Carnitine palmitoyltransferase activities: effects of serum albumin, acyl-CoA binding protein and fatty acid binding protein

The carnitine palmitoyltransferase activity of various subcellular preparations measured with octanoyl-CoA as substrate was markedly increased by bovine serum albumin at low microM concentrations of octanoyl-CoA. However, even a large excess (500 microM) of this acyl-CoA did not inhibit the activity of the mitochondrial outer carnitine palmitoyltransferase, a carnitine palmitoyltransferase isoform that is particularly sensitive to inhibition by low microM concentrations of palmitoyl-CoA. This bovine serum albumin stimulation was independent of the salt activation of the carnitine palmitoyltransferase activity. The effects of acyl-CoA binding protein (ACBP) and the fatty acid binding protein were also examined with palmitoyl-CoA as substrate. The results were in line with the findings of stronger binding of acyl-CoA to ACBP but showed that fatty acid binding protein also binds acyl-CoA esters. Although the effects of these proteins on the outer mitochondrial carnitine palmitoyltransferase activity and its malonyl-CoA inhibition varied with the experimental conditions, they showed that the various carnitine palmitoyltransferase preparations are effectively able to use palmitoyl-CoA bound to ACBP in a near physiological molar ratio of 1:1 as well as that bound to the fatty acid binding protein. It is suggested that the three proteins mentioned above affect the carnitine palmitoyltransferase activities not only by binding of acyl-CoAs, preventing acyl-CoA inhibition, but also by facilitating the removal of the acylcarnitine product from carnitine palmitoyltransferase. These results support the possibility that the acyl-CoA binding ability of acyl-CoA binding protein and of fatty acid binding protein have a role in acyl-CoA metabolism in vivo.     

Effects of simvastatin on the metabolism of polyunsaturated fatty acids and on glycerolipid, cholesterol, and de novo lipid synthesis in THP-1 cells

In the monocytic THP-1 cells, the 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor simvastatin (5 microM) enhances the conversion of exogenous linoleic (18:2 n-6) and eicosapentaenoic (20:5 n-3) acids to their long-chain polyunsaturated fatty acid (LC-PUFA) derivatives, and this effect is associated with changes in the desaturation steps. In addition, formation of monounsaturated fatty acids from endogenously synthesized precursors is increased. These metabolic changes lead to elevated LC-PUFA and fatty acid (FA) unsaturation in cells. The effects of simvastatin on FA metabolism are associated with increased synthesis of triglycerides from glycerol. The dose-effect relationships for the activity of simvastatin on total linoleic acid (LA) conversion and cholesterol synthesis reveal that enhancement of PUFA metabolism is already maximal at 0.5 microM simvastatin, whereas cholesterol synthesis is further inhibited by concentrations of simvastatin up to 5 microM. The effects of 5 microM simvastatin on PUFA metabolism are partially prevented by mevalonate (1 mM) and geranylgeraniol (5 microM) but not by farnesol (10 microM). These data indicate that HMG-CoA inhibitors have profound effects on PUFA metabolism, and that the pathways for cholesterol and PUFA synthesis are mutually modulated.     

Effects of curcumin on P-glycoprotein in primary cultures of rat hepatocytes

Curcumin is a natural phenolic compound found in the rhizomes of Curcuma longa and endowed with beneficial biological activities including antioxidant, anticarcinogenic and hepatoprotective effects. In this study curcumin was tested for its potential ability to interact in vitro with hepatic P-glycoprotein (Pgp), in a model system represented by primary cultures of rat hepatocytes, in which spontaneous overexpression of multidrug resistance (mdr) genes occurs. In both freshly-plated hepatocytes, containing low levels of Pgp, and 72 hour-cultured hepatocytes, containing high levels of Pgp, the Rhodamine-123 (R-123) efflux, which represents a specific functional test for Pgp-mediated transport, was inhibited by curcumin in a dose-dependent manner. Western blot analysis showed that 25microM curcumin, when included in the culture medium throughout the experimental observation (72 hours), was able to significantly lower the increase of mAb C219-immunoreactive protein spontaneously occurring in the cells during culture. Curcumin, at doses ranging from 50 to 150microM was cytotoxic for freshly-plated hepatocytes, as shown by the strong decrease in the cell ability to exclude trypan blue 24 hours later, but it was significantly less cytotoxic when added to 24 or 48 hour-cultured cells. The resistance to curcumin, progressively acquired by cells during culture, was significantly reduced by high concentrations of dexamethasone (DEX) or dimethyl-sulfoxide (DMSO), culture conditions known to inhibit the spontaneous overexpression of Pgp. In addition, in a concentration-dependent manner, verapamil reverted curcumin resistance in Pgp overexpressing hepatocytes. In photoaffinity labeling studies, curcumin competed with azidopine for binding to Pgp, suggesting a direct interaction with glycoprotein. These results suggest that curcumin is able to modulate in vitro both expression and function of hepatic Pgp and support the hypothesis that curcumin, a chemopreventive phytochemical, could reveal itself also as a compound endowed with chemosensitizing properties on mdr phenotype.     

Unsaturated fatty acids induce inhibition of the A1-adenosine receptor in rat brain membranes

We examined the effect of fatty acids (FA) and some other bioactive lipids on A1-adenosine receptor (A1-AR) binding in rat brain membranes using the selective agonist (3H)-N6-cyclohexyladenosine [(3H)CHA]. A significant reduction in ligand binding was observed at micromolar concentrations of unsaturated fatty acid with the following potency: oleic < arachidonic < decosaenoic < linoleic < linolenic acid. The other tested compounds: lysophospholipids, platelet-activating factor (PAF) and GM1-ganglioside were without effect on A1-AR expression. The inhibition with unsaturated FA was non-competitive and partially reversed by albumin. The A1-AR agonist binding inhibition evoked by unsaturated FA in many respects is similar to that observed previously following ischaemia and may be contributory to the increased excitability of post-ischaemic brain.     

The effect of curcumin on human B-cell immortalization by Epstein-Barr virus

Cyclosporine is a commonly used immunosuppressant in solid-organ transplantation. It is, however, associated with an increased incidence of Epstein-Barr virus (EBV)induced post-transplant lymphoproliferative disorder (PTLD). In this study, human B lymphocytes isolated from healthy volunteers were immortalized in vitro with EBV. The effect of oxidative stress mediated by cyclosporine A or hydrogen peroxide on in vitro B cell immortalization was studied by coculturing immortalized B cells with cyclosporine A and hydrogen peroxide. Curcumin, a phenolic extract of the spice turmeric, was then used to observe its effect on this process. We found that in vitro B-cell immortalization with EBV was promoted by the oxidative stress induced by cyclosporine A and hydrogen peroxide, with the maximum effect seen at concentrations of 500 ng/ml and 100 microM, respectively. Curcumin blocked the B-cell immortalization in a dose-dependent fashion with nearly complete inhibition at 20 microM. We conclude that, because both hydrogen peroxide and cyclosporine A strongly promote in vitro B-cell immortalization with EBV (the putative process responsible for PTLD) and curcumin, an extract of a common spice is an effective inhibitor of this process; curcumin may be an effective adjunct in the prevention of PTLD in the patients undergoing therapy with cyclosporine A.     

Fatty acids stimulate trehalose synthesis in trophocytes of the cockroach (Periplaneta americana) fat body

Trophocytes from the disaggregated fat body of the cockroach (Periplaneta americana) respond to synthetic hypertrehalosemic hormone (HTH) by increasing the rate of trehalose synthesis. The cells give a similar response when incubated with stearic, oleic, linoleic, or arachidonic acid. A maximal increase in trehalose synthesis was obtained with 1-10 microM fatty acids. Synthesis of trehalose by the trophocytes was also increased by 1 microM prostaglandin F2alpha to nearly the same extent as that evoked by HTH. Furthermore, the data show that the trophocytes are capable of converting linoleic acid into arachidonic acid. This suggests that the cells may convert arachidonic acid, formed from the linoleic acid released by the action of HTH, to a prostaglandin which serves as an integral part of the hypertrehalosemic mechanism.     

Fatty acids suppress voltage-gated Na+ currents in HEK293t cells transfected with the alpha-subunit of the human cardiac Na+ channel

Studies have shown that fish oils, containing n-3 fatty acids, have protective effects against ischemia-induced, fatal cardiac arrhythmias in animals and perhaps in humans. In this study we used the whole-cell voltage-clamp technique to assess the effects of dietary, free long-chain fatty acids on the Na+ current (INa,alpha) in human embryonic kidney (HEK293t) cells transfected with the alpha-subunit of the human cardiac Na+ channel (hH1alpha). Extracellular application of 0.01 to 30 microM eicosapentaenoic acid (EPA, C20:5n-3) significantly reduced INa,alpha with an IC50 of 0.51 +/- 0.06 microM. The EPA-induced suppression of INa,alpha was concentration- and voltage-dependent. EPA at 5 microM significantly shifted the steady-state inactivation relationship by -27.8 +/- 1.2 mV (n = 6, P < 0.0001) at the V1/2 point. In addition, EPA blocked INa,alpha with a higher "binding affinity" to hH1alpha channels in the inactivated state than in the resting state. The transition from the resting state to the inactivated state was markedly accelerated in the presence of 5 microM EPA. The time for 50% recovery from the inactivation state was significantly slower in the presence of 5 microM EPA, from 2.1 +/- 0.8 ms for control to 34.8 +/- 2.1 ms (n = 5, P < 0.001). The effects of EPA on INa,alpha were reversible. Furthermore, docosahexaenoic acid (C22:6n-3), alpha-linolenic acid (C18:3n-3), conjugated linoleic acid (C18:2n-7), and oleic acid (C18:1n-9) at 5 microM and all-trans-retinoic acid at 10 microM had similar effects on INa,alpha as EPA. Even 5 microM of stearic acid (C18:0) or palmitic acid (C16:0) also significantly inhibited INa, alpha. In contrast, 5 microM EPA ethyl ester did not alter INa,alpha (8 +/- 4%, n = 8, P > 0.05). The present data demonstrate that free fatty acids suppress INa,alpha with high "binding affinity" to hH1alpha channels in the inactivated state and prolong the duration of recovery from inactivation.     

Linoleic acid peroxidation by Solanum tuberosum lipoxygenase was activated in the presence of human 5-lipoxygenase-activating protein

The present investigation describes the ability of human 5-lipoxygenase-activating protein (FLAP) to activate a plant 5-lipoxygenase. The presence of an active recombinant human FLAP in the 100000xg membrane fraction of infected Sf9 cells led to a specific increase in 9-hydroperoxyoctadecadienoic acid (9-HPOD) synthesis (+68%) or in 5-hydroperoxyeicosatetraenoic acid (5-HPETE) synthesis (+68%), after action of Solanum tuberosum tuber 5-lipoxygenase (S.t.LOX) on linoleic acid (natural plant lipoxygenase substrate) or on arachidonic acid. On the contrary, the presence of non-transfected membranes obtained from non-infected Sf9 cells led to an inhibition of lipoxygenase activity. MK-886, a potent inhibitor of leukotriene biosynthesis, blocked the FLAP dependent S.t.LOX activation after preincubation with FLAP transfected membranes. In conclusion, this study demonstrates that a recombinant human FLAP can stimulate a lipoxygenase other than mammalian 5-lipoxygenase (S.t.LOX) by using different polyunsaturated fatty acids as substrates.     

Distinct effects of fatty acids on translocation of gamma- and epsilon-subspecies of protein kinase C

Effects of fatty acids on translocation of the gamma- and epsilon-subspecies of protein kinase C (PKC) in living cells were investigated using their proteins fused with green fluorescent protein (GFP). gamma-PKC-GFP and epsilon-PKC-GFP predominated in the cytoplasm, but only a small amount of gamma-PKC-GFP was found in the nucleus. Except at a high concentration of linoleic acid, all the fatty acids examined induced the translocation of gamma-PKC-GFP from the cytoplasm to the plasma membrane within 30 s with a return to the cytoplasm in 3 min, but they had no effect on gamma-PKC-GFP in the nucleus. Arachidonic and linoleic acids induced slow translocation of epsilon-PKC-GFP from the cytoplasm to the perinuclear region, whereas the other fatty acids (except for palmitic acid) induced rapid translocation to the plasma membrane. The target site of the slower translocation of epsilon-PKC-GFP by arachidonic acid was identified as the Golgi network. The critical concentration of fatty acid that induced translocation varied among the 11 fatty acids tested. In general, a higher concentration was required to induce the translocation of epsilon-PKC-GFP than that of gamma-PKC-GFP, the exceptions being tridecanoic acid, linoleic acid, and arachidonic acid. Furthermore, arachidonic acid and the diacylglycerol analogue (DiC8) had synergistic effects on the translocation of gamma-PKC-GFP. Simultaneous application of arachidonic acid (25 MicroM) and DiC8 (10 microM) elicited a slow, irreversible translocation of gamma-PKC- GFP from the cytoplasm to the plasma membrane after rapid, reversible translocation, but a single application of arachidonic acid or DiC8 at the same concentration induced no translocation. These findings confirm the involvement of fatty acids in the translocation of gamma- and epsilon-PKC, and they also indicate that each subspecies has a specific targeting mechanism that depends on the extracellular signals and that a combination of intracellular activators alters the target site of PKCs.     

Antiproliferative effect of curcumin (diferuloylmethane) against human breast tumor cell lines

Pharmacologically safe compounds that can inhibit the proliferation of tumor cells have potential as anticancer agents. Curcumin, a diferuloylmethane, is a major active component of the food flavor turmeric (Curcuma longa) that exhibits anticarcinogenic properties in vivo. In vitro, it suppressed c-jun/Ap-1 and NF-kappaB activation and type 1 human immunodeficiency virus long-terminal repeat-directed gene expression. We examined the antiproliferative effects of curcumin against several breast tumor cell lines, including hormone-dependent and -independent and multidrug-resistant (MDR) lines. Cell growth inhibition was monitored by [3H]thymidine incorporation, Trypan blue exclusion, crystal violet dye uptake and flow cytometry. All the cell lines tested, including the MDR-positive ones, were highly sensitive to curcumin. The growth inhibitory effect of curcumin was time- and dose-dependent, and correlated with its inhibition of ornithine decarboxylase activity. Curcumin preferentially arrested cells in the G2/S phase of the cell cycle. Curcumin-induced cell death was neither due to apoptosis nor to any significant change in the expression of apoptosis-related genes, including Bcl-2, p53, cyclin B and transglutaminase. Overall our results suggest that curcumin is a potent antiproliferative agent for breast tumor cells and may have potential as an anticancer agent.     

Effect on the partition equilibrium of various drugs by the formation of mixed bile salt/phosphatidylcholine/fatty acid micelles. A characterization by micellar affinity capillary electrophoresis. Part IV

Mixed micelles, which mimic the bile containing fatty acids in the gastrointestinal tract, were used as a pseudostationary phase in capillary electrophoresis. The mixed micellar system studied contained the dihydroxy bile salts sodium glycodeoxycholate or sodium taurodeoxycholate or the trihydroxy bile salt sodium taurocholate, in association with different sodium salts of fatty acids including lauric, myristic, palmitic, oleic, stearic and linoleic acid and lecithin or dipalmitoylphosphatidylcholine as phospholipid. The determination of the changing mobilities of ionic analytes in the presence of mixed micelles reflected interactions between the used drugs and the mixed micelles. These were determined as dependence on the fatty acid concentration in the bile salt/fatty acid micelles and the mixed bile salt/phosphatidylcholine/fatty acid micelles. The capacity factor kappa MMC, for the partition between mixed micellar and aqueous phase was calculated. The partition equilibrium of basic and acidic drugs depends considerably on shape and charge of the mixed micelles (dependent on the fatty acid concentration) as well as on the acid-base properties of the drug. The mobility of the micelle aggregates was determined as an important reference value to the calculations of kappa MMC. This paper also describes the use of laser-induced fluorescence detection and electrospray mass spectrometry and tandem mass spectrometry for the characterization of the mixed micelle composition.     

Fatty acid binding protein. Role in esterification of absorbed long chain fatty acid in rat intestine

Fatty acid binding protein (FABP) is a protein of 12,000 mol wt found in cytosol of intestinal mucosa and other tissues, which exhibits high affinity for long chain fatty acids. It has been suggested that FABP (which may comprise a group of closely related proteins of 12,000 mol wt) participates in cellular fatty acid transport and metabolism. Although earlier findings were consistent with this concept, the present studies were designed to examine its physiological function more directly. Everted jejunal sacs were incubated in mixed fatty acid-monoglyceride-bile acid micelles, in the presence or absence of equimolar concentrations of either of two compounds which inhibit oleate binding to FABP:flavaspidic acid-N-methyl-glucaminate and alpha-bromopalmitate. Oleate uptake, mucosal morphology, and oxidation of [14C]acetate remained unaffected by these agents, but oleate incorporation into triglyceride was inhibited by 62-64% after 4 min. The inhibition by flavaspidic acid was reversible with higher oleate concentrations. The effect of these compounds on enzymes of triglyceride biosynthesis was examined in intestinal microsomes. Neither flavaspidic acid nor alpha-bromopalmitate inhibited acyl CoA:monoglyceride acyl-transferase. Fatty acid:coenzyme A ligase activity was significantly enhanced in the presence of partially purified FABP, probably reflecting a physical effect on the fatty acid substrate or on the formation of the enzyme-substrate complex. Activity of the enzyme in the presence of 0.1 mM oleate was only modestly inhibited by equimolar flavaspidic acid and alpha-bromopalmitate, and this effect was blunted or prevented by FABP. We conclude that in everted gut sacs, inhibition of triglyceride synthesis by flavaspidic acid and alpha-bromopalmitate could not be explained as an effect on fatty acid uptake or on esterifying enzymes in the endoplasmic reticulum but rather can be interpreted as reflecting inhibition of fatty acid binding to FABP. These findings lend further support to the concept that FABP participates in cellular fatty acid transport and metabolism. It is also possible that FABP, by effecting an intracellular compartmentalization of fatty acids and acyl CoA, may play a broader role in cellular lipid metabolism.     

Sensitivity of Staphylococcus aureus to unsaturated fatty acids

Some unsaturated fatty acids were found to inhibit the growth of Staphylococcus aureus. Their effectiveness was related both to the degree of unsaturation and to the configuration of the molecule about the double bonds. Both linoleic acid and linolenic acid increased the proportion of plasmid-negative bacteria in a growing culture of bacteria containing a penicillinase plasmid. This was not due to a 'curing' effect of the fatty acids but was the result of greater sensitivity of the growth of bacteria containing penicillinase plasmid to inhibition by the unsaturated fatty acids. The presence of a plasmid conferring resistance to tetracycline, however, did not make the bacterium more sensitive to inhibition by linoleic or linolenic acids.     

A new version of the "token test" for aphasics: a concrete objects form

The preparation of PC with the 14C-fatty acids palmitic, stearic, oleic and linoleic acid at OH-group in position 2 is described. The starting material is egg yolk lecithin. By the attack of snake venom phospholiphase lyso-PC is produced which is reacylated by the appropriate fatty acid anhydride. In comparison with the methods published up to now this preparation has the advantage of higher yields and greater simplicity. By means of in vivo synthesis it is impossible to get PC species with only one fatty acid in a defined position. Working with radioactive fatty acids the specific radioactivity can be adapted to the requirements. The procedure can be made on a semi-technical scale.     

Exogenous fatty acids modulate the functional and cytotoxic responses of cultured pulmonary artery endothelial cells to oxidant stress

We previously reported that supplementation with exogenous fatty acids modulated the susceptibility of cultured pulmonary artery endothelial cells (PAEC) to oxidant-mediated cytotoxicity. The current study investigates the effects of fatty acids with increasing degrees of unsaturation on oxidant-mediated dysfunction and cytotoxicity in cultured porcine pulmonary artery and aortic endothelial cells (AEC). Monolayers supplemented with 0.1 mmol/L oleic (18:1), linoleic (18:2), or gamma-linolenic (18:3) acids were exposed to oxidant stress (100 mumol/L hydrogen peroxide (H2O2)) or to control conditions for 30 minutes. Gas chromatographic analysis of the PAEC fatty acids confirmed incorporation of supplemental fatty acids into PAEC lipids. Cytotoxicity, measured as the release of intracellular lactate dehydrogenase (LDH), and PAEC monolayer barrier function, assessed by measuring the monolayer clearance of Evans blue dye bound to albumin, were determined for 1 to 3 hours after oxidant stress. The PAEC and AEC demonstrated comparable responses to H2O2. Hydrogen peroxide caused increases in monolayer permeability and detachment of cells from the monolayer that were most attenuated by supplementation with 18:2 or 18:3, and to a lesser degree with 18:1. In contrast, H2O2-mediated LDH release was attenuated by supplementation with 18:1, whereas 18:2 and 18:3 potentiated cytotoxicity after exposure to H2O2. These results indicate that the relationship between PAEC lipid composition and oxidant susceptibility is complex and that the extent of fatty acid unsaturation does not predict the functional or cytotoxic responses of PAEC to oxidant stress. Furthermore, these results suggest that functional derangements may not correlate with traditional assays of cytotoxicity induced by oxidant injury in cultured endothelium.     

Fatty acid composition of plasma phosphatidylcholine and levels of lipids and lipoproteins in hyperlipoproteinemia. II. Relation with B lipoproteins

BACKGROUND: Epidemiological trials provided evidence that the cholesterol concentration in lipoproteins B, i.e. VDL, IDL and LDL, correlate significantly with the incidence of ischaemic heart disease (IHD). The objective of the present study was to assess how the fatty acid composition in plasma phosphatidyl choline affects the total and LDL cholesterol, triglyceride and apolipoprotein B concentrations in subjects with primary hyperlipoproteinaemia and dyslipidaemia. METHODS AND RESULTS: In a group of 142 subjects with primary hyperlipoproteinaemia and dyslipidaemia the concentrations of plasma lipids, lipoproteins apolipoproteins and fatty acids in plasma phosphatidyl choline (PC) were assessed. The authors provided evidence by discriminant analysis where the dependent variables were the lower quintiles (Q1 + Q2) and the upper quintiles (Q4 + Q5) of concentrations of total cholesterol, triglycerides, LDL-cholesterol and apolipoprotein B and the independent variables were FA concentrations in plasma PC, that the total cholesterol concentration was inversely associated with the concentration of docosahexaenic acid (22:6n-3). The concentration of LDL-cholesterol correlated inversely with the concentration of palmitoleic acid (16:1n-7). Triglyceridaemia was inversely associated with the linoleic acid concentration (18:2n-6). The concentration of apolipoprotein B correlated positively with myristic acid (14:0) and negatively with concentrations of oleic acid (18:1n-9) and linoleic acid (18:2n-6). CONCLUSIONS: The submitted results indicate that the fatty acid concentrations of PC in plasma are significantly and markedly correlated with concentrations of total cholesterol, triglycerides, LDL-cholesterol and apolipoprotein B. It is possible that atherogenic lipoproteins may be favourably influenced not only by the amount of fat but also by a suitable fatty acid composition.     

The effect of different molecular species of sphingomyelin on phospholipase C delta 1 activity

Bovine brain sphingomyelin was separated into different molecular species using a reverse phase column. PLC delta 1 was inhibited by all molecular species of sphingomyelin. The extent of this inhibition was dependent on the hydrophobicity. Based on fatty acid analysis, we conclude that the inhibition of PLC delta 1 depends on the chain length and degree of unsaturation of the fatty acid moiety of SM. N-palmitoyl-D-sphingomyelin and N-stearoyl-D-sphingomyelin inhibited PLC delta 1 less then N-oleoyl-D-sphingomyelin. In the absence of Ca2+ (1 mM EGTA) all tested molecular species of SM inhibited weakly the enzyme. The sensitivity of PLC delta 1 to inhibition by SM increased with increasing Ca2+ concentration. The shape of calcium curve differed for molecular species with saturated and unsaturated fatty acids. Inhibition of PLC delta 1 by N-palmitoyl-D-sphingomyelin and N-stearoyl-D-sphingomyelin reached a maximum at 0.2 microM Ca2+, while inhibition by N-oleoyl-D-sphingomyelin reached maximum at 2 microM Ca2+. PLC delta 1 is more sensitive to inhibition by SM when it is maximally activated by spermine and calcium and the extent of this inhibition depends on the length and degree of fatty acid unsaturation of the molecular species.     

The rabbit 15-lipoxygenase preferentially oxygenates LDL cholesterol esters, and this reaction does not require vitamin E

The oxidation of low density lipoprotein (LDL) by mammalian 15-lipoxygenases (15-LOX) was implicated in early atherogenesis. We investigated the molecular mechanism of 15-LOX/LDL interaction and found that during short term incubations, LDL cholesterol esters are oxygenated preferentially by the enzyme. Even when the LDL particle was loaded with free linoleic acid, cholesteryl linoleate constituted the major LOX substrate. In contrast, only small amounts of free oxygenated fatty acid isomers were detected, and re-esterification of oxidized fatty acids into the LDL ester lipid fraction was ruled out. When LDL was depleted from alpha-tocopherol, specific oxygenation of the cholesterol esters was not prevented, and the product pattern was not altered. Similar results were obtained at low (LDL/LOX ratio of 1:1) and high LOX loading (LDL/LOX ratio of 1:10) of the LDL particle. During long term incubations (up to 24 h), a less specific product pattern was observed. However, when the hydroperoxy lipids formed by the 15-LOX were immediately reduced by the phospholipid hydroperoxide glutathione peroxidase, when the reaction was carried out with vitamin E-depleted LDL, or when the assay sample was diluted, the specific pattern of oxygenation products was retained over a long period of time. These data suggest that mammalian 15-LOX preferentially oxidize LDL cholesterol esters, forming a specific pattern of oxygenation products. During long term incubations, free radical-mediated secondary reactions, which lead to a more unspecific product pattern, may become increasingly important. These secondary reactions appear to be suppressed when the hydroperoxy lipids formed are immediately reduced, when alpha-tocopherol-depleted LDL was used, or when the incubation sample was diluted. It may be concluded that 15-LOX-initiated LDL oxidation constitutes a dual-type oxygenase reaction with an initial enzymatic and a subsequent nonenzymatic phase. The biological relevance of this dual-type reaction for atherogenesis will be discussed.     

Imidazolyl carboxylic acids as mechanistic probes of flavocytochrome P-450 BM3

omega-Imidazolyl carboxylic acids (C10-C12) have been used as probes of the active site and catalytic mechanism of the fatty acid hydroxylase P-450 BM3 from Bacillus megaterium. These compounds are the most potent inhibitors of P-450 BM3 yet reported. All are mixed inhibitors, increasing the Km and decreasing the kcat for laurate oxidation. All ligate the P-450 BM3 ferric heme iron, inducing a type II shift in the Soret absorbance band from 419 to 424 nm. Binding to the ferrous form is much weaker. 10-(Imidazolyl)decanoic acid was the best inhibitor (Kic = 0.9 microM, Kiu = 5.7 microM), while 12-(imidazolyl)dodecanoic acid (Kic = 1.35 microM, Kiu = 6.9 microM) was superior to 11-(imidazolyl)undecanoic acid (Kic = 7.5 microM, Kiu = 16 microM). Dissociation constants for binding to oxidized P-450 BM3 heme iron were determined spectrophotometrically as 8 microM (C12 azole) and 27 microM (C11 azole). The binding of 10-(imidazolyl)decanoic acid was too tight for an absolute Kd to be determined spectrophotometrically, but this value is <0.2 microM. The binding of different fatty acids to the enzyme was found to have distinct effects on the Kd for the azoles. Laurate induced tighter binding (Kd for the C12 azole lowered to 4.7 microM), while arachidonate weakened the affinity (Kd increased to 23 microM). Arachidonate diminished the affinity for the C10 azole sufficiently that a Kd could be determined by spectrophotometric titration (11 microM). Affinity for the C12 azole was decreased in active-site-mutants R47G (R47 tethers the fatty acid carboxylate group) and F87Y but increased in mutant F87G-indicating an important role for this residue in determining heme accessibility. The C10 azole binds much more weakly to the spin-state-insensitive F87Y (32. 2 microM), suggesting that the inhibitors may bind preferentially to different conformers of P-450 BM3. NADP+ binding in the reductase also tightened affinity of these inhibitors for P-450 BM3 (Kd for the C12 azole decreased to 2.7 microM), but this effect was not observed for FMN-deficient mutant W574D, suggesting that the interdomain effect of NADP+ on inhibitor binding was mediated via flavin mononucleotide. Resonance Raman spectroscopy indicates that the inhibitors form low-spin complexes with P-450 BM3 and that their binding induces movements of the heme vinyls relative to the ring.     

Fatty acids and retinoids bind independently and simultaneously to beta-lactoglobulin

beta-Lactoglobulin (Big) binds 1 mol of a fatty acid spin-label analog, 5-doxylstearic acid (5-DSA), per mole of protein with a dissociation constant Kd = 0.8 microM for the strongest binding site. There are also several weaker sites for this ligand. Blg saturated with either retinol or retinoic acid binds 5-DSA with essentially equal affinity (Kd = 0.6 and 1 microM, respectively). Palmitic acid and SDS displace bound 5-DSA from Blg. However, unlike palmitic acid, 5-DSA binding does not enhance the structural stability of Blg to urea denaturation. The spin-labeled fatty acid also binds to the protein at low pH, presumably at secondary fatty acid binding sites. These results suggest that Blg binds at least two different types of hydrophobic ligands simultaneously.