Studies of the fatty acid-binding site of rat liver fatty acid-binding protein using fluorescent fatty acids

Rat liver fatty acid-binding protein (FABP) is a 14.3-kDa cytosolic protein which binds long chain free fatty acids (ffa) and is believed to participate in intracellular movement and/or distribution of ffa. In the studies described here fluorescently labeled ffa were used to examine the physical nature of the ffa-binding site on FABP. The fluorescent analogues were 16- and 18-carbon ffa with an anthracene moiety covalently attached at eight different points along the length of the hydrocarbon chain (AOffa). Emission maxima of all FABP-bound AOffa were found to be considerably blue-shifted with respect to emission of phospholipid membrane-bound AOffa, suggesting a high degree of motional constraint for protein-bound ffa. Large fluorescence quantum yields and long excited state life-times indicate that the FABP-binding site for ffa is highly hydrophobic. Analysis of rotational correlation times for the FABP-bound AOffa suggest that the ffa are tightly bound to the protein. Variation of the quantum yield with attachment site suggests that the carboxylic acid group of the fatty acyl chain is located near the aqueous surface of the FABP. The rest of the ffa hydrocarbon chain is buried within the protein in a hydrophobic pocket and is particularly constrained at the midportion of the acyl chain.     

The fatty acid transport function of fatty acid-binding proteins

The intracellular fatty acid-binding proteins (FABPs) comprise a family of 14-15 kDa proteins which bind long-chain fatty acids. A role for FABPs in fatty acid transport has been hypothesized for several decades, and the accumulated indirect and correlative evidence is largely supportive of this proposed function. In recent years, a number of experimental approaches which more directly examine the transport function of FABPs have been taken. These include molecular level in vitro modeling of fatty acid transfer mechanisms, whole cell studies of fatty acid uptake and intracellular transfer following genetic manipulation of FABP type and amount, and an examination of cells and tissues from animals engineered to lack expression of specific FABPs. Collectively, data from these studies have provided strong support for defining the FABPs as fatty acid transport proteins. Further studies are necessary to elucidate the fundamental mechanisms by which cellular fatty acid trafficking is modulated by the FABPs.     

Intersubunit transfer of fatty acyl groups during fatty acid reduction

Fatty acid reduction in Photobacterium phosphoreum is catalyzed in a coupled reaction by two enzymes: acyl-protein synthetase, which activates fatty acids (+ATP), and a reductase, which reduces activated fatty acids (+NADPH) to aldehyde. Although the synthetase and reductase can be acylated with fatty acid (+ATP) and acyl-CoA, respectively, evidence for acyl transfer between these proteins has not yet been obtained. Experimental conditions have now been developed to increase significantly (5-30-fold) the level of protein acylation so that 0.4-0.8 mol of fatty acyl groups are incorporated per mole of the synthetase or reductase subunit. The acylated reductase polypeptide migrated faster on sodium dodecyl sulfate-polyacrylamide gel electrophoresis than the unlabeled polypeptide, with a direct 1 to 1 correspondence between the moles of acyl group incorporated and the moles of polypeptide migrating at this new position. The presence of 2-mercaptoethanol or NADPH, but not NADP, substantially decreased labeling of the reductase enzyme, and kinetic studies demonstrated that the rate of covalent incorporation of the acyl group was 3-5 times slower than its subsequent reduction with NADPH to aldehyde. When mixtures of the synthetase and reductase polypeptides were incubated with [3H] tetradecanoic acid (+ATP) or [3H]tetradecanoyl-CoA, both polypeptides were acylated to high levels, with the labeling again being decreased by 2-mercaptoethanol or NADPH. These results have demonstrated that acylation of the reductase represents an intermediate and rate-limiting step in fatty acid reduction. Moreover, the activated acyl groups are transferred in a reversible reaction between the synthetase and reductase proteins in the enzyme mechanism.     

Structure-function correlation of fatty acyl-CoA dehydrogenase and fatty acyl-CoA oxidase

We have employed a new pseudosubstrate, beta-(2-furyl)propionyl coenzyme A (FPCoA), to study the functional properties of two enzymes, fatty acyl-CoA dehydrogenase from porcine liver and fatty acyl-CoA oxidase from Candida tropicalis, involved in the oxidation of fatty acids. Previous studies from our laboratory have shown that the dehydrogenase exhibits oxidase activity at the rate of dissociation of the product charge-transfer complex. This raises the question of the difference in functionality between these two flavoproteins. To investigate these differences, we have compared the pH dependence of product formation, the isotope effects using tetradeuterio-FPCoA, and the spectral properties and chemical reactivity of the product charge-transfer complexes formed with the two enzymes. The pH dependencies of the reaction of FPCoA with electron-transfer flavoprotein (ETF) for the dehydrogenase and of the reaction of FPCoA with O2 for the oxidase are quite similar. Both reactions proceed more rapidly at basic pH values while substrate binds more tightly at acidic pH values. These data for both enzymes are consistent with a mechanism in which enzyme is involved in protonation of the carbonyl group of substrate followed by base-catalyzed removal of the C-2 proton from substrate. The C-2 anion of substrate may then serve as the active species in reduction of enzyme-bound flavin. The deuterium isotope effects for both enzyme systems are primary across the entire pH range, assuring that the chemically important step of substrate oxidation is rate limiting in these steady-state kinetic experiments. The two enzymes differ in the chemical reactivity of their product charge-transfer complexes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Production of hydroxy fatty acids by microbial fatty acid-hydroxylation enzymes

Hydroxy fatty acids are widely used in chemical, food, and cosmetic industries as starting materials for the synthesis of polymers and as additives for the manufacture of lubricants, emulsifiers, and stabilizers. They have antibiotic, anti-inflammatory, and anticancer activities and therefore can be applied for medicinal uses. Microbial fatty acid-hydroxylation enzymes, including P450, lipoxygenase, hydratase, 12-hydroxylase, and diol synthase, synthesize regio-specific hydroxy fatty acids. In this article, microbial fatty acid-hydroxylation enzymes, with a focus on region-specificity and diversity, are summarized and the production of mono-, di-, and tri-hydroxy fatty acids is introduced. Finally, the production methods of regio-specific and diverse hydroxy fatty acids, such as gene screening, protein engineering, metabolic engineering, and combinatory biosynthesis, are suggested.     

Role of fatty acid-binding protein in cardiac fatty acid oxidation

Summary Although abundant in most biological tissues and chemically well characterized, the fatty acid-binding protein (FABP) was until recently in search of a function. Because of its strong affinity for long chain fatty acids and its cytoplasmic origin, this protein was repeatedly claimed in the literature to be the transcytoplasmic fatty acid carrier. However, techniques to visualize and quantify the movements of molecules in the cytoplasm are still in their infancy. Consequently the carrier function of FABP remains somewhat speculative. However, FABP binds not only fatty acids but also their CoA and carnitine derivatives, two typical molecules of mitochondrial origin. Moreover, it has been demonstrated and confirmed that FABP is not exclusively cytoplasmic, but also mitochondrial. A function for FABP in the mitochondrial metabolism of fatty acids plus CoA and carnitine derivatives would therefore be anticpated. Using spin-labelling techniques, we present here evidence that FABP is a powerful regulator of acylcarnitine flux entering the mitochondrial -oxidative system. In this perspective FABP appears to be an active link between the cytoplasm and the mitochondria, regulating the energy made available to the cell. This active participation of FABP is shown to be the consequence of its gradient-like distribution in the cardiac cell, and also of the coexistence of multispecies of this protein produced by self-aggregation.     

Coupling of the biosynthesis of fatty acids and fatty alcohols.

A cell-free system for the biosynthesis of fatty alcohols in the pink portion of the rabbit harderian gland is described. The radiolabeled substrates for the fatty acid reductase were generated using soluble fatty acid synthase from the gland in the presence of acetyl-CoA, malonyl-CoA, and NADPH. Harderian gland microsomes, ATP, and Mg²⁺ were absolute requirements for the synthesis of fatty alcohols and if any of these components were deleted from the assay mixture, no alcohols were detected. We were also unable to detect formation of fatty alcohols if acyl-CoAs were substituted for fatty acid synthase with either NADPH or NADH as reducing agents. The reductase was localized in the microsomal fraction and appears to be on the cytosol-membrane interface of the vesicles, as indicated in experiments using detergents and trypsin. The fatty alcohols formed by the system had the same chain length distribution as the fatty acids made by the fatty acid synthase. The alkyl moieties of the ether lipids in the harderian gland are exclusively saturated and the properties of the alcohol-synthesizing system described in this report can account for the observed exclusion of unsaturated alkyl moieties from the ether lipids of this gland.     

Specific inactivation of hepatic fatty acid hydroxylases by acetylenic fatty acids

The terminal acetylenic analogue of lauric acid, 11-dodecynoic acid (11-DDYA), specifically inactivates hepatic cytochrome P-450 enzymes that catalyze omega- and omega-1-hydroxylation of lauric acid. The inactivation, as required for a suicidal process, is NADPH- and time-dependent and follows pseudo-first order kinetics. In contrast, 11-DDYA causes no measurable change in the spectroscopically-measured concentration of cytochrome P-450 or in the N-demethylation of benzphetamine or N-methyl p-chloroaniline. 10-Undecynoic acid is as effective a suicide substrate for fatty acid hydroxylases as 11-DDYA but 11-dodecenoic acid is much less effective. 11-DDYA is able to completely inhibit omega-hydroxylation but suppresses no more than 50% of omega-1-hydroxylation despite the fact that both activities are completely inactivated by 1-aminobenzotriazole. At least three hepatic cytochrome P-450 fatty acid hydroxylases, one omega-hydroxylase and two omega-1-hydroxylases, are required by these results. The construction of suicide substrates that specifically inactivate cytochrome P-450 fatty acid hydroxylases provides a new experimental probe of the physiological role of this process.     

Free fatty acid mobilization in the development of cerium-induced fatty liver

Following cerium injection to female rats: (1) Plasma free fatty acids (FFA) concentration increases during the first 24 hours, then remains constant up to 48 hours. (2) Adipose tissue lipolytic activity increases tremendously during the first 12 hours (+380%), maintaining high values throughout the study (48hrs). These modifications are followed by a time-dependent increase of total liver lipids consisting mainly of triglycerides and to a less extent of cholesterol. (3) Adrenalectomy prevented the development of cerium-induced fatty liver: plasma FFA and lipolysis failed to increase in adrenalectomized cerium-treated animals. Thus, our study demonstrates the involvement of adrenergic stimulation of adipose tissue lipase as an obligatory step in the development of cerium-induced fatty liver.     

Insights into binding of fatty acids by fatty acid binding proteins

Members of the phylogenetically related intracellular lipid binding protein (iLBP) are characterized by a highly conserved tertiary structure, but reveal distinct binding preferences with regard to ligand structure and conformation, when binding is assessed by the Lipidex method (removal of unbound ligand by hydrophobic polymer) or by isothermal titration calorimetry, a true equilibrium method. Subfamily proteins bind retinoids, subfamily II proteins bind bulky ligands, examples are intestinal bile acid binding protein (I-BABP) and liver fatty acid binding protein (L-FABP) which binds 2 ligand molecules, preferably monounsaturated and n-3 fatty acids. Subfamily III intestinal fatty acid binding protein (I-FABP) binds fatty acid in a bent conformation. The fatty acid bound by subfamily IV FABPs has a U-shaped conformation; here heart (H-) FABP preferably binds n-6, brain (B-) FABP n-3 fatty acids. The ADIFAB-method is a fluorescent test for fatty acid in equilibrium with iLBP and reveals some correlation of binding affinity to fatty acid solubility in the aqueous phase; these data are often at variance with those obtained by the other methods. Thus, in this review published binding data are critically discussed, taking into account on the one hand binding increments calculated for fatty acid double bonds on the basis of the solubility hypothesis, on the other hand the interpretation of calorimetric data on the basis of crystallographic and solution structures of iLBPs.     

Characterization of fatty amides produced by lipase-catalyzed amidation of multi-hydroxylated fatty acids

Novel multi-hydroxylated primary fatty amides produced by direct amidation of 7,10-dihydroxy-8(E)-octadecenoic acid and 7,10,12-trihydroxy-8(E)-octadecenoic acid were characterized by GC-MS and NMR. The amidation reactions were catalyzed by immobilized Pseudozyma (Candida) antarctica lipase B (Novozym 435) in organic solvent with ammonium carbamate. The mass spectra of the underivatized products exhibited characteristic primary amide peaks at m/z 59 and m/z 72 that differed in peak intensities. Other peaks present were consistent with cleavage next to the hydroxyl groups. The mass spectra of the silylated amidation products showed the correct molecular weight and the typical fragmentation pattern of silylated hydroxy compounds. The mass spectra, together with proton and 13C NMR data, suggest that the products of lipase-catalyzed direct amidation of 7,10-dihydroxy-8(E)-octadecenoic acid and 7,10,12-trihydroxy-8(E)-octadecenoic acid are, 7,10-dihydroxy-8(E)-octadecenamide and 7,10,12-trihydroxy-8(E)-octadecenamide acid, respectively. Amidation of multi-hydroxylated fatty acids had increased the melting point, but reduced the surface active property of the resulting primary amides.     

Partition of fatty acids

The partition ratios of radioactive fatty acids between n-heptane and a physiological buffer at 37 degrees C were measured. The fatty acids included the saturated acids with an even number of carbons from 10 to 18 and the unsaturated acids oleic, linoleic, and linolenic. In addition, the partition ratios of decanoate, myristate, and palmitate were determined over a wide pH range. Any single plot of partition ratio vs. aqueous concentration of an acid gave a nearly straight line, a finding consistent with very little association in the aqueous phase. In the case of the acids with 16 and 18 carbon atoms, however, comparison of the constants calculated from these plots with the assumption of no aqueous phase association revealed several inconsistencies. These inconsistencies cannot be resolved completely by assuming the existence of fatty acid association in the aqueous solution. We believe that at least some of the deviations are due to the presence of trace quantities of radioactive impurities in the labeled fatty acids. For example, purification of a sample of supposedly pure [1-(14)C]myristate by a series of solvent extractions increased the partition ratio by a factor of 1.5. Although all of the observations cannot be explained by this interpretation, we believe that our studies suggest that there is no appreciable association of fatty acids under the usual physiological conditions.