Determination of regioisomeric distribution in carbohydrate fatty acid monoesters by LC-ESI-MS

A new LC-ESI-MS method for characterizing the regioisomeric distribution in carbohydrate monoesters with long-chain fatty acids is described. Sucrose monolaurate mixtures were used for development of the method. The surfactant nature and high polarity of these compounds make them appropriate analytes for being detected by electrospray-ionization mass spectrometry (ESI-MS). Despite the structural similarity of regioisomers, an excellent resolution of all regioisomers present in the different samples studied (sucrose monodecanoates, sucrose monolaurates, sucrose monopalmitates and melezitose monolaurates) is achieved. Reversed-phase liquid chromatography with isocratic acetonitrile-water mixtures was used for a proper separation of the analytes. Finally, the superiority of this chromatographic method for determining the regioselectivity in enzymatic carbohydrate acylation reactions, with respect to the typical methodology based on routine 13C NMR spectroscopy, is also discussed.     

N-d-Gluco-N-methylalkanamide compounds, a new class of non-ionic detergents for membrane biochemistry

N-d-Gluco-N-methylalkanamide detergents have been synthesized. The detergents, which were produced in high yield and at low cost, compared favourably in biochemical studies with commonly used non-ionic detergents, including a chemically related n-alkyl glucoside. The ease of removal by dialysis, high solubilizing power and non-denaturing properties of this new class of detergents make them valuable reagents for membrane research.     

Docosahexaenoic acid: membrane properties of a unique fatty acid

Docosahexaenoic acid (DHA) with 22-carbons and 6 double bonds is the extreme example of an omega-3 polyunsaturated fatty acid (PUFA). DHA has strong medical implications since its dietary presence has been positively linked to the prevention of numerous human afflictions including cancer and heart disease. The PUFA, moreover, is essential to neurological function. It is remarkable that one simple molecule has been reported to affect so many seemingly unrelated biological processes. Although details of a molecular mode of action remain elusive, DHA must be acting at a fundamental level common to many tissues that is related to the high degree of conformational flexibility that the multiple double bonds have been identified to confer. One likely target for DHA action is at the cell membrane where the fatty acid is known to readily incorporate into membrane phospholipids. Once esterified into phospholipids DHA has been demonstrated to significantly alter many basic properties of membranes including acyl chain order and "fluidity", phase behavior, elastic compressibility, permeability, fusion, flip-flop and protein activity. It is concluded that DHA's interaction with other membrane lipids, particularly cholesterol, may play a prominent role in modulating the local structure and function of cell membranes.     

The biochemistry of hypo- and hyperlipidemic fatty acid derivatives: metabolism and metabolic effects.

A selection of amphipatic hyper- and hypolipidemic fatty acid derivatives (fibrates, thia- and branched chain fatty acids) are reviewed. They are probably all ligands for the peroxisome proliferation activation receptor (PPARalpha) which has a low selectivity for its ligands. These compounds give hyper- or hypolipidemic responses depending on their ability to inhibit or stimulate mitochondrial fatty acid oxidation in the liver. The hypolipidemic response is explained by the following metabolic effects: Lipoprotein lipase is induced in liver where it is normally not expressed. Apolipoprotein CIII is downregulated. These two effects in liver lead to a facilitated (re)uptake of chylomicrons and VLDL, thus creating a direct transport of fatty acids from the gut to the liver. Fatty acid metabolizing enzymes in the liver (CPT-I and II, peroxisomal and mitochondrial beta-oxidation enzymes, enzymes of ketogenesis, and omega-oxidation enzymes) are induced and create an increased capacity for fatty acid oxidation. The increased oxidation of fatty acids "drains" fatty acids from the body, reduces VLDL formation, and ultimately explains the antiadiposity and improved insulin sensitivity observed after administration of peroxisome proliferators.     

Control of fatty acid incorporation in membrane phospholipids. X-ray-induced changes in fatty acid uptake by tumor cells

Lymphosarcoma cells isolated from the spleens of tumor-bearing mice were used to study the effect of a low dose of X-rays (5 Gy) on the incorporation of [³H]palmitate and [¹⁴C]arachidonate into the lipids of the tumor cells. Palmitate and arachidonate were rapidly incorporated especially into the phospholipids of the cells. Between one and three hours after the start of the incubation with radiactive palmitate 80–90% of the label of the total lipids was found in the phospholipid fraction. Already after a few minutes of incubation with radioactive arachidonate, about 95% of the label was incorporated in the phospholipids. Irradiation caused a small but significant increase in the rate of fatty acid incorporation for both fatty acids. Concomitantly, a significantly increased amount of fatty acid was removed from the medium by the cells as a result of the irradiation, and the specific radioactivity of the free fatty acids in the cells was found to be enhanced. The radiation effect on the tumor cells could be mimicked by a hypotonic treatment. The magnitude of the radiation-induced stimulation of the fatty acid incorporation was similar to that of the hypotonically induced effect. Cells which had received a hypotonic treatment before the irradiation, did not show an additional radiation-induced enhancement of fatty acid incorporation into the cellular lipids. When the cells were incubated with serum albumin loaded with a relatively large (non-physiological) amount of complexed fatty acids (fatty acid: albumin molar ratio, $\text{ν}\text{= 3.7}$), no radiation effect on the fatty acid incorporation could be detected. It is concluded that hypotonic treatment, irradiation, and increased supply of exogenous fatty acids all lead to an enhanced flux of fatty acids into the cells. These results confirm our previous suggestion that the uptake of fatty acids through the plasma membrane is the rate-limiting step in the fatty acid incorporation into the phospholipids and that ionizing radiation is one of the means to enhance fatty acid uptake through the plasma membrane leading to an increased incorporation into the phospholipids.     

Essential fatty acids: biochemistry, physiology and pathology

Essential fatty acids (EFAs), linoleic acid (LA), and alpha-linolenic acid (ALA) are essential for humans, and are freely available in the diet. Hence, EFA deficiency is extremely rare in humans. To derive the full benefits of EFAs, they need to be metabolized to their respective long-chain metabolites, i.e., dihomo-gamma-linolenic acid (DGLA), and arachidonic acid (AA) from LA; and eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) from ALA. Some of these long-chain metabolites not only form precursors to respective prostaglandins (PGs), thromboxanes (TXs), and leukotrienes (LTs), but also give rise to lipoxins (LXs) and resolvins that have potent anti-inflammatory actions. Furthermore, EFAs and their metabolites may function as endogenous angiotensin-converting enzyme and 3-hdroxy-3-methylglutaryl coenzyme A reductase inhibitors, nitric oxide (NO) enhancers, anti-hypertensives, and anti-atherosclerotic molecules. Recent studies revealed that EFAs react with NO to yield respective nitroalkene derivatives that exert cell-signaling actions via ligation and activation of peroxisome proliferator-activated receptors. The metabolism of EFAs is altered in several diseases such as obesity, hypertension, diabetes mellitus, coronary heart disease, schizophrenia, Alzheimer's disease, atherosclerosis, and cancer. Thus, EFAs and their derivatives have varied biological actions and seem to be involved in several physiological and pathological processes.     

Rapid synthesis of long chain fatty acid esters of steroids in ionic liquids with microwave irradiation: Expedient one-pot procedure for estradiol monoesters

We report the rapid synthesis (1 min) in high yield of fatty acid ester (FAE) derivatives of several steroids under microwave irradiation in an ionic liquid (IL). An expedient regioselective hydrolysis at C-3 of estradiol diesters is also reported.     

Shifts in membrane fatty acid profiles associated with acid adaptation of Streptococcus mutans

Cells of Streptococcus mutans UA159 physiologically adapted to acidification during growth at pH 5 in glucose-limited chemostat cultures were enriched in mono-unsaturated and longer chain fatty acids compared with unadapted cells grown under the same conditions but at pH 7. Ratios of unsaturated to saturated fatty acids in the cells were, respectively, 1.2 and 0.3. Cyclopropane fatty acids were not detected. Streptococcus sobrinus 6715, which is known to have minimal acid-adaptive capacity, showed only minimal change in membrane fatty acids.     

Multiple fatty acid sensing mechanisms operate in enteroendocrine cells: novel evidence for direct mobilization of stored calcium by cytosolic fatty acid

Fatty acids (FA) with at least 12 carbon atoms increase intracellular Ca(2+) ([Ca(2+)](i)) to stimulate cholecystokinin release from enteroendocrine cells. Using the murine enteroendocrine cell line STC-1, we investigated whether candidate intracellular pathways transduce the FA signal, or whether FA themselves act within the cell to release Ca(2+) directly from the intracellular store. STC-1 cells loaded with fura-2 were briefly (3 min) exposed to saturated FA above and below the threshold length (C(8), C(10), and C(12)). C(12), but not C(8) or C(10), induced a dose-dependent increase in [Ca(2+)](i), in the presence or absence of extracellular Ca(2+). Various signaling inhibitors, including d-myo-inositol 1,4,5-triphosphate receptor antagonists, all failed to block FA-induced Ca(2+) responses. To identify direct effects of cytosolic FA on the intracellular Ca(2+) store, [Ca(2+)](i) was measured in STC-1 cells loaded with the lower affinity Ca(2+) dye magfura-2, permeabilized by streptolysin O. In permeabilized cells, again C(12) but not C(8) or C(10), induced release of stored Ca(2+). Although C(12) released Ca(2+) in other permeabilized cell lines, only intact STC-1 cells responded to C(12) in the presence of extracellular Ca(2+). In addition, 30 min exposure to C(12) induced a sustained elevation of [Ca(2+)](i) in the presence of extracellular Ca(2+), but only a transient response in the absence of extracellular Ca(2+). These results suggest that at least two FA sensing mechanisms operate in enteroendocrine cells: intracellularly, FA (>/=C(12)) transiently induce Ca(2+) release from intracellular Ca(2+) stores. However, they also induce sustained Ca(2+) entry from the extracellular medium to maintain an elevated [Ca(2+)](i).     

Modification of membrane lipid: physical properties in relation to fatty acid structure.

Differential scanning calorimetry (DSC) and electron spin resonance (ESR) measurements were made to characterize how modifications in the fatty acid composition of Escherichia coli affected the thermotropic phase transition(s) of the membrane lipd. When the fatty acid composition contained between 20 and 60% saturated fatty acids, the DSC curves for isolated phospholipids and cytoplasmic membranes showed a broad (15-25 degree C) gel to liquid-crystalline phase transition, the position of which depended on the particular fatty acid composition. Utilizing multiple lipid mutants, enrichment of the membrane phospholipids with a single long-chain cis-monoenoic fatty acid in excess of that possible in a fatty acid levels less than 20% and gradually replaced the broad peak as the cis-monoenoic fatty acid content increased. These results were obtained with phospholipids, cytoplasmic membranes, and whole cells. With these same phopholipids, plots of 2,2,6,6-tetramethylpiperidinyl-1-oxy partitioning and ESR order parameters vs. 1/T revealed discontinuities at temperatures 40-60 degrees C above the calorimetrica-ly measured gel to liquid-crystalline phase transitions. Moreover, when the membrane phospholipids were enriched with certain combinations of cis-monenoic fatty acids (e.g., cis-delta 9-16:1 plus cis-delta 11-18:1) the DSC curve showed a broad gel to liquid crystalline phase change below 0 degrees C but the ESR studies revealed no discontinuities at temperatures above those of the gel to liquid-crystalline transition. These results demonstrated that enrichment of the membrane lipids with molecules in which both fatty acyl chains are identical cis-monoenoic residues led to a distinct type of liquid-crystalline phase. Furthermore, a general conclusion from this study is that Escherichia coli normally maintains a heterogeneous mixture of lipid molecules and, by so doing, prevents strong lipid-lipid associations that lead to the formation of lipid domains in the membrane.     

Detergents as tools in membrane biochemistry.

Detergents are invaluable tools for studying membrane proteins. However, these deceptively simple, amphipathic molecules exhibit complex behavior when they self-associate and interact with other molecules. The phase behavior and assembled structures of detergents are markedly influenced not only by their unique chemical and physical properties but also by concentration, ionic conditions, and the presence of other lipids and proteins. In this minireview, we discuss the various aggregate forms detergents assume and some misconceptions about their structure. The distinction between detergents and the membrane lipids that they may (or may not) replace is emphasized in the most recent high resolution structures of membrane proteins. Detergents are clearly friends and foes, but with the knowledge of how they work, we can use the increasing variety of detergents to our advantage.     

A novel function for fatty acid translocase (FAT)/CD36: involvement in long chain fatty acid transfer into the mitochondria

Fatty acid translocase (FAT)/CD36 is a long chain fatty acid transporter present at the plasma membrane, as well as in intracellular pools of skeletal muscle. In this study, we assessed the unexpected presence of FAT/CD36 in both subsarcolemmal and intermyofibril fractions of highly purified mitochondria. Functional assessments demonstrated that the mitochondria could bind (14)C-labeled palmitate, but could only oxidize it in the presence of carnitine. However, the addition of sulfo-N-succinimidyl oleate, a known inhibitor of FAT/CD36, resulted in an 87 and 85% reduction of palmitate oxidation in subsarcolemmal and intermyofibril fractions, respectively. Further studies revealed that maximal carnitine palmitoyltransferase I (CPTI) activity in vitro was inhibited by succinimidyl oleate (42 and 48% reduction). Interestingly, CPTI immunoprecipitated with FAT/CD36, indicating a physical pairing. Tissue differences in mitochondrial FAT/CD36 protein follow the same pattern as the capacity for fatty acid oxidation (heart > red muscle > white muscle). Additionally, chronic stimulation of hindlimb muscles (7 days) increased FAT/CD36 expression and also resulted in a concomitant increase in mitochondrial FAT/CD36 content (46 and 47% increase). Interestingly, with acute electrical stimulation of hindlimb muscles (30 min), FAT/CD36 expression was not altered, but there was an increase in the mitochondrial content of FAT/CD36 compared with the non-stimulated control limb (35 and 37% increase). Together, these data suggest a role for FAT/CD36 in mitochondrial long chain fatty acid uptake and demonstrate system flexibility to match FAT/CD36 mitochondrial content with an increased capacity for fatty acid oxidation, possibly involving translocation of FAT/CD36 to the mitochondria.     

Microbial production of a novel trihydroxy unsaturated fatty acid from linoleic acid

A bacterium isolated from a dry soil sample collected from McCalla, AL, USA, converted linoleic acid to a novel compound, 12,13,17-trihydroxy-9 (Z)-octadecenoic acid (THOA). The organism is a Gram-positive, non-motile rod (0.5 μ m × 2 μ m). It was identified as a species of Clavibacter ALA2. The product was purified by high pressure liquid chromatography, and its structure was determined by ¹H and ¹³C nuclear magnetic resonance and Fourier transform infrared spectroscopies, and by mass spectrometer. Maximum production of THOA with 25% conversion of the substrate was reached after 5–6 days of reaction. THOA was not further metabolized by strain ALA2. This is the first report of a 12,13,17-trihydroxy unsaturated fatty acid and its production by microbial transformation. Some dihydroxy intermediates were also detected. THOA has a structure similar to those of known plant self-defense substances. Received 13 January 1997/ Accepted in revised form 05 May 1997     

Origin of membrane dipole potential: contribution of the phospholipid fatty acid chains

The large intrinsic membrane dipole potential, phi(d), is important for protein insertion and functioning as well as for ion transport across natural and model membranes. However, the origin of phi(d) is controversial. From experiments carried out with lipid monolayers, a significant dependence on the fatty acid chain length is suggested, whereas in experiments with lipid bilayers, the contribution of additional -CH(2)-groups seems negligibly small compared with that of the phospholipid carbonyl groups and lipid-bound water molecules. To compare the impact of the -CH(2)-groups of dipalmitoylphosphatidylcholine (DPPC) near and far from the glycerol backbone, we have varied the structure of DPPC by incorporation of sulfur atoms in place of methylene groups in different positions of the fatty acid chain. The phi(d) of symmetric lipid bilayers containing one heteroatom was obtained from the charge relaxation of oppositely charged hydrophobic ions. We have found that the substitution for a S-atom of a -CH(2)-group decreases phi(d). The effect (deltaphi(d) = -22.6 mV) is most pronounced for S-atoms near the lipid head group while a S-atom substitution in the C(13)- or C(14)-position of the hydrocarbon chain does not effect the bilayer dipole potential. Most probably deltaphi(d) does not originate from an altered dipole potential of the acyl chain containing an heteroatom but is mediated by the disruption of chain packing, leading to a decreased density of lipid dipoles in the membrane.     

Use of detergents in two-dimensional crystallization of membrane proteins

Structure determination at high resolution is actually a difficult challenge for membrane proteins and the number of membrane proteins that have been crystallized is still small and far behind that of soluble proteins. Because of their amphiphilic character, membrane proteins need to be isolated, purified and crystallized in detergent solutions. This makes it difficult to grow the well-ordered three-dimensional crystals that are required for high resolution structure analysis by X-ray crystallography. In this difficult context, growing crystals confined to two dimensions (2D crystals) and their structural analysis by electron crystallography has opened a new way to solve the structure of membrane proteins. However, 2D crystallization is one of the major bottlenecks in the structural studies of membrane proteins. Advances in our understanding of the interaction between proteins, lipids and detergents as well as development and improvement of new strategies will facilitate the success rate of 2D crystallization. This review deals with the various available strategies for obtaining 2D crystals from detergent-solubilized intrinsic membrane proteins. It gives an overview of the methods that have been applied and gives details and suggestions of the physical processes leading to the formation of the ordered arrays which may be of help for getting more proteins crystallized in a form suitable for high resolution structural analysis by electron crystallography.