proteins extracted with organic solvents. Thus, IRBP may function as an intercellular fatty acid carrier and may depend on the covalently bound fatty acids for anchoring in the outer leaflet of cell membranes.     

Covalently bound fatty acids in the gastrointestinal epithelial cell membrane.

Myristic, palmitic, stearic, oleic and linoleic acids have been identified as the covalently bound fatty acids in the monkey gastrointestinal mucosal membrane proteins and among them palmitolation was predominant. Distribution studies in various regions of the gastrointestinal mucosa showed no significant difference in the content and composition of covalently bound fatty acids in these membrane and most of the fatty acids were found to be ester linked. Total membranes from isolated crypt and villus enterocytes and colonocytes had similar composition of these fatty acids. Covalently bound fatty acid levels were higher in the small intestinal brush border membrane. As suggested for the mucus glycoproteins, covalently bound fatty acids in the intestinal epithelial cell membrane may protect these membranes from proteolytic damage from the luminal proteases.     

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.     

Presence of covalently attached fatty acids in rat apolipoprotein B via thiolester linkages

Thiolester linked lipids in rat apolipoprotein B (ApoB) were examined by incubating reduced and carboxymethylated ApoB in 6 M urea buffer with [14C]methylamine at pH 8.5, 30 degrees C. It was observed that [14C]methylamine was covalently incorporated into ApoB, and there was a [14C]methylamine modified product which was lipid in nature. After extraction with organic solvents, the [14C]methylamine labeled product showed its Rf on TLC to be similar to that of the synthetic N-methyl fatty acyl amide. After purification on TLC and transesterification with 3 N methanolic HC1, methyl esters of C16:0, C18:0 and C18:1 fatty acids were identified by gas-liquid chromatography. These results suggest that rat ApoB, similar to human ApoB, contained covalently linked fatty acids through the high energy, labile thiolester bonds.     

The effects of delipidation on the major antigenic determinant of purified human intestinal mucin

To investigate whether the antigenicity of purified human intestinal mucin was dependent on the presence of associated lipid, native mucin (purified by equilibrium density gradient centrifugation in CsCl (twice) and gel filtration on Sepharose 2B) was extracted five times with organic solvents to remove any noncovalently bound lipid and, subsequently, treated with hydroxylamine to release any covalently bound fatty acids. The first organic extract contained cholesterol, phosphatidylethanolamine, and phosphatidylserine, with lesser amounts of phosphatidylcholine, triglycerides, fatty acids, sphingomyelin, and glycolipids. In total, this noncovalently bound lipid amounted to less than 5% by weight of the native mucin preparation. Further organic extracts were free of lipid. Removal of noncovalently bound lipid had essentially no effect on mucin antigenicity, as assessed by radioimmunoassay. Treatment of the delipidated mucin with hydroxylamine caused no detectable changes in mucin antigenicity or composition and the release of covalently (ester) bound fatty acids could not be demonstrated. We therefore conclude that although purified human intestinal mucin contains small amounts of noncovalently bound lipid this lipid is not involved in mucin antigenicity.     

Isolation and Characterization of the cis-trans-Unsaturated Fatty Acid Isomerase of Pseudomonas oleovorans GPo12

Pseudomonas oleovorans contains an isomerase which catalyzes the cis-trans conversion of the abundant unsaturated membrane fatty acids 9-cis-hexadecenoic acid (palmitoleic acid) and 11-cis-octadecenoic acid (vaccenic acid). We purified the isomerase from the periplasmic fraction of Pseudomonas oleovorans. The molecular mass of the enzyme was estimated to be 80 kDa under denaturing conditions and 70 kDa under native conditions, suggesting a monomeric structure of the active enzyme. N-terminal sequencing showed that the isomerase derives from a precursor with a signal sequence which is cleaved from the primary translation product in accord with the periplasmic localization of the enzyme. The purified isomerase acted only on free unsaturated fatty acids and not on esterified fatty acids. In contrast to the in vivo cis-trans conversion of lipids, this in vitro isomerization of free fatty acids did not require the addition of organic solvents. Pure phospholipids, even in the presence of organic solvents, could not serve as substrate for the isomerase. However, when crude membranes from Pseudomonas or Escherichia coli cells were used as phospholipid sources, a cis-trans isomerization was detectable which occurred only in the presence of organic solvents. These results indicate that isolated membranes from Pseudomonas or E. coli cells must contain factors which, activated by the addition of organic solvents, enable and control the cis-trans conversion of unsaturated acyl chains of membrane phospholipids by the periplasmic isomerase.     

Effect of temperature acclimatization on the fatty acid composition of goldfish intestinal lipids

1. The fatty acid composition of whole goldfish, whole-intestinal mucosa, intestinal mucosal membranes and individual phospholipids extracted from mucosal membranes were measured, fish adapted to different temperatures being used. 2. Alterations of the adaptation temperature did not noticeably affect the fatty acid composition of the whole-fish lipids, but there were marked changes in the fatty acids of lipids extracted from homogenates of goldfish intestinal mucosa. These changes were more pronounced in a membrane fraction prepared from these homogenates. Raising the adaptation temperature by 20 degrees C halved the percentage of C(20:1), C(20:4) and C(22:6) fatty acids and nearly doubled the percentage of C(18:0) and C(20:3) fatty acids recovered. 3. Choline phosphoglycerides constituted about one-half and ethanolamine phosphoglycerides about one-quarter of the total membrane phospholipids. 4. The fatty acids of choline and ethanolamine phosphoglycerides were more susceptible to temperature-dependent changes than were the phosphoglycerides of inositol or serine. 5. The increase in C(18:0) fatty acid that occurred in membranes of warm-adapted fish was greatest for ethanolamine phosphoglycerides, but increases also occurred in other phospholipid fractions and in membrane neutral lipids.     

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.     

Protein fatty acylation: a novel mechanism for association of proteins with membranes and its role in transmembrane regulatory pathways

A wide range of proteins of cellular and viral origin have been shown to be modified covalently by long-chain fatty acids. Recent studies have revealed at least two distinct types of protein fatty acylation which involve different fatty acyltransferases. The abundant fatty acid, palmitate, is incorporated post-translationally through a thiol ester linkage into a variety of cell surface glycoproteins and non-glycosylated intracellular proteins. In contrast, the rare fatty acid, myristate, is incorporated co-translationally through an amide linkage into numerous intracellular proteins. Identification of proteins that contain covalent fatty acids has revealed that this modification is common to a broad array of proteins that play important roles in transmembrane regulatory pathways. For many of these proteins, the fatty acid moiety appears to play an important role in directing the polypeptide to the appropriate membrane and in mediating protein-protein interactions within the membrane. This review will summarize recent studies that define different pathways for protein fatty acylation and will consider the potential functions for this unique covalent modification of proteins.     

Acylation of cellular proteins with endogenously synthesized fatty acids

A number of cellular proteins contain covalently bound fatty acids. Previous studies have identified myristic acid and palmitic acid covalently linked to protein, the former usually attached to proteins by an amide linkage and the latter by ester or thio ester linkages. While in a few instances specific proteins have been isolated from cells and their fatty acid composition has been determined, the most frequent approach to the identification of protein-linked fatty acids is to biosynthetically label proteins with fatty acids added to intact cells. This procedure introduces possible bias in that only a selected fraction of proteins may be labeled, and it is not known whether the radioactive fatty acid linked to the protein is identical with that which is attached to the protein when the fatty acid is derived from endogenous sources. We have examined the distribution of protein-bound fatty acid following labeling with [3H]acetate, a general precursor of all fatty acids, using BC3H1 cells (a mouse muscle cell line) and A431 cells (a human epidermoid carcinoma). Myristate, palmitate, and stearate account for essentially all of the fatty acids linked to protein following labeling with [3H]acetate, but at least 30% of the protein-bound palmitate in these cells was present in amide linkage. In BC3H1 cells, exogenous palmitate becomes covalently bound to protein such that less than 10% of the fatty acid is present in amide linkage. These data are compatible with multiple protein acylating activities specific for acceptor protein fatty acid chain length and linkage.(ABSTRACT TRUNCATED AT 250 WORDS)     

Indentification and Localization of the Fatty Acids in Haemophilus parainfluenzae

Haemophilus parainfluenzae was capable of synthesizing 22 fatty acids. These fatty acids were equivalent to 4% of the bacterial dry weight. These fatty acids were localized in the membrane-wall complex, which contained the respiratory pigments, the quinone, and the phospholipids. The fatty acids which could be extracted with organic solvents comprised 86% of the total fatty acids of the cell. These fatty acids were distributed as 98% in the phospholipids and 1.9% in the neutral lipids, of which 0.5% were free fatty acids. Palmitic, palmitoleic, oleic, and vaccenic acids comprised 72% of the total fatty acids and were found almost exclusively in the phospholipids. The phospholipids also contained the cyclopropane fatty acids. The neutral lipids contained significant proportions of the odd-numbered branched and straight-chain fatty acids. The principal free fatty acids were n-dodecanoic and pentadecenoic acids. The nonextractable wall complex contained 14% of the total fatty acids. These wall fatty acids were rendered soluble only after saponification. The wall fraction contained all of the beta-hydroxymyristic acid and most of the myristoleic and pentadecenoic acids. The significance of the distribution of fatty acids between nonesterified, neutral lipid, phospholipid, and nonextractible wall remains to be determined.     

Removal of off-flavors and isolation of fatty acids from boiled anchovies using supercritical carbon dioxide

Herein, we extracted boiled anchovies with supercritical carbon dioxide (SCO₂) at various temperatures (40 to 50°C) and pressures (7.7 to 35 MPa), which successfully removed trimethylamine and dimethyl disulfide, the main odorous compounds that produce off-flavors in fish. We extracted the following fatty acids from boiled anchovies: myristic acid, palmitic acid, stearic acid, palmitoleic acid, EPA (eicosapentaenoic acid), and DHA (docosahexaenoic acid). In addition, we observed a higher concentration of polyunsaturated fatty acids (PUFAs), such as EPA and DHA, in SCO₂-extracted anchovy oil than in organic solvent-extracted oil.     

Lipid and fatty acid composition of Gluconobacter oxydans before and after intracytoplasmic membrane formation

Gluconobacter oxydans differentiates by forming quantities of intracytoplasmic membranes at the end of exponential growth, and this formation occurs concurrently with a 60% increase in cellular lipid. The present study was initiated to determine whether this newly synthesized lipid differed from that extracted before intracytoplasmic membrane synthesis. Undifferentiated exponential-phase cells were found to contain 30% phosphatidylcholine, 27.1% caridolipin, 25% phosphatidylethanolamine, 12.5% phosphatidylglycerol, 0.4% phosphatidic acid, 0.2% phosphatidylserine, and four additional unidentified lipids totaling less than 5%. The only change detected after formation of intracytoplasmic membranes was a slight decrease in phosphatidylethanolamine and a corresponding increase in phosphatidylcholine. An examination of lipid hydrolysates revealed 11 different fatty acids in the lipids from each cell type. Hexadecanoic acid and monounsaturated octadecenoic accounted for more than 75% of the total fatty acids for both cell types. Proportional changes were noted in all fatty acids except octadecenoate. Anteiso-pentadecanoate comprised less than 1% of the fatty acids from undifferentiated cells but more than 13% of the total fatty acids from cells containing intracytoplasmic membranes. These results suggest that anteiso-pentadecanoate formation closely parallels the formation of intracytoplasmic membranes. Increased concentrations of this fatty acid may contribute to the fluidity necessary for plasma membrane convolution during intracytoplasmic membrane development.     

A microtubule-binding protein of Trypanosoma brucei which contains covalently bound fatty acid

Covalently linked fatty acids are increasingly recognized as an important type of post-translational protein modification. Many of such acylated proteins are found associated with cellular membranes. The membrane skeleton of the parasitic hemoflagellate Trypanosoma brucei consists of a regular array of microtubules which are tightly bound to the overlying cell membrane. A microtubule-binding protein (p41) has been identified within this structure which carries covalently bound fatty acid. The fatty acid linkage is sensitive to hydroxylamine treatment. After chemical transesterification, the released radioactivity co-migrates with fatty acid methyl esters in thin-layer chromatograms, establishing that the fatty acid was covalently bound to p41 via an ester (thioester) linkage. Upon detergent extraction of trypanosomes, p41 remains tightly bound to the cytoskeleton as long as Ca2+ ions are present. It can selectively be released from this structure by the addition of excess EGTA. Conversely, p41 binds to isolated cytoskeletons and to purified microtubules in vitro, the reaction again being entirely Ca2+-dependent.     

Transport of fatty acids across membranes by the diffusion mechanism

In early research on fatty acid transport, passive diffusion seemed to provide an adequate explanation for movement of fatty acids through the membrane bilayer. This simple hypothesis was later challenged by the discovery of several proteins that appeared to be membrane-related fatty acid transporters. In addition, some biophysical studies suggested that fatty acids moved slowly through the simple model membranes (phospholipid bilayers), which would provide a rationale for protein-assisted transport. Furthermore, it was difficult to rationalize how fatty acids could diffuse passively across the bilayer as anions. Newer studies have shown that fatty acids are present in membranes in the un-ionized as well as the ionized form, and that the un-ionized form can cross a protein-free phospholipid bilayer quickly. This flip-flop mechanism has been validated in cells by intracellular pH measurements. The role of putative fatty acid transport proteins remains to be clarified.     

Design, synthesis, and fluorescence studies of fluorenyl fatty acids as new depth-dependent fluorescent probes for membranes: getting over the looping-back problem

Fluorescent fatty acids have proved very useful in studying the membrane hydrophobic core. They readily partition into membranes or can be converted to phospholipids, which form integral components of membranes. By attaching the fluorescent chromophore to different positions along the alkyl chain of fatty acids, e.g., an anthroyloxy group attached via an ester linkage to n-hydroxystearic acid, membranes have been probed at different depths. While this is an interesting approach and has been extensively used, relatively little attention has been paid to the molecular design of these probes in order to have minimal membrane perturbation. In the present study we have looked into the general problem of design of such depth-dependent membrane probes. We report here a series of fluorenyl fatty acids with varying fatty acid chain lengths, i.e., (2-fluorenyl)acetic acid, -butyric acid, -hexanoic acid, and -octanoic acid, in order to obtain information at different depths in the membrane hydrophobic core. To see the effect of attachment of a hydrophobic tail on the orientation of such fatty acids in membranes, an n-butyl group was linked to the C-7 position of fluorene in (2-fluorenyl)butyric acid to get 4-(7-n-butylfluoren-2-yl)butyric acid. Further, to assess their ability to act as depth-dependent fluorescent probes, these fatty acids were incorporated in vesicles prepared from egg phosphatidylcholine, and their fluorescence quenching was studied with potassium iodide, Cu(II), 9,10-dibromostearic acid, and 12-bromostearic acid.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and analysis of the protoplast membrane of Bacillus megaterium

1. Optimum conditions were found for the lysis of Bacillus megaterium KM by lysozyme. The age of culture, density of suspension and concentration of lysozyme affected the rate of lysis. 2. Protoplast membranes were isolated by centrifugation of lysates and were exhaustively washed. 3. Treatment with chloroform removed some lipid from the membranes, but about half of the total membrane lipid could be extracted only after partial acid hydrolysis. 4. The defatted membranes consisted of protein together with variable amounts of RNA; carbohydrate was almost absent. 5. Lipid accounted for 23% of the weight of the membrane, and included both neutral lipid and phospholipid. In both classes, branched-chain C(15) acids made up about 80% of the total fatty acid. 6. The phospholipid was a kephalin, and contained small quantities of several amino acids.     

Thermally induced heterogeneity in microsomal membranes of fatty acid-supplemented Tetrahymena: lipid composition, fluidity and enzyme activity

Thermally induced phase separation was observed to occur in microsomal membranes of the ciliate Tetrahymena pyriformis, using the technique of freeze-fracture electron microscopy. In the present study, we attempted to fractionate the phase-separated membranes which were produced by chilling cells by sucrose density gradient centrifugation. When Tetrahymena was grown in the presence of palmitic acid, cells rapidly incorporated the fatty acid into their phospholipids. The resulting endoplasmic reticulum containing a high level of palmitic acid was more susceptible to thermotropic phase separation. Despite the profound alterations in the fatty acid composition, the cells retained normal growth rate, appearance and cell motility. Smooth microsomes isolated from palmitic acid-supplemented Tetrahymena cells were sonicated and then fractionated into three major subfractions. Fraction-I with lower buoyant density was rich in phospholipids and saturated fatty acids, while Fraction-III with higher density was rather rich in proteins and contained more unsaturated fatty acids in the phospholipids. A significant change was also observed in the polar head composition of phospholipids in these fractions. ESR analysis demonstrated that the extracted lipids from Fraction-III were more fluid than those from Fraction-I. In addition, the motion of the spin probe in the native membranes was more restricted than in extracted lipids. These results indicate that the lipid phase separation causes "squeezing out" of the membrane proteins from the less fluid to the fluid areas. Furthermore, we examined the temperature dependence of the activities of glucose-6-phosphatase and palmitoyl CoA desaturase.     

Isolation and characterization of outer membranes of Bacteroides thetaiotaomicron grown on different carbohydrates.

To determine whether certain outer membrane proteins are associated with growth of Bacteroides thetaiotaomicron on polysaccharides, we developed a procedure for separating outer membranes from inner membranes by sucrose density centrifugation. Cell extracts in 10% (wt/vol) sucrose-10 mM HEPES buffer (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) (pH 7.4) were separated into two fractions on a two-step (37 and 70% [wt/vol]) sucrose gradient. These fractions were further resolved into outer membranes (p = 1.21 g/cm3) and inner membranes (p = 1.14 g/cm3) on sucrose gradients. About 20 to 26% of the total 3-hydroxy fatty acids from lipopolysaccharide and 2 to 3% of the total cellular succinate dehydrogenase activity were recovered in the outer membrane preparation. The inner membrane preparation contained 22 to 49% of the total succinate dehydrogenase activity and 2 to 3% of the total 3-hydroxy fatty acids from lipopolysaccharide. Outer membranes contained a lower concentration of protein (0.34 mg/mg [dry weight]) than did the inner membranes (0.68 mg/mg [dry weight]). Molecular weights of inner membrane polypeptides ranged from 11,000 to 133,000. The most prominent polypeptides had molecular weights ranging from 11,000 to 26,000. In contrast, the molecular weights of outer membrane polypeptides ranged from 17,000 to 117,000. The most prominent polypeptides had molecular weights ranging from 42,000 to 117,000. There were several polypeptides in the outer membranes of bacteria grown on polysaccharides (chondroitin sulfate, arabinogalactan, or polygalacturonic acid) which were not detected or were not as prominent in outer membranes of bacteria grown on monosaccharide components of these polysaccharides.     

13种微藻的脂肪酸组成分析

分析了13种微藻(包括7种绿藻,5种杂色藻和1种红藻)的总脂含量和脂肪酸组成,结果表明,不同门类微藻的脂肪酸组成差异较大:绿藻的脂肪酸组成以C16和C18为主;杂色藻类的脂肪酸组成相近,金藻门含有14:0、16:0、18:1、18:4等特征脂肪酸,三角褐指藻主要的脂肪酸为14:0、16:0、16:1、16:3和20:5,而粉核油球藻的脂肪酸以14:0、16:0、20:5为主;紫球藻的脂肪酸组成以16:0、20:4和20:5为主。在测试的13种微藻中,杜氏盐藻的亚麻酸含量最高,占总脂肪酸的60.9%;等鞭金藻的十八碳四烯酸含量最高,占总脂肪酸的19.6%;紫球藻和粉核油球藻中花生四烯酸与二十碳五烯酸(EPA)含量分别占总脂肪酸的17.1%和20.9%。