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1.
Summary The fluorescent fatty acids,trans-parimaric andcis-parinaric acid, were used as analogs of saturated and unsaturated fatty acids in order to evaluate binding of fatty acids to liver plasma membranes isolated from normal fed rats. Insulin (10–8 to 10–6 m) decreasedtrans-parinaric acid binding 7 to 26% whilecis-parinaric acid binding was unaffected. Glucagon (10–6 m) increasedtrans-parinaric acid binding 44%. The fluorescence polarization oftrans-parinarate,cis-parinarate and 1,6-diphenyl-1,3,5-hexatriene was used to investigate effects of triiodothyronine, insulin and glucagon on the structure of liver plasma membranes from normal fed rats or from rats treated with triiodothyronine or propylthiouracil. The fluorescence polarization oftrans-parinarate,cis-parinarate, and 1,6-diphenyl-1,3,5-hexatriene was 0.300±0.004, 0.251±0.003, and 0.302±0.003, respectively, in liver plasma membranes from control rats and 0.316±0.003, 0.276±0.003 and 0.316±0.003, respectively, in liver plasma membranes from hyperthyroid rats (p<0.025,n=5). Propylthiouracil treatment did not significantly alter the fluorescence polarization of these probe molecules in the liver plasma membranes. Thus, liver plasma membranes from hyperthyroid animals appear to be more rigid than those of control animals. The effects of triiodothyronine, insulin and glucagon addedin vitro to isolated liver plasma membrane preparations were also evaluated as follows: insulin (10–10 m) and triiodothyronine (10–10 m) increased fluorescence polarization oftrans-parinaric acid,cis-parinaric acid and 1,6-diphenyl-1,3,5-hexatriene in liver plasma membranes while glucagon (10–10 m) had no effects. These hormonal effects on probe fluorescence polarization in liver plasma membranes were abolished by pretreatment of the rats for 7 days with triiodothyronine. Administration of triiodothyronine (10–10 m)in vitro increased the fluorescence polarization of trans-parinaric acid in liver plasma membranes from propylthiouracil-treated rats. Thus, hyperthyroidism appeared to abolish thein vitro increase in polarization of probe molecules in the liver plasma membranes. Temperature dependencies in Arrhenius plots of absorption-corrected fluorescence and fluorescence polarization oftrans-parinaric acid,cis-parinaric acid and 1,6-diphenyl-1,3,5-hexatriene were noted near 25°C in liver plasma membranes from triiodothyronine-treated rats and near 18°C in liver plasma membranes from propylthiouracil-treated rats. In summary, hormones such as triiodothyronine, insulin and glucagon may at least in part exert their biological effects on metabolism by altering the structure of the liver plasma membranes.  相似文献   

2.
The interactions of fatty acids with porcine and bovine β-lactoglobulins were measured using tryptophan fluorescence enhancement. In the case of bovine β-lactoglobulin, the apparent binding constants for most of the saturated and unsaturated fatty acids were in the range of 10?7 M at neutralpH. Bovine β-lactoglobulin displays only one high affinity binding site for palmitate with an apparent dissociation constant of 1·10?7 M. The strength of the binding was decreasing in the following way: palmitate > stearate > myristate > arachidate > laurate. Caprylic and capric acids are not bound at all. The affinity of β-lactoglobulin for palmitate decreased as thepH of the incubation medium was lowered and BLG/palmitate complex was not observed atpH's lower than 4.5. Surprisingly, chemically modified bovine β-lactoglobulin and porcine β-lactoglobulin did not bind fatty acids in the applied conditions.  相似文献   

3.
Binding sites of bile acids on human serum albumin were studied using various probes: dansylsarcosine (site I probe), 7-anilinocoumarin-4-acetic acid (ACAA, site II probe), 5-dimethylaminonaphthelene-1-sulfonamide (DNSA, site III probe), cis-parinaric acid (probe for fatty acid binding site) and bilirubin. Bile acids competitively inhibited the binding of dansylsarcosine to human serum album whereas bile acids enhanced the binding of ACAA, DNSA, cis-parinaric acid and bilirubin. Considering the concentrations of bile acids required to inhibit the binding of dansylsarcosine to human serum albumin, the secondary binding site of bile acids may correspond to site I. Dissociation constants (Kd) of the primary binding sites of lithocholic and chenodeoxycholic acid to human serum albumin were approximately 0.2 and 4 μM, respectively, which was measured by equilibrium dialysis at 37° C. All the bile acids and their sulfates and glucuronides inhibited the binding of chenodeoxycholic acid to human serum albumin. Lithocholic and chenodeoxycholic acid and their sulfates and glucuronides exhibited more inhibition than cholic acid and its conjugates. In conclusion, bile acids may bind to a novel binding site on human serum albumin.  相似文献   

4.
A computer-centered spectrofluorimeter was used to examine the physicochemical properties of hepatic microsomes and microsomal lipids obtained from isolated rat livers perfused with medium containing palmitate or oleate. The fatty acid composition and degree of unsaturation of the liver microsomal lipids reflected that the fatty acid present in the perfusate. The absorption corrected fluorescence, relative fluorescence efficiency, polarization, and fluorescence anisotropy of several fluorescent probe molecules were measured to determine if their different microenvironments may be altered by the type of fatty acid infused. The probe molecules β-parinaric acid and 1.6-diphenyl-1,3,5-hexatriene had higher values for each of these parameters when incorporated into microsomes obtained from livers perfused with a medium containing palmitate than with oleate. The same parameters measured for cholesta-5,7,9(11)-trien-3β-ol and N-phenyl-1-naphthylamine were not altered. These differences appeared to be primarily due to alterations in microviscosity of the probe microenvironments since the rotational correlation time of 1,6-diphenyl-1,3,5-hexatriene was 25% lower in the microsomes from livers perfused with oleate as compared to livers perfused with palmitate. Thermal discontinuities in Arrhenius plots were noted in the intact microsomes but not in the isolated microsomal lipids with the fluorescence probe molecule β-parinaric acid. Break points occurred at 10°C and 26°C for microsomes from livers perfused with palmitate and at 12°C and 17°C for microsomes from livers perfused with oleate containing medium. These results suggest that the physicochemical properties of liver microsomes were determined in part by the fatty acid in the perfusate.  相似文献   

5.
Phenytoin (PHT) modified the fluorescent characteristics of anthroyloxy-fatty acids in synaptosomal membranes. Association of PHT with synaptosomal membranes caused the greatest change when the fluorescent probe was located at the 6-carbon position of N-(anthroyloxy)stearic acid and was incorporated into the membranes. Phenytoin and 6-(anthroyloxy)stearic acid compete for high affinity binding regions which are probably lipid domains within the membrane. Phenytoin has a weaker association with the sites than the fluorescent fatty acids. Divalent cations, e.g. Mg2+ or Ca2+, are required to observe maximal change in polarization of fluorescence of fatty acid probes in the presence of PHT. It is proposed that the membrane lipid bilayer reorganizes to accommodate exogenous compounds, such as phenytoin or the fatty acid probe in order to permit the most efficient packing of lipids. This reorganization of the lipid bilayer may influence membrane enzyme activities and ion channels.  相似文献   

6.
The cis-isomer of parinaric acid, a naturally occurring C-18 polyene fatty acid, was incubated with brain subcellular fractions and the polarization of fluorescence increased in a time dependent manner. Greatest increases occurred in synaptosomal and microsomal membranes. This increase in polarization of fluorescence was found with the cis, but not the trans, isomer of parinaric acid and required Mg2+ or Ca2+ and was stimulated by coenzyme A and ATP. Synaptosomes were incubated with cis-parinaric acid and lipids were extracted and examined by high performance liquid chromatography. The highest incorporations of cis-parinaric acid were found in phosphatidylcholine (71%) and phosphatidylethanolamine (20%) while only traces were found in phosphatidylserine and phosphatidylinositol. [3H]Oleic acid was also incorporated into membrane phospholipids and unlabeled oleic acid blocked incorporation of cis-parinaric acid. It is proposed that cis-parinaric acid, like fatty acids normally found in brain, is incorporated into membrane phospholipids by an acyl-CoA acyltransferase. The presence of this enzyme in nervous tissue may make it possible to easily introduce fluorescent fatty acid probes into membrane phospholipids and to thereby facilitate study of membrane-mediated processes.  相似文献   

7.
Fatty acids (FA) are important nutrients that the body uses to regulate the storage and use of energy resources. The predominant mechanism by which long-chain fatty acids enter cells is still debated widely as it is unclear whether long-chain fatty acids require protein transporters to catalyze their transmembrane movement. We use stopped-flow fluorescence (millisecond time resolution) with three fluorescent probes to monitor different aspects of FA binding to phospholipid vesicles. In addition to acrylodan-labeled fatty acid binding protein, a probe that detects unbound FA in equilibrium with the lipid bilayer, and cis-parinaric acid, which detects the insertion of the FA acyl chain into the membrane, we introduce fluorescein-labeled phosphatidylethanolamine as a new probe to measure the binding of FA anions to the outer membrane leaflet. We combined these three approaches with measurement of intravesicular pH to show very fast FA binding and translocation in the same experiment. We validated quantitative predictions of our flip-flop model by measuring the number of H+ delivered across the membrane by a single dose of FA with the probe 6-methoxy-N-(3-sulfopropyl) quinolinium. These studies provide a framework and basis for evaluation of the potential roles of proteins in binding and transport of FA in biological membranes.  相似文献   

8.
The binding of cAMP to the chemotactic cAMP receptor in intact Dictyostelium discoideum cells and isolated membranes is strongly inhibited by unsaturated fatty acids. In isolated membranes, cis-unsaturated fatty acids decreased the number of accessible cAMP binding sites, without significantly altering their affinity. Most potent were C18 and C20 cis-poly unsaturated fatty acids, like arachidonic acid, linoleic acid and linolenic acid. Trans-unsaturated fatty acid was less potent than its cis isomer, while saturated fatty acids did not affect the binding of cAMP to receptors at all. Oxidation reactions were not important for the effect of unsaturated fatty acids. When membranes were preincubated with millimolar concentrations of Ca2+, the effect of unsaturated fatty acids was strongly diminished. Mg2+ was ineffective. Ca2+, if presented after the incubation of membranes with unsaturated fatty acids, did not reverse the inhibitory effect. The specificity of the fatty acid effect, and the interference with Ca2+, but not Mg2+, suggest that the properties of the cAMP receptor are changed as a result of alterations in the lipid bilayer structure of the membrane.  相似文献   

9.
We present a method by which it is possible to describe the binding of fatty acids to phospholipid bilayers. Binding constants for oleic acid and a number of fatty acids used as spectroscopic probes are deduced from electrophoresis measurements. There is a large shift in pK value for the fatty acids on binding to the phospholipid bilayers, consistent with stronger binding of the uncharged form of the fatty acid. For dansylundecanoic acid, fluorescence titrations are consistent with the binding constants derived from the electrophoresis experiments. For 12-(9-anthroyloxy)stearic acid, fluorescence and electrophoresis data are inconsistent, and we attribute this to quenching of fluorescence at high molar ratios of 12-anthroylstearic acid to phospholipid in the bilayer.  相似文献   

10.
The rate of hydrolysis of phosphatidylcholine bilayers by soluble phospholipase A2 (PLA2) is greatly enhanced by the presence in the bilayer of a threshold mole fraction of the reaction products: fatty acid and lysophosphatidylcholine (lyso-PC). The threshold requirement of these products appears to vary as a function of vesicle and calcium concentration. To further identify the roles of myristic acid, lyso-PC, and calcium in promoting optimal PLA2 activity, we have quantified the various interactions among these components and dimyristoylphosphatidylcholine large unilamellar vesicles. The bilayer/water partition coefficient for myristic acid was obtained by competition of vesicles for the binding of the fatty acid to an acrylodan conjugate of an intestinal fatty acid binding protein as monitored by the acrylodan fluorescence emission spectrum. The partition coefficient for lyso-PC was obtained by a similar procedure using the tryptophan emission spectrum of bovine serum albumin. The effect of calcium concentration on these interactions was also quantified. These results were incorporated into an empirical model to describe the threshold requirements for these products in the bilayer. This information is vital for elucidating the mechanism of activation of PLA2 by the hydrolysis products.  相似文献   

11.
The α-helical from of poly(L -glutamic acid) [α-poly(Glu)] gives rise to the same amide I and III lines as α-poly(γ-benzyl-L -glutamate) at 1652 and 1296 cm?1, respectively. The latter is a superposition of the amide III line near 1290 cm?1 and a line deu to vibrational made of CH2 groups of the side chain near 1300 cm?1. A line at 924 cm?1 is tentatively identified as characteristics of α-poly(Glu). Both the β1- and β2- forms of poly(Glu) give rise to characteristic of β-amide. III frequencies that are similar because of their similar backbone structures. Differences in the conformations of their side chains and in the environments of the backbone are reflected in the region 800–1200 cm?1 and in the amide I. A line at 1042 cm?1 and a pair at 1021 and 1059 cm?1 are tentatively assigned as characteristic of β1-poly(Glu) and β2-poly(Glu), respectively. The α-β2 transition in poly(L -Glu78L -Val22) is shown by the appearance of all the β2-characteristic lines in the thermally transformed sample. The same features observed in poly(L -Glu95L -Val5) also indicate that the α-β2 transition of poly(Glu) is facilitated by the presence of L -valine and that the content of L -valine is not critical for this purpose. Investigation of the Raman spectra of the calcium, strontium, barium and sodium slats of poly(Glu) shows that these salts, under the conditions of preparation used, all the have random-coil conformations.  相似文献   

12.
A simple and selective spectrofluorimetric method for the detection of chlortetracycline (CTC) was studied. In pH 7.4 buffer medium l ‐tryptophan (l ‐Trp), applied as the fluorescence probe, interacted with CTC resulting in fluorescence quenching of the probe. CTC was detected with maximum excitation and emission wavelengths at λex/λem = 275/350 nm. Notably, quenching of fluorescence intensities was positively proportional to the CTC concentration over the range of 0.65–30 μmol L?1 and the limit of detection was 0.2 μmol L?1. Effect of temperature shown in Stern?Volmer plots, absorption spectra and fluorescence lifetime determination, indicated that fluorescence quenching of l ‐Trp by CTC was mainly by static quenching. The proposed study used practical samples analysis satisfactorily.  相似文献   

13.
Highly purified coupling factor 1 (CF1) from chloroplasts was found to contain 3.6 mol tryptophan/mol of enzyme. Although the α, β, γ, and δ subunits of the enzyme are devoid of tryptophan, the ? subunit was found to contain two tryptophans per mole. These results support a stoichiometry of two ? per mole of CF1. Two classes of tyrosine and tryptophan were detected in CF1 and evidence for a correlation between activation of the ATPase activity of CF1 and a quenching of tryptophan fluorescence is given. Tryptophan should be a useful marker for the ? subunit and its fluorescence and modification should provide a probe for its function.  相似文献   

14.
The iturinic antibiotics, which contain long chain β-amino acids, are produced by Bacillus subtilis. Screening these strains for the presence of a possible precursor of the iturinic antibiotics, we isolated a lipopeptide containing β-hydroxy fatty acids. The structure of this compound was studied and it appears to be identical or structurally very similar to surfactin. The carbon chain of its β-hydroxy fatty acids was n C16, iso C16, iso C15 or anteiso C15. The percentages of each β-hydroxy fatty acids varied according to the strain producing iturinic antibiotics and were influenced by addition of branched-chain α-amino acids to the culture medium. These results demonstrate for the first time that iso C14 β-hydroxy fatty acid is a constituent present in such a surfactin like lipopeptide. Besides, the presence of radioactive β-hydroxy fatty acids in the phospholipids when the strains were grown in the presence of sodium [14C]acetate seems also characterize the different strains producing iturinic antibiotics.  相似文献   

15.
The conformation of basic fatty acid binding protein from chicken liver and the binding properties of the apo protein toward 11-dansylamino-undecanoic acid were investigated by CD and fluorescence spectroscopy. In one set of experiments the binding process was followed by the appearance of induced optical activity in the absorption region of the dansyl chromophore. In a second set of experiments the binding process was followed by the large enhancement of emission fluorescence of the dansyl fluorophore. From the saturation curves, the stoichiometry of the complex and the binding constant of the fatty acid to the protein were precisely determined. The values of the dissociation constant determined with the two methods were in excellent agreement: we obtained KD = (1.0 ± 0.1) · 10?6M in a 0.9 : 1 stoichiometry. The native conformation of the protein is remarkably stable in a variety of solvent systems, including acetonitrile–water, ethylene glycol–water, and dicxane–water of various compositions. The CD results also showed that the binding of the fatty acid does not induce any appreciable change in the protein conformation. In a mixture of water and 2,2,2-trifluoroethanol 1 : 9 (v/v), the native conformation collapses and a new ordered structure is formed, characterized by a high amount of α-helix. © 1994 John Wiley & Sons, Inc.  相似文献   

16.
The effect of synthetic anti-oxidant potassium phenosan (PP, potassium salt of β-(4-hydroxy-3,5-ditretbutil-phenyl)-propionic acid) on the structural state of the surface (8 Å) and deep (20–22 Å) lipid regions of plasma membranes of mice liver cells was studied by spin probes method in vitro in a wide range of concentrations (10?5–10?21 M). Two stable free radicals, 5- and 16-doxyl-stearic acids (C5 and C16), were used as spin probes. The nonlinear polymodal dose-effect dependences were obtained for parameters that characterize the microviscosity of the lipid bilayer (τc) in the site of localization of the probe C16, and the order parameter (S), which characterizes the stiffness of the surface layers of lipids in the site of localization of the probe C5. Statistically a reliable increase was observed for parameter τc after addition of PP at concentrations 10?5–10?7 M and 10?18–10?19 M, and for parameter S after addition of PP at concentrations 10?6–10?7 M and 10?13–10?15 M. Peaks on both dose-effect curves were separated by the intervals of concentrations where PP had no effect on the studied physico-chemical characteristics of biomembranes. For PP concentrations which caused maximal changes in τc and S, we investigated thermal dependence of these parameters and determined the thermally induced structural transitions. Comparing with control, ultra-low doses of PP (10?13–10?15 M) and (10?18–10?19 M) caused an appearance of additional thermally induced structural transition in the surface and deep regions of plasma membrane lipids. The possible role of the interaction of PP molecules with specific binding sites on plasma membranes and formation of nanoparticles of PP in very dilute aqueous solutions are discussed.  相似文献   

17.
Liver fatty acid-binding protein (FABP) binds a variety of non-polar anionic ligands including fatty acids, fatty acyl CoAs, and bile acids. Previously we prepared charge reversal mutants and demonstrated the importance of lysine residues within the portal region in ligand and membrane binding. We have now prepared several tryptophan-containing mutants within the portal region, and one tryptophan at position 28 (L28W) has proved remarkably effective as an intrinsic probe to further study ligand binding. The fluorescence of the L28W mutant was very sensitive to fatty acid and bile acid binding where a large (up to 4-fold) fluorescence enhancement was obtained. In contrast, the binding of oleoyl CoA reduced tryptophan fluorescence. Positive cooperativity for fatty acid binding was observed while detailed information on the orientation of binding of bile acid derivatives was obtained. The ability of bound oleoyl CoA to reduce the fluorescence of L28W provided an opportunity to demonstrate that fatty acyl CoAs can compete with fatty acids for binding to liver FABP under physiological conditions, further highlighting the role of fatty acyl CoAs in modulating FABP function in the cell.  相似文献   

18.
Abstract: Amyloid β-peptide (Aβ) aggregates are one of the key neuropathological characteristics of Alzheimer's disease. Aβ belongs to a group of proteins that aggregate and form β-sheets, and some of these proteins bind cholesterol and other lipids. The purpose of the experiments reported here was to determine if cholesterol, fatty acids, and phosphatidylcholine (PC) would bind to Aβ1–40 and if such binding would be dependent on aggregation of Aβ1–40. Lipid binding was determined using fluorescent-labeled lipids. Incubation of Aβ1–40 for 0, 1, 3, 6, 21, and 24 h resulted in aggregation of the peptide with formation of dimers, trimers (1–24 h), and polymers (6–24 h) as determined by sodium dodecyl sulfate-gel electrophoresis. No change in the fluorescence of the lipids was observed when lipids were added to Aβ1–40 that had been incubated for 0, 1, or 3 h. However, the fluorescence intensities of cholesterol, saturated fatty acids, and PC were significantly increased (p < 0.0001) when added to Aβ1–40 that had been incubated for 6, 21, and 24 h in which Aβ1–40 polymers were detected. The binding affinity of cholesterol to Aβ1–40 polymers (KD of 3.24 ± 0.315 × 10?9M) was markedly higher as compared with the other lipids (stearic acid, 9.42 ± 0.41 × 10?8M; PC, 7.07 ± 0.12 × 10?7M). The results of this study indicate that Aβ1–40 polymers bind lipids and have a higher affinity for cholesterol than PC or saturated fatty acids. Aggregated Aβ1–40 may affect lipid transport between cells or remove specific lipids from membranes, and such effects could contribute to neuronal dysfunction.  相似文献   

19.
Murine fibroblasts, LM cells, were cultured in suspension with laurate (12:0), myristate (14:0), palmitate (16:0), palmitoleate (16:1), or palmitate + palmitoleate (16:0 + 16:1) bound to fatty acid-free bovine serum albumin. Supplementation with saturated fatty acids decreased the ratio of unsaturated/saturated fatty acids in membrane phospholipids as much as 3.4-fold (palmitate-enriched cells). Concomitantly fluorescence polarization, absorption-corrected fluorescence, and relative fluorescence efficiency of the fluorescence probe molecule, β-parinaric acid, increased 1.5-, 2.9-, and 1.8-fold, respectively, in the membrane phospholipids. Unsaturated fatty acid (palmitoleate) increased the unsaturated/saturated fatty acid ratio by 20% but did not significantly alter the fluorescence parameters. When the cells were fed mixtures of palmitate and palmitoleate, the unsaturated/saturated fatty acid ratio of the membrane phospholipids and the above fluorescence parameters had values intermediate between those if each fatty acid had been fed separately. All fatty acid supplements caused a loss of two characteristic temperatures in Arrhenius plots of relative fluorescence efficiency. However, no shifts or appearance of new characteristic temperatures occurred. The break points at approximately 42, 37, and 22 °C were essentially un-altered. The data were consistent with the possibility that LM cells were unable to maintain constant fluidity, as indicated by fluorescence polarization, when supplemented with different fatty acids. A good correlation could be made between the phospholipid unsaturated/ saturated fatty ratio, the fluorescence polarization, and the toxicity elicited by different fatty acid supplements.  相似文献   

20.
A series of phosphatidylcholines and phosphatidylethanolamines was synthesized containing two acyl chains of the following polyunsaturated fatty acids: linoleic acid (18:2), linolenic acid (18:3), arachidonic acid (20:4) and docosahexaenoic acid (22:6). In addition two phospholipids with mixed acid composition were synthesized: 16:0/18:1c phosphatidylcholine and 16:0/18:1c phosphatidylethanolamine. The structural properties of these lipids in aqueous dispersions in the absence and in the presence of equimolar cholesterol were studied using 31P-NMR, freeze fracturing and differential scanning calorimetry (DSC).The phosphatidylcholines adopt a bilayer configuration above 0°C. Incorporation of 50 mol% of cholesterol in polyunsaturated species induces a transition at elevated temperatures into structures with 31P-NMR characteristics typical of non-bilayer organizations. When the acyl chains contain three or more double bonds, this non-bilayer organization is most likely the hexagonal HII phase, 16:0/15:1c phosphatidylethanolamine shows a bilayer to hexagonal transition temperature of 75°C. The polyunsaturated phosphatidylethanolamines exhibit a bilayer to hexagonal transition temperature below 0°C which decreases with increasing unsaturation and which is lowered by approximately 10°C upon incorporation of 50 mol% of cholesterol. Finally, it was found that small amounts of polyunsaturated fatty acyl chains in a phosphatidylethanolamine disproportionally lower its bilayer to hexagonal transition temperature.  相似文献   

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