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• W2022983144 abstract "Hepatocytes expressing liver fatty acid binding protein (L-FABP) are known to be more resistant to oxidative stress than those devoid of this protein. The mechanism for the observed antioxidant activity is not known. We examined the antioxidant mechanism of a recombinant rat L-FABP in the presence of a hydrophilic (AAPH) or lipophilic (AMVN) free radical generator. Recombinant L-FABP amino acid sequence and its amino acid oxidative products following oxidation were identified by MALDI quadrupole time-of-flight MS after being digested by endoproteinase Glu-C. L-FABP was observed to have better antioxidative activity when free radicals were generated by the hydrophilic generator than by the lipophilic generator. Oxidative modification of L-FABP included up to five methionine oxidative peptide products with a total of ∼80 Da mass shift compared with native L-FABP. Protection against lipid peroxidation of L-FABP after binding with palmitate or α-bromo-palmitate by the AAPH or AMVN free radical generators indicated that ligand binding can partially block antioxidant activity. We conclude that the mechanism of L-FABP's antioxidant activity is through inactivation of the free radicals by L-FABP's methionine and cysteine amino acids. Moreover, exposure of the L-FABP binding site further promotes its antioxidant activity. In this manner, L-FABP serves as a hepatocellular antioxidant. Hepatocytes expressing liver fatty acid binding protein (L-FABP) are known to be more resistant to oxidative stress than those devoid of this protein. The mechanism for the observed antioxidant activity is not known. We examined the antioxidant mechanism of a recombinant rat L-FABP in the presence of a hydrophilic (AAPH) or lipophilic (AMVN) free radical generator. Recombinant L-FABP amino acid sequence and its amino acid oxidative products following oxidation were identified by MALDI quadrupole time-of-flight MS after being digested by endoproteinase Glu-C. L-FABP was observed to have better antioxidative activity when free radicals were generated by the hydrophilic generator than by the lipophilic generator. Oxidative modification of L-FABP included up to five methionine oxidative peptide products with a total of ∼80 Da mass shift compared with native L-FABP. Protection against lipid peroxidation of L-FABP after binding with palmitate or α-bromo-palmitate by the AAPH or AMVN free radical generators indicated that ligand binding can partially block antioxidant activity. We conclude that the mechanism of L-FABP's antioxidant activity is through inactivation of the free radicals by L-FABP's methionine and cysteine amino acids. Moreover, exposure of the L-FABP binding site further promotes its antioxidant activity. In this manner, L-FABP serves as a hepatocellular antioxidant. Liver fatty acid binding protein (L-FABP) is a low molecular weight protein (14–15 kDa) that belongs to a family of lipid binding proteins (1Glatz J.F. van der Vusse G.J. Cellular fatty acid-binding proteins: their function and physiological significance.Prog. Lipid Res. 1996; 35: 243-282Crossref PubMed Scopus (480) Google Scholar). The protein was first discovered by Ockner et al. in 1972 (2Ockner R.K. Manning J.A. Poppenhausen R.B. Ho W.K. A binding protein for fatty acids in cytosol of intestinal mucosa, liver, myocardium, and other tissues.Science. 1972; 177: 56-58Crossref PubMed Scopus (518) Google Scholar) and was originally named Z-protein. Since that time, there has been an explosive growth in information regarding the role of L-FABP in cellular homeostasis. While FABPs are present in many tissues, such as heart, brain, intestinal, skin, adipose, muscle, epidermal, ileal, myelin, and testis, L-FABP is found in abundance in hepatocytes where it accounts for ∼2% of the total cellular protein. Although L-FABP is abundant in the liver, it also is present in tissues such as murine alveolar macrophages (3Schachtrup C. Scholzen T.E. Grau V. Luger T.A. Sorg C. Spener F. Kerkhoff C. L-FABP is exclusively expressed in alveolar macrophages within the myeloid lineage: evidence for a PPARalpha-independent expression.Int. J. Biochem. Cell Biol. 2004; 36: 2042-2053Crossref PubMed Scopus (30) Google Scholar), kidney (4Yamamoto T. Noiri E. Ono Y. Doi K. Negishi K. Kamijo A. Kimura K. Fujita T. Kinukawa T. Taniguchi H. et al.Renal L-type fatty acid–binding protein in acute ischemic injury.J. Am. Soc. Nephrol. 2007; 18: 2894-2902Crossref PubMed Scopus (277) Google Scholar), and intestine (5Neeli I. Siddiqi S.A. Siddiqi S. Mahan J. Lagakos W.S. Binas B. Gheyi T. Storch J. Mansbach 2nd, C.M. Liver fatty acid-binding protein initiates budding of pre-chylomicron transport vesicles from intestinal endoplasmic reticulum.J. Biol. Chem. 2007; 282: 17974-17984Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). L-FABP is made up of 127 amino acids that compose 10 antiparallel β-strands. These stands are organized into two five-stranded β-sheets and two α-helices. The two α-helices are hypothesized to function as a portal that controls entry and release of ligands from the binding pocket created by the β-strands (6Thompson J. Winter N. Terwey D. Bratt J. Banaszak L. The crystal structure of the liver fatty acid-binding protein. A complex with two bound oleates.J. Biol. Chem. 1997; 272: 7140-7150Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar). A large interior water-filled cavity forms the confines of the β-strands, which serve as the hydrophobic ligand-binding site. L-FABP is able to bind and translocate many lipophilic substrates throughout the cytosol. Some of these substrates include long-chain fatty acids (7Norris A.W. Spector A.A. Very long chain n-3 and n-6 polyunsaturated fatty acids bind strongly to liver fatty acid-binding protein.J. Lipid Res. 2002; 43: 646-653Abstract Full Text Full Text PDF PubMed Google Scholar, 8Martin G.G. Atshaves B.P. McIntosh A.L. Mackie J.T. Kier A.B. Schroeder F. Liver fatty acid-binding protein gene-ablated female mice exhibit increased age-dependent obesity.J. Nutr. 2008; 138: 1859-1865Crossref PubMed Scopus (35) Google Scholar, 9Thumser A.E. Storch J. Liver and intestinal fatty acid-binding proteins obtain fatty acids from phospholipid membranes by different mechanisms.J. Lipid Res. 2000; 41: 647-656Abstract Full Text Full Text PDF PubMed Google Scholar), bile acids (10Martin G.G. Atshaves B.P. McIntosh A.L. Mackie J.T. Kier A.B. Schroeder F. Liver fatty-acid-binding protein (L-FABP) gene ablation alters liver bile acid metabolism in male mice.Biochem. J. 2005; 391: 549-560Crossref PubMed Scopus (56) Google Scholar), eicosanoids (11Raza H. Pongubala J.R. Sorof S. Specific high affinity binding of lipoxygenase metabolites of arachidonic acid by liver fatty acid binding protein.Biochem. Biophys. Res. Commun. 1989; 161: 448-455Crossref PubMed Scopus (72) Google Scholar), and hypolipidemic drugs (12Jefferson J.R. Slotte J.P. Nemecz G. Pastuszyn A. Scallen T.J. Schroeder F. Intracellular sterol distribution in transfected mouse L-cell fibroblasts expressing rat liver fatty acid-binding protein.J. Biol. Chem. 1991; 266: 5486-5496Abstract Full Text PDF PubMed Google Scholar). Transfer of these ligands from L-FABP to membranes is thought to occur by a diffusive process; i.e., the ligand first dissociates from the binding pocket and then diffuses to its site of action, while transfer of ligands from other FABPs, such as I-FABP, is thought to occur by a direct collisional interaction (13Hsu K.T. Storch J. Fatty acid transfer from liver and intestinal fatty acid-binding proteins to membranes occurs by different mechanisms.J. Biol. Chem. 1996; 271: 13317-13323Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). It seems that L-FABP also plays a role in transferring bound ligands into the nucleus. These ligands could activate the peroxisome proliferator-activated receptor α nuclear receptors and thus initiate transcriptional activity that could affect lipid and glucose metabolism among other effects (14Storch J. Thumser A.E. The fatty acid transport function of fatty acid-binding proteins.Biochim. Biophys. Acta. 2000; 1486: 28-44Crossref PubMed Scopus (433) Google Scholar, 15Schroeder F. Petrescu A.D. Huang H. Atshaves B.P. McIntosh A.L. Martin G.G. Hostetler H.A. Vespa A. Landrock D. Landrock K.K. et al.Role of fatty acid binding proteins and long chain fatty acids in modulating nuclear receptors and gene transcription.Lipids. 2008; 43: 1-17Crossref PubMed Scopus (180) Google Scholar, 16Hostetler H.A. Kier A.B. Schroeder F. Very-long-chain and branched-chain fatty acyl-CoAs are high affinity ligands for the peroxisome proliferator-activated receptor alpha (PPARalpha).Biochemistry. 2006; 45: 7669-7681Crossref PubMed Scopus (78) Google Scholar, 17Gossett R.E. Frolov A.A. Roths J.B. Behnke W.D. Kier A.B. Schroeder F. Acyl-CoA binding proteins: multiplicity and function.Lipids. 1996; 31: 895-918Crossref PubMed Scopus (114) Google Scholar, 18Binas B. Erol E. FABPs as determinants of myocellular and hepatic fuel metabolism.Mol. Cell. Biochem. 2007; 299: 75-84Crossref PubMed Scopus (35) Google Scholar). A unique property of L-FABP that has escaped detection is its antioxidant effect. L-FABP is known to bind polyunsaturated fatty acids (19Ek B.A. Cistola D.P. Hamilton J.A. Kaduce T.L. Spector A.A. Fatty acid binding proteins reduce 15-lipoxygenase-induced oxygenation of linoleic acid and arachidonic acid.Biochim. Biophys. Acta. 1997; 1346: 75-85Crossref PubMed Scopus (55) Google Scholar) and long-chain fatty acid peroxidation products (11Raza H. Pongubala J.R. Sorof S. Specific high affinity binding of lipoxygenase metabolites of arachidonic acid by liver fatty acid binding protein.Biochem. Biophys. Res. Commun. 1989; 161: 448-455Crossref PubMed Scopus (72) Google Scholar). By binding polyunsaturated fatty acids, L-FABP modulates the availability of these fatty acids to intracellular oxidative pathways (19Ek B.A. Cistola D.P. Hamilton J.A. Kaduce T.L. Spector A.A. Fatty acid binding proteins reduce 15-lipoxygenase-induced oxygenation of linoleic acid and arachidonic acid.Biochim. Biophys. Acta. 1997; 1346: 75-85Crossref PubMed Scopus (55) Google Scholar) and in this manner controls the amount of reactive oxygen species (ROS) released within the cell from this pathway. In addition to these well-known functions, recent studies have shown that L-FABP plays a further role in the cellular antioxidant defense mechanism (20Wang G. Gong Y. Anderson J. Sun D. Minuk G. Roberts M.S. Burczynski F.J. Antioxidative function of L-FABP in L-FABP stably transfected Chang liver cells.Hepatology. 2005; 42: 871-879Crossref PubMed Scopus (140) Google Scholar, 21Wang G. Shen H. Rajaraman G. Roberts M.S. Gong Y. Jiang P. Burczynski F. Expression and antioxidant function of liver fatty acid binding protein in normal and bile-duct ligated rats.Eur. J. Pharmacol. 2007; 560: 61-68Crossref PubMed Scopus (48) Google Scholar, 22Rajaraman G. Wang G.Q. Yan J. Jiang P. Gong Y. Burczynski F.J. Role of cytosolic liver fatty acid binding protein in hepatocellular oxidative stress: effect of dexamethasone and clofibrate treatment.Mol. Cell. Biochem. 2007; 295: 27-34Crossref PubMed Scopus (44) Google Scholar). Using an L-FABP cDNA transfection model Wang et al. (20Wang G. Gong Y. Anderson J. Sun D. Minuk G. Roberts M.S. Burczynski F.J. Antioxidative function of L-FABP in L-FABP stably transfected Chang liver cells.Hepatology. 2005; 42: 871-879Crossref PubMed Scopus (140) Google Scholar) reported that hepatocytes containing L-FABP were associated with much lower levels of ROS compared with hepatocytes devoid of L-FABP. Using a bile-duct ligated model of cholestasis, this group further showed that clofibrate increased L-FABP levels were associated with improved hepatic function and reduced lipid peroxidation products (21Wang G. Shen H. Rajaraman G. Roberts M.S. Gong Y. Jiang P. Burczynski F. Expression and antioxidant function of liver fatty acid binding protein in normal and bile-duct ligated rats.Eur. J. Pharmacol. 2007; 560: 61-68Crossref PubMed Scopus (48) Google Scholar). The antioxidative function of L-FABP is thought to be due to its amino acid composition. L-FABP contains one cysteine and several methionine groups that are known to take part in cellular redox cycling. Thus, although the actual mechanism for the L-FABP antioxidant property is not known, it is likely to involve these amino acids. Also unknown is whether L-FABP preferentially inactivates ROS released in lipophilic environments, such as membranes, or in the hydrophilic cytosolic environment. In this study, we investigated the mechanism and efficiency of the recombinant L-FABP antioxidant property when free radicals were released into the aqueous or lipid environments. Plasmid pGEX-6P-2 was purchased from GE Healthcare Bio-Sciences (Baie d'Urfé, Québec, Canada). Restriction enzymes, DNA polymerase, and T4 DNA ligase were obtained from Roche (Laval, Quebec, Canada). The 2,2′-azobis(2,4-dimethylvaleronitrile) and 2,2′-azobis(2-amidinopropane) dihydrochloride were purchased from Wako (Osaka, Japan). All other chemicals were obtained from Sigma Chemical (Oakville, ON, Canada). The expression vector pGEX-6P-2 was digested with BamHI and XhoI for the insertion of a full-length rat L-FABP cDNA, which was cloned by a pair of gene-specific PCR primers designed by the Oligo 5.1 computer software according to the GenBank sequence (BC086947). The primers contained BamHI and XhoI, respectively, and were synthesized by Invitrogen. PCR cloning of rat L-FABP was performed according to our previous publication (20Wang G. Gong Y. Anderson J. Sun D. Minuk G. Roberts M.S. Burczynski F.J. Antioxidative function of L-FABP in L-FABP stably transfected Chang liver cells.Hepatology. 2005; 42: 871-879Crossref PubMed Scopus (140) Google Scholar). The plasmid pGEX-6P-2 with L-FABP was constructed according to the pGEX-6P-2 instruction manual, and successful ligation of L-FABP with pGEX-6P-2 was confirmed by restriction enzyme digestion and DNA sequencing. Rat L-FABP was expressed by transformation into bacteria DH5α and amplified by addition of 0.1 nM isopropyl-β-d-thio-galactoside according to standard protocol. After expression in DH5α, L-FABP was purified according to the manual of pGEX-6p-2. The fusion protein GST/L-FABP was first associated with the glutathione-Sepharose 4B beads, and then the GST/L-FABP was eluted by addition of elution buffer or L-FABP digested from GST by addition of 10 units PreScission Protease and cleavage buffer. Both GST/L-FABP and L-FABP were analyzed by SDS-PAGE and MALDI-quadrupole time-of-flight (QqTOF) MS, respectively. The resolved proteins were either stained with Coomassie blue for SDS-PAGE or transferred to Nitroplus-2000 membranes for Western immunoblotting as previously outlined by our group (23Wang G. Chen Q.M. Minuk G.Y. Gong Y. Burczynski F.J. Enhanced expression of cytosolic fatty acid binding protein and fatty acid uptake during liver regeneration in rats.Mol. Cell. Biochem. 2004; 262: 41-49Crossref PubMed Scopus (23) Google Scholar). For the Western blot, a 15% SDS-polyacrylamide gel was used. Once proteins were separated, bands were transferred onto Nitroplus-2000 membrane (Micron Separations, Westborough, MA). Nonspecific antibody binding was blocked by preincubation of membranes in 1× TBS containing 5% skim milk for 1 h at room temperature. Membranes were then incubated overnight at 4°C with a polyclonal antibody raised against rat L-FABP as described previously (22Rajaraman G. Wang G.Q. Yan J. Jiang P. Gong Y. Burczynski F.J. Role of cytosolic liver fatty acid binding protein in hepatocellular oxidative stress: effect of dexamethasone and clofibrate treatment.Mol. Cell. Biochem. 2007; 295: 27-34Crossref PubMed Scopus (44) Google Scholar) in 1× TBS containing 5% skim milk. After being washed, membranes were incubated with donkey anti-rabbit IgG (Amersham) at 1:500 dilution for 1 h (room temperature). Bands were visualized using an enhanced chemiluminescence kit (ECL system; GE Healthcare Bio-Sciences. L-FABP proteins (∼5 ug) were digested with 50 ng sequencing grade trypsin or endoprotease Glu-C (Roche Diagnostic) in 25 mM ammonium bicarbonate and the solution incubated at 37°C for 6 h. Analyses of the proteolytic peptides were performed on Applied Biosystems/MDS Sciex QStar XL QqTOF mass spectrometer by MALDI at positive ionization mode. The instrument was equipped with a MALDI II source and a UV nitrogen laser operating at 337 nm. Samples were prepared at the ratio of 1:1 (v/v) of the peptide digest to matrix (i.e., 2,5-dihydroxybenzoic acid) in 50% acetonitrile/water and subsequently dried on a stainless steel MALDI plate at room temperature. After MALDI MS mapping, the individual peptide sequences were identified by MS/MS measurements using argon as the collision gas. The intact mass of L-FABP was determined by MALDI and also confirmed by nanospray ESI TOF mass spectrometry using the same instrument. In the case of ESI, the protein sample was dissolved in 50% methanol/0.1% formic acid solution. Peptide fingerprinting masses were searched by MS-Fit program against the National Center for Biotechnology Information database using ProteinProspector at the UCSF web site (http://prospector.ucsf.edu), whereas the MS/MS ions search on each tandem mass spectrum was performed through the Mascot search engine (MatrixScience; http://www.matrixscience.com). These searches take account of up to three missed enzyme cleavage sites and the modifications of methionine oxidation, asparagine and glutamine deamidation to aspartic acid and glutamic acid, and N-terminal pyroglutamation. Mass tolerance between calculated and observed masses used for database search was considered at the range of ±100 ppm for MS peaks and ±0.2 Da for MS/MS fragment ions. If no result was retrieved by the automated database search, then manual data interpretation was conducted on the spectrum based on the L-FABP predicted sequence. As described previously, 2′,7′-dichlorofluorescin-diacetate (DCFH-DA) was used for determining free radical levels released by H2O2. Briefly, DCFH-DA was deesterified to generate the oxidation substrate DCFH by mixing 125 μl of a 1.5 mM DCFH-DA solution in ethanol with 0.5 ml of 0.01 N NaOH for 30 min at room temperature in the dark. The mixture was neutralized with 2.5 ml of 20 mM sodium phosphate buffer (pH 7.0) to give a final concentration of 60 μM of the activated DCFH dye stock solution. The deesterified DCFH-DA could then be oxidized by free radicals to a highly fluorescent dichlorofluorescein (DCF) whose absorbance could be quantitated at 504 nm spectrophotometrically. Oxidation reactions were carried out in 96-well CoStar plates using 10–30 μM dye stock solution with 200 μM hydrogen peroxide (H2O2) and different concentrations of L-FABP. Plasma fractions of <1.21 densities were separated from fresh human plasma by ultracentrifugation in the presence of 1 mM EDTA. After dialysis, plasma lipoproteins were applied on a lysine-Sepharose 4B affinity chromatography column. Unbound lipoproteins were used to prepare Lp(a)-free LDL (density 1.019–1.063) using ultracentrifugation. Lipoproteins were stored in sealed tubes filled with nitrogen and kept in the dark at 4°C to prevent oxidization during storage. Protein concentrations of LDL were measured by a BCA protein kit (Fisher Scientific). Lipoprotein peroxidation was induced by two oxygen-derived free radical generators: a hydrophilic radical generator, AAPH, and a lipophilic radical generator, AMVN. All the free radical generators were prepared fresh and preincubated with LDL (1 mg cholesterol/ml) for 90 min at 37°C. AAPH was dissolved in deionized water, whereas AMVN was dissolved in 95% ethanol. The lipid peroxidation product (malonaldehyde, MDA) was determined by the thiobarbituric acid-reactive substance (TBARS) method (24Buege J.A. Aust S.D. Microsomal lipid peroxidation.Methods Enzymol. 1978; 52: 302-310Crossref PubMed Scopus (10776) Google Scholar). Briefly, the TBARS assay was used to demonstrate reactive aldehydes from lipid peroxidation, which has been widely accepted as a general marker of free radical production. After incubation for 90 min, the reaction was terminated by addition of 1 ml TBA reagent (0.67%, w/v, TBA in a 15%, w/v, trichloroacetic acid solution and 0.25 N HCl). The reaction mixture was heated to 100°C for 15 min and then cooled on ice. Samples were then centrifuged, resulting in the development of a pink chromogen whose absorbance was measured at 535 nm in a spectrophotometer. Freshly diluted malondialdehyde bis(dimethyl acetal 1,1,3,3-tetramethoxypropane) was used as a reference standard. Thiobarbituric acid reactive substances were expressed as MDA equivalents. Results are expressed as mean ± SD. Appropriate statistical analysis included Student's t-test (unpaired) where two groups were compared. Two-way ANOVA was used for multiple comparisons. Statistical significance was considered at P < 0.05. The n value refers to the number of experimental assays in each study. The cDNA fragment encoding the complete rat L-FABP sequence was cloned into the pGEX-6P-2 plasmid downstream of the hybrid glutathione S-transferase (GST) tag promoter to allow for the inducible and efficient intracellular expression of rat GST/L-FABP in E. coli. Following sonication and removal of the bacterial cell pellet, the soluble GST/L-FABP complex was isolated from bacterial cytosolic proteins. Optimal expression time was evaluated by determining the extent of protein accumulation and degradation using SDS-PAGE analysis of cellular lysates over 1–8 h. Following 6 h of induction, the GST/L-FABP tag reached a peak intracellular concentration of ∼80% of total E. coli intracellular protein, and 0.1 mM isopropyl-β-d-thio-galactoside was sufficient to induce a maximum level of GST/L-FABP expression. GST/L-FABP was purified to homogeneity by three successive elution steps using GST 4B beads, which precipitated 85% of the GST/L-FABP. This purification method had the highest protein recovery and optimal elution characteristics for GST/L-FABP separation. Following elution, ∼50–60 mg of GST/L-FABP was obtained from a 500 ml culture. Impurities were not present as observed by SDS-PAGE on 15% polyacrylamide gels. Thus, contaminants could be effectively removed from the GST/L-FABP complex by GST 4B beads. Purified recombinant L-FABP was successfully isolated after incubating the GST/L-FABP complex with PreScission Protease and cleavage buffer. SDS-PAGE results showed that our recombinant L-FABP had the same molecular weight (∼14 kDa) as L-FABP isolated from hepatoma cells. The DCF fluorescence assay was used as a convenient screening method for assessing the extent of the antioxidative potential of our recombinant L-FABP. Since intracellular L-FABP has been reported to play a major role in suppressing oxidative stress (20Wang G. Gong Y. Anderson J. Sun D. Minuk G. Roberts M.S. Burczynski F.J. Antioxidative function of L-FABP in L-FABP stably transfected Chang liver cells.Hepatology. 2005; 42: 871-879Crossref PubMed Scopus (140) Google Scholar, 22Rajaraman G. Wang G.Q. Yan J. Jiang P. Gong Y. Burczynski F.J. Role of cytosolic liver fatty acid binding protein in hepatocellular oxidative stress: effect of dexamethasone and clofibrate treatment.Mol. Cell. Biochem. 2007; 295: 27-34Crossref PubMed Scopus (44) Google Scholar), a reduction in ROS by L-FABP would be observed in an in vitro DCF oxidation assay. As shown in Fig. 1, our recombinant L-FABP inhibited H2O2-induced free radical levels as observed by a decrease in DCF fluorescence intensity. Moreover, inhibition of the released free radicals was increased with increased L-FABP concentration. Suppression of free radical release was greatest at 200 μM L-FABP, which was observed to be 66 ± 1% of control values (P < 0.001). To determine the possibility that L-FABP quenched the DCF fluorescence by binding DCF or by some other unknown mechanism, Rajaraman et al. (22Rajaraman G. Wang G.Q. Yan J. Jiang P. Gong Y. Burczynski F.J. Role of cytosolic liver fatty acid binding protein in hepatocellular oxidative stress: effect of dexamethasone and clofibrate treatment.Mol. Cell. Biochem. 2007; 295: 27-34Crossref PubMed Scopus (44) Google Scholar) investigated the quenching potential of L-FABP. They reported that fluorescence was not affected by L-FABP concentration. Thus, results in this study support the notion that the associated decrease in DCF fluorescence was in fact due to L-FABP inactivation of free radicals. To compare the effectiveness of antioxidants in both lipophilic and hydrophilic free radical generating systems, it was necessary to examine the effects of AAPH and AMVN free radical generators in a LDL solution. Levels of the lipid peroxidation product (MDA) were first examined as a function of varying concentrations of AMVN or AAPH (Fig. 2A). The lipid-soluble peroxyl radical generator (AMVN) induced greater MDA production in LDL than the water-soluble generator (AAPH). Thus, for subsequent comparison, it was necessary to use AAPH and AMVN concentrations that induced similar amounts of MDA production. The same amount of MDA was produced by 10 mM AMVN and 40 mM AAPH. Therefore, those concentrations were used in all in vitro studies. To compare the difference of antioxidant activity between L-FABP and ascorbic acid or α-tocopherol, LDL was incubated with 40 mM AAPH or 10 mM AMVN in the absence or the presence of different concentrations of ascorbic acid or α-tocopherol (Fig. 3A, B). These two antioxidants inhibited the oxidation of LDL in a dose-dependent manner. Using the AAPH generator, 1 mM ascorbic acid was able to reduce MDA production by 60% (P < 0.001) (Fig. 3A), whereas no significant protective effect against lipid peroxidation was observed in the 0–40 mM concentration range in the AMVN assay. At a concentration of 5 μM, α-tocopherol reduced >70% of MDA formation (Fig. 3A). The same concentration of α-tocopherol inhibited MDA production by <50% in the AMVN-induced lipid peroxidation assay (Fig. 3B), showing that α-tocopherol has a weaker effect in the inhibition of LDL oxidation by AMVN. We were interested in knowing whether L-FABP is more effective as a hydrophilic or lipophilic free radical scavenger. Since L-FABP is water soluble, it is thought that most of the antioxidant properties may be directed in the cytosol. However, as a protein it also imparts some lipophilic properties, and as such some of the antioxidant property may be directed at the lipophilic or cell membrane environment. Thus, we investigated the LDL oxidation levels mediated by 40 mM AAPH or 10 mM AMVN in the absence or presence of different L-FABP concentrations using an in vitro system. Figure 2B shows a dose-dependent inhibition in oxidized LDL by 1–20 µM L-FABP on AAPH- and AMVN-induced MDA production. Using AAPH to produce free radicals, a concentration of 1 μM L-FABP was able to reduce MDA formation by 40% (P < 0.001), and a 90% (P < 0.001) reduction in MDA formation was obtained when the concentration of L-FABP was increased to 20 μM. Figure 3A shows that 10 μM L-FABP inhibited a similar amount of MDA production as 10 mM ascorbic acid but inhibited more MDA production than 10 μM α-tocopherol (P < 0.01). At a concentration of 20 μM L-FABP, L-FABP protected against free radical damage much more than either 20 μM α-tocopherol (P < 0.001) or 20 mM ascorbic acid (P < 0.001). L-FABP was less potent against MDA production when free radicals were induced by AMVN. Figure 3B shows that 20 μM L-FABP provided greater antioxidant effect against lipid peroxidation induced by AAPH (90%) than that of AMVN (55%). Figure 3B also shows that 10 μM L-FABP inhibited 50% of MDA production and had a similar effect to that of 10 μM α-tocopherol in the AMVN-induced lipophilic free radical generating system. Thus, the antioxidant activity of L-FABP was comparable to α-tocopherol and much greater than ascorbic acid at the same molar concentrations. To understand whether long-chain fatty acid binding to L-FABP influences its antioxidant activity, LDL was incubated with 40 mM AAPH or 10 mM AMVN in the absence or presence of 10 μM L-FABP, which was preincubated with 30 μM α-bromo-palmitate (α-bromo-palmitate is not metabolized) or 30 μM palmitate (reversible binding). In the AAPH-induced lipid peroxidation system, 10 µM L-FABP was able to significantly reduce MDA production by 70 ± 2% (P < 0.001), while α-bromo-palmitate and palmitate partially blocked the L-FABP antioxidative activity by 29 ± 2% and 19 ± 1%, respectively (see Fig. 4; compared with L-FABP no binding; P < 0.001). Figure 4 also shows that in the AMVN-induced lipid peroxidation system, 10 µM L-FABP was able to reduce MDA production by 50 ± 1%, while α-bromo-palmitate and palmitate blocked L-FABP antioxidative activity by 43 ± 1% and 11 ± 1%, respectively (compared with L-FABP no binding; P < 0.001). These results indicated that by blocking the L-FABP binding cavity some of the L-FABP antioxidant activity was eliminated. Recombinant L-FABP was analyzed following separation using chromatography by MALDI QqTOF MS. The amino acid sequence of our recombinant rat L-FABP (Table 1) was identical with that of L-FABP isolated from rat liver (25She Y.M. Wang G.Q. Loboda A. Ens W. Standing K.G. Burczynski F.J. Sequencing of rat liver cytosolic proteins by matrix-assisted laser desorption ionization-quadrupole time-of-flight mass spectrometry following electrophoretic separation and extraction.Anal. Biochem. 2002; 310: 137-147Crossref PubMed Scopus (13) Google Scholar). MALDI-TOF analysis showed presence of a major mass peak at m/z of 15,275.9 (Fig. 5A). Total measured mass (15,274.9 Da) of L-FABP was greater than that predicted based on the amino acid sequence (14,272.450 Da). The increas" @default.
• W2022983144 created "2016-06-24" @default.
• W2022983144 creator A5010253703 @default.
• W2022983144 creator A5023894216 @default.
• W2022983144 creator A5046208806 @default.
• W2022983144 creator A5064284123 @default.
• W2022983144 creator A5067416656 @default.
• W2022983144 creator A5076862230 @default.
• W2022983144 date "2009-12-01" @default.
• W2022983144 modified "2023-10-17" @default.
• W2022983144 title "Molecular mechanism of recombinant liver fatty acid binding protein's antioxidant activity" @default.
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