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.     

Characterization and quantitation of fatty acids covalently bound to erythrocyte membrane proteins: anion transporter contains 1 mol of fatty acid thiol ester

Human erythrocyte membranes which had been thoroughly extracted with organic solvents contained 20 nmol of fatty acids/mg dry wt. The major fatty acids were palmitic and stearic with their monoethenoic derivatives as minor constituents. No other fatty acids were detected. When solvent-extracted membranes were digested with Pronase about 90% of the original content of fatty acids was retained in the insoluble residue. Fatty acids were linked to membrane proteins through alkali-labile bonds of which 30% were of a thiol ester and the remainder of an O-ester type. This conclusion is based on differential liberation of fatty acids by hydroxylamine at pH 7.0 and pH 11.0. Two extracts of membranes enriched in peripheral proteins (bands 1, 2, 5 and 2.1, 4.1, 4.2, 6) were prepared and extracted with organic solvents but each contained about six times less fatty acids than the parent solvent-extracted membranes. Glycophorin A contains little if any covalently bound fatty acids. Anion transporter (band 3) contains about 1 mol of thiol ester of fatty acid. This accounts for about half of the thiol ester-linked fatty acids in the parent solvent-extracted membranes. Most of the O-ester-linked fatty acids are linked to an undisclosed membrane protein.     

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.     

Influence of dietary partially hydrogenated fat high in trans fatty acids on lipid composition and function of intestinal brush border membrane in rats

The effect of dietary hydrogenated fat (Indian vanaspati) high in trans fatty acids (6 en%) on lipid composition, fluidity and function of rat intestinal brush border membrane was studied at 2 and 8 en% of linoleic acid. Three groups of weanling rats were fed rice-pulse based diet containing 10% fat over a ten week period: Group I (groundnut oil), Group II (vanaspati), Group III (vanaspati + safflower oil). The functionality of the brush border membrane was assessed by the activity of membrane bound enzymes and transport of D-glucose and L-leucine. The levels of total cholesterol and phospholipids were similar in all groups. The data on fatty acid composition of membrane phospholipids showed that, at 2 en% of linoleic acid in the diet, trans fatty acids lowered arachidonic acid and increased linoleic acid contents indicating altered polyunsaturated fatty acid metabolism. Alkaline phosphatase activity was increased while the activities of sucrase, gamma-glutamyl transpeptidase and transport of D-glucose and L-leucine were not altered by dietary trans fatty acids. However at higher intake of linoleic acid in the diet, trans fatty acids have no effect on polyunsaturated fatty acid composition and alkaline phosphatase activity of intestinal brush border membrane. These data suggest that feeding dietary fat high in trans fatty acids is associated with alteration in intestinal brush border membrane polyunsaturated fatty acid composition and alkaline phosphatase activity only when the dietary linoleic acid is low.     

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.     

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.     

The relationship between plasma membrane lipid composition and physical-chemical properties. II. Effect of phospholipid fatty acid modulation on plasma membrane physical properties and enzymatic activities

The fatty acid composition of plasma membrane phospholipids of the murine T lymphocyte tumor EL4 were systematically modified in an attempt to understand the relationship between lipid bilayer composition and plasma membrane physical and biological properties. Two plasma membrane enzyme activities, adenylate cyclase and ouabain-sensitive (Na+ + K+)-ATPase, were measured in normal and fatty acid-substituted EL4 plasma membrane fractions. The fatty acid effect on enzyme activities was similar to previously reported effects of fatty acids on cytotoxic T cell function. The activity of both enzymes was inhibited by saturated fatty acids, while unsaturated fatty acids had a moderate enhancing effect on both enzyme activities. Using two different nitroxide derivatives of stearic acid, the order parameter and approximate rotational correlation times were calculated from ESR spectra of normal and fatty acid-modified plasma membranes. No significant differences was found in either parameter in these membranes. These results, in conjunction with earlier data from our laboratory and others, suggest that caution should be exercised in inferring changes in membrane 'fluidity' based on lipid modulation of biological membranes.     

Lipid composition of the isolated rat intestinal microvillus membrane

1. Rat intestinal microvillus plasma membranes were prepared from previously isolated brush borders and the lipid composition was analysed. 2. The molar ratio of cholesterol to phospholipid was greatest in the membranes and closely resembled that reported for myelin. 3. Unesterified cholesterol was the major neutral lipid. However, 30% of the neutral lipid fraction was accounted for by glycerides and fatty acid. 4. Five phospholipid components were identified and measured, including phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, sphingomyelin and lysophosphatidylcholine. Though phosphatidylethanolamine was the chief phospholipid, no plasmalogen was detected. 5. In contrast with other plasma membranes in the rat, the polar lipids of the microvillus membrane were rich in glycolipid. The cholesterol:polar lipid (phospholipid+glycolipid) ratio was about 1:3 for the microvillus membrane. Published data suggest that this ratio resembles that of the liver plasma membrane more closely than myelin or the erythrocyte membrane. 6. The fatty acid composition of membrane lipids was altered markedly by a single feeding of safflower oil. Membrane polar lipids did not contain significantly more saturated fatty acids than cellular polar lipids. Differences in the proportion of some fatty acids in membrane and cellular glycerides were noted. These differences may reflect the presence of specific membrane glycerides.     

Plasma and cellular zinc levels and membrane lipid composition in streptozotocin diabetic rats

1. Lipid and zinc analyses were conducted on liver mitochondrial and microsomal membranes as well as erythrocyte ghosts from streptozotocin (STZ) treated animals. 2. In STZ animals, analysis of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) fatty acids revealed an increase in palmitic acid and a corresponding decrease in stearic acid. 3. Polyunsaturated fatty acids were also affected, with an increase in 18:2, decrease in 20:4 and an increase in 22:6 in STZ animals compared to controls. 4. The change in fatty acid composition was observed in all three membrane fractions. 5. Plasma zinc levels in STZ animals were elevated while no difference was observed in membrane bound zinc. 6. Thus, while there appears to be both altered trace metal as well as membrane lipid metabolism in STZ treated animals, membrane bound zinc does not seem to be affected.     

Fatty Acid Composition of Myelin Proteolipid Protein During Vertebrate Evolution

The hydrophobic myelin proteolipid protein (PLP) contains covalently bound long-chain fatty acids which are attached to intracellular cysteine residues via thioester linkages. To gain insight into the role of acylation in the structure and function of myelin PLP, the amount and pattern of acyl groups attached to the protein during vertebrate evolution was determined. PLP isolated from brain myelin of amphibians, reptiles, birds and several mammals was subjected to alkaline methanolysis and the released methyl esters were analyzed by gas-liquid chromatography. In all species studied, PLP contained approximately the same amount of covalently bound fatty acids (3% w/w), and palmitic, palmitoleic, oleic and stearic acids were always the major acyl groups. Although the relative proportions of these fatty acids changed during evolution, the changes did not necessarily follow the variations in the acyl chain composition of the myelin free fatty acid pool, suggesting fatty acid specificity. The phylogenetic conservation of acylation suggests that this post-translational modification is critical for PLP function.     

Rat proximal small intestinal Golgi membranes: lipid composition and fluidity

The present studies were conducted to examine and characterize the lipid composition and physical state of the membrane lipids of rat proximal small intestinal Golgi membranes. Golgi membranes were purified from isolated enterocytes; lipids were extracted from these membranes and analyzed by thin-layer and gas-liquid chromatography. The 'static' and 'dynamic' components of fluidity of Golgi membranes and their liposomes were assessed by steady-state fluorescence polarization techniques utilizing r infinity and S values of 1,6-diphenyl-1,3,5-hexatriene and r values of DL-2-(9-anthroyl)- and DL-12-(9-anthroyl)stearic acid, respectively. Additional studies were also performed on these membranes, using benzyl and methyl alcohol, to examine the relationship between alterations in lipid fluidity and glycosphingolipid glycosyltransferase activities. The results of these studies demonstrated that: (1) the principal phospholipids and neutral lipids of intestinal Golgi membranes, respectively, were phosphatidylcholine, phosphatidylethanolamine and sphingomyelin, and unesterified cholesterol and fatty acids; (2) the major fatty acids of Golgi membranes were palmitic (16:0), stearic (18:0), linoleic (18:2), arachidonic (20:4) and oleic (18:1) acids; (3) fluorescence polarization studies using diphenylhexatriene detected a thermotropic transition at 24-26 degrees C in Golgi membranes and liposomes prepared from lipid extracts of these membranes; (4) benzyl alcohol (25 and 50 mM) but not methyl alcohol (50 mM) significantly increased the fluidity of these membranes; and (5) at these same concentrations, benzyl alcohol was also found to increase significantly the specific activity of UDP-galactosyllactosylceramide galactosyltransferase but not CMP-acetylneuraminic acid: lactosylceramide sialyltransferase. Methyl alcohol was not found to influence either enzyme's activity in these membranes.     

Protein acylation in normoxic and ischemic/reperfused cardiac tissue.

In addition to a prominent role in tissue energy conversion, fatty acids are involved in signal transduction and modulation of cellular protein localization and function. The latter is accomplished by acylation of specific cellular proteins. In the present study the amount of fatty acyl moieties covalently bound to cardiac proteins and the effect of myocardial ischemia and reperfusion on the degree and relative fatty acyl composition of cardiac proteins have been investigated in isolated rat hearts. In the normoxic heart about 0.32% of the cellular fatty acyl pool is covalently bound to proteins. Approximately 90% of these fatty acyl chains are thio-esterified, whereas a relatively minor part is attached to cardiac proteins through amide linkage. Thio-esterified fatty acyl chains are derived from palmitic, stearic, oleic, linoleic, arachidonic and docosahexaenoic acid. In contrast, amide linked protein acylation shows a preference for myristic acyl chains. Acute ischemia and reperfusion inflicted upon the isolated rat heart did enhance significantly the content of (unesterified) fatty acids, but did neither affect the degree of protein acylation nor the relative fatty acyl composition of acylated proteins in cardiac tissue.     

Alterations in the physical state and composition of brush border membrane lipids of rat enterocytes during differentiation

The physical state of the membrane lipid of brush border membranes, prepared from rat small intestinal villus and crypt cells, was examined by steady-state fluorescence polarization using three lipid-soluble fluorophors. Membranes prepared from crypt cells were found to possess a higher lipid fluidity than those of villus cells with each probe. Analysis of the composition of these membranes revealed that those from crypt cells had lower ratios of cholesterol/phospholipid (mol/mol), protein/lipid (w/w), and saturated fatty acyl chains/unsaturated chains (w/w). Alterations in the levels of stearic (18:0) and oleic (18:1) acids were responsible for differences in the latter ratio. The results, therefore, demonstrate that alterations in the lipid composition and fluidity of brush border membranes of enterocytes occur during the process of differentiation.     

Changes in fatty acid composition during cell differentiation in the small intestine of suckling piglets.

Alterations of phospholipid fatty acid composition in the renewing intestine were studied in the infant piglet. Newborn piglets were fed from birth to 2 weeks of age a concentrated cow's milk which defined a standard supply of dietary fatty acids. Phospholipids were isolated from the whole mucosa, isolated intestinal cells and purified brush border membranes. Intestinal cells were isolated according to their position along the crypt-villus axis and cell phospholipids were extracted at each step of differentiation. Changes in fatty acid composition of cell phospholipids were related to those of lactase activity in the corresponding cell homogenates. In cell phospholipids, the relative content of linoleic and linoleic acids increased about 2-fold from crypt base to villus tip. Substantial contents of alkenylacyl glycerophospholipids (plasmalogens) were found in crypt cell phospholipids and in purified brush border membrane phosphatidylethanolamine (11 and 14% of alkenyl groups by weight of total fatty acids, respectively). The proportion of alkenylacyl glycerophospholipids decreased as cells ascended the villus column and became more differentiated. The results show that fatty acid compositional changes in differentiating cell phospholipids occurred in the immature intestine (before weaning) and suggest that these alterations might be related to the appearance of specific functions.     

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)     

Changes in the fatty acid composition of blood cell membranes in children with inflammatory diseases

Fatty acid composition of erythrocyte and leukocyte membranes has been studied in children (with normal body mass and obesity) with inflammatory diseases of the gastrointestinal tract and children with bronchial asthma in comparison with basically healthy children. Fifty seven children aged from 7 to 14 years were examined: 13 with inflammatory diseases of the gastrointestinal tract (IDGIT) (eosophagitis, gastroduodenitis, stomach ulcer), 25 with obesity stages (I–III) complicated by IDGIT, 9 with bronchial asthma and 10 basically healthy children. The study revealed that both IDGIT and bronchial asthma caused significant and similar changes in fatty acid composition of cell membranes. These included accumulation of ω3 eicosapentaenoic acid (EPA) and the decrease of docosahexaenoic acid (DHA); this phenomenon observed in both erythrocyte and leukocyte membranes suggests a common feature of the detected changes in fatty acid composition of cell membranes in inflammation. There was a significant decrease in the level of membrane ω6 polyunsaturated fatty acids (PUFA), first of all arachidonic acid and total ω6 PUFA. Consequently, EPA accumulation in membranes may be a compensatory response to low dietary arachidonic acid supply and/or its increased loss for the synthesis of pro-inflammatory eicosanoids (prostaglandins, leukotriens, thromboxans) during inflammatory process.     

Temperature adaptation of biological membranes: differential homoeoviscous responses in brush-border and basolateral membranes of carp intestinal mucosa

The effects of temperature acclimation of carp upon the hydrocarbon order of intestinal membranes has been determined. A fractionation technique has been developed for the simultaneous purification of brush-border and basolateral membrane fractions from the intestinal mucosa. The specific activity of alkaline phosphatase in the brush-border fraction was enhanced 6.4-fold over that of the initial homogenate, whilst the (Na(+)-K+)-stimulated ATPase was enhanced 5.8-fold in the basolateral fraction. The specific activities of NADPH-cytochrome-c reductase, succinate-cytochrome-c reductase and acid phosphatase were not increased in these two fractions. Membrane hydrocarbon order in membranes from 10 and 30 degrees C-acclimated carp has been compared by measuring the steady-state fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene over a range of temperatures. In the brush-border fraction, polarization was identical in both cold- and warm-acclimated groups, whilst large differences were observed in the basolateral fraction sufficient to offset approx. 75% of the temperature-induced ordering effects of cold. The fatty acid composition of the major phosphoglyceride fractions in the brush-border fraction was also largely unaffected by thermal acclimation, whilst the basolateral fraction showed significant increases in the proportion of unsaturated fatty acids in the cold. It is concluded that whilst the basolateral membrane of intestinal mucosa displays a large homoeoviscous response that correlates with a shift in lipid composition, the brush-border membrane does not. These findings are consistent with evidence of functional adaptations of the basolateral membrane during thermal acclimation (Gibson, J.S., Ellory, J.C. and Cossins, A.R. (1985) J. Exp. Biol. 114, 355-364).     

The relatioship between plasma membrane lipid composition and physical-chemical properties II. Effect of phospholipid fatty acid modulation on plasma membrane physical properties and enzymatic activities

The fatty acid composition of plasma membrane phospholipids of the murine T lymphocyte tumor EL4 were systematically modified in an attempt to understand the relationship between lipid bilayer composition and plasma membrane physical and biological properties. Two plasma membrane enzyme activities, adenylate cyclase and ouabain-sensitive ($\text{Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}$)-ATPase, were measured in normal and fatty acid-substituted EL4 plasma membrane fractions. The fatty acid effect on enzyme activities was similar to previously reported effects of fatty acids on cytotoxic T cell function. The activity of both enzymes was inhibited by saturated fatty acids, while unsaturated fatty acids had a moderate enhancing effect on both enzyme activities. Using two different nitroxide derivatives of stearic acid, the order parameter and approximate rotational correlation times were calculated from ESR spectra of normal and fatty acid-modified plasma membranes. No significant difference was found in either parameter in these membranes. These results, in conjunction with earlier data from our laboratory and others, suggest that caution should be exercised in inferring changes in membrane ‘fluidity’ based on lipid modulation of biological membranes.     

Dietary triacylglycerol modulates sodium-dependent D-glucose transport, fluidity and fatty acid composition of rat small intestinal brush-border membrane

Rats were maintained on nutritionally complete diets enriched in unsaturated (menhaden fish oil) or saturated (butter fat) triacylglycerols. After 4 weeks, the animals were killed, proximal small intestinal brush-border membranes were prepared, and examined and compared with respect to their lipid composition, molecular species of phosphatidylcholine, lipid fluidity and sodium-dependent D-glucose transport. Membranes prepared from the two dietary groups were found to possess similar ratios of cholesterol/phospholipid (mol/mol), sphingomyelin/phosphatidylcholine (mol/mol), and protein/lipid (w/w). In contrast to these findings, however, striking differences were noted in the total fatty acid compositions of these membranes. Plasma membranes prepared from animals fed the fish oil diet possessed higher percentages of saturated fatty acids as well as (n - 3) unsaturated fatty acids and lower percentages of monounsaturated and (n - 6) unsaturated fatty acids than those prepared from animals fed the butter fat diet. Analysis of the molecular species of phosphatidylcholine by HPLC, moreover, revealed that membranes from rats fed fish oil had higher levels of 16:0-20:5, 16:0-22:6 and 18:0-20:5 and lower levels of 18:0-18:2 and 16:0-18:1 than their butter fat counterparts. As assessed by steady-state fluorescence polarization, differential polarized phase fluorometric and excimer/monomer fluorescence intensity techniques using various fluorophores, the lipid fluidity of membranes from rats fed fish oil was also found to be significantly lower compared to membranes from rats fed butter fat. Finally, comparison of the kinetic parameters of Na+-dependent D-glucose transport revealed that fish oil-membrane vesicles had a higher maximum velocity (Vmax) than butter fat membrane vesicles but a similar Km for glucose.     

Fatty acids covalently bound to erythrocyte proteins undergo a differential turnover in vivo

Recently, covalently bound fatty acids have been identified on a variety of proteins. Many of these acyl proteins are physiologically important, and the lipid modification often appears to be essential for their function. In this investigation mature erythrocytes have been used to study in detail the metabolic behavior of protein-bound fatty acids. Although deficient in protein synthesis, these cells are still able to covalently attach [3H]palmitic acid to proteins located at the plasma membrane and its associated cytoskeleton. Linkage analyses demonstrated that the labeled polypeptides contained ester- or thioester-bound fatty acids. The covalent binding of fatty acid was rapidly reversible. Half-lives of the protein-bound fatty acid molecules ranged from less than 30 min to more than 3 h. The deacylation reaction was not due to a chemically labile linkage of protein and fatty acid but appeared to be physiologically induced. Differences in the fatty acid turnover rates between the acyl proteins suggested an independent regulation of their lipid turnover. A number of proteins underwent dynamic fatty acid acylation, indicating that palmitylated proteins undergoing fatty acid turnover are not a rare exception.