scholarly journals The Antioxidant Activity of Recombinant Rat Hepatic Fatty Acid Binding Protein T94A Variant

2018 ◽  
Vol 21 (1s) ◽  
pp. 309s-324s
Author(s):  
Tyson Le ◽  
Yuewen Gong ◽  
Neal M Davies ◽  
Ryan Lillico ◽  
Ted Lakowski ◽  
...  
Keyword(s):  
Lipid Peroxidation ◽  
Fatty Acids ◽  
Antioxidant Activity ◽  
Fatty Acid ◽  
Allele Frequency ◽  
Fatty Acid Binding Protein ◽  
Binding Protein ◽  
Fatty Acid Binding ◽  
Acid Binding Protein ◽  
Long Chain

PURPOSE: Liver fatty acid binding protein (FABP1) is a cytoplasmic polypeptide that transports substrates throughout the cytosol and functions as an antioxidant. A common polymorphic variant, FABP1 T94A has a minor allele frequency of 26-38%, 8.3±1.9% homozygous in the human population. The purpose of this study was to mutate and isolate recombinant rat FABP1 to the T94A variant to evaluate the mutant’s antioxidant activity using in vitro studies. METHODS: Site-directed mutagenesis was used to generate a mutation in rat cDNA within a pGEX-6p-2 vector. This plasmid was transformed into competent cells and cultured for expression of FABP1 T94A mutant. The mutated protein was purified using GSTrap Fastflow columns within an ÄKTA FPLC system. A 2,7-dichlorofluorescein (DCF) assay was used to screen the T94A variant antioxidant activity. Additionally, Thiobarbituric Acid Reactive Substances (TBARS) assay was used in determining T94A mutant antioxidant activity in hydrophilic and lipophilic environments through the use of the azo compounds AAPH and MeO-AMVN, respectively and in the presence and absence of the long-chain fatty acid palmitate and α-bromo palmitate. RESULTS: Although the FABP1 T94A (20 μM) mutant significantly reduced DCF fluorescence compared to control (no protein; P< 0.001), there were no significant difference when compared to the wild-type (WT) FABP1. T94A was able to diminish the formation of malondialdehyde (MDA) in both lipophilic and hydrophilic systems. There were significant differences between T94A mutant and WT FABP1 at concentrations 1 and 10 μM (P< 0.05) in the hydrophilic milieu, however, this was not seen at 20 μM and also not seen in the lipophilic milieu at all concentrations. When T94A was pre-incubated with the long-chain fatty acids palmitate or α -bromo palmitate, MDA formation was decreased in both lipid peroxidation systems. There were no statistical differences between the WT FABP1 and T94A bound with fatty acids in both lipid peroxidation systems, however, there was a slight statistical difference when the T94A and WT FABP1 bound α-Br-PA in the AAPH lipid peroxidation system only. CONCLUSIONS: The T94A has antioxidant activity in both hydrophilic and lipophilic environments. The T94A variant of FABP1 does not have a loss of function in regard to acting as an antioxidant but the extent of function may be influenced by ligand binding. We conclude that populations having the minor T94A allele frequency would have similar ROS scavenging potential as those with nascent FABP1.

Comparisons of the effects of temperature on the liver fatty acid binding proteins from hibernator and nonhibernator mammals

1998 ◽  
Vol 76 (4) ◽  
pp. 593-599 ◽  
Author(s):  
J M Stewart ◽  
T E English ◽  
K B Storey
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Ground Squirrel ◽  
Fatty Acid Binding Protein ◽  
Unsaturated Fatty Acids ◽  
Binding Protein ◽  
Liver Fatty Acid ◽  
Fatty Acid Binding ◽  
Acid Binding Protein ◽  
Long Chain

Hibernating mammals rely heavily on lipid metabolism to supply energy during hibernation. We wondered if the fatty acid binding protein from a hibernator responded to temperature differently than that from a nonhibernator. We found that the Kd for oleate of the liver fatty acid binding protein (1.5 micromolar) isolated from ground squirrel (Spermophilus richardsonii) was temperature insensitive over 5-37°C, while the rat liver fatty acid binding protein was affected with the Kd at 37°C being about half (0.8 micromolar) that found at lower temperatures. This same trend was observed when comparing the specificity of various fatty acids of differing chain length and degree of unsaturation for the two proteins at 5 and 37°C. At the lower temperature, ground squirrel protein bound long-chain unsaturated fatty acids, particularly linoleate and linolenate, at least as well as at the higher temperature and matched requirements for these fatty acids in the diet. The most common long-chain fatty acid, palmitate, was a more effective ligand for ground squirrel liver fatty acid binding protein at 5°C than at 37°C, with the opposite occurring in the eutherm. Rat protein was clearly not adapted to function optimally at temperatures lower than the animal's body temperature.Key words: fatty acid binding protein, temperature, hibernation.

Titration and Exchange Studies of Liver Fatty Acid-Binding Protein with13C-Labeled Long-Chain Fatty Acids†

Biochemistry ◽  
2002 ◽  
Vol 41 (17) ◽  
pp. 5453-5461 ◽  
Author(s):  
Hsin Wang ◽  
Yan He ◽  
Christopher D. Kroenke ◽  
Sarala Kodukula ◽  
Judith Storch ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Fatty Acid Binding Protein ◽  
Binding Protein ◽  
Long Chain Fatty Acids ◽  
Liver Fatty Acid ◽  
Fatty Acid Binding ◽  
Acid Binding Protein ◽  
Long Chain ◽  
Chain Fatty Acids

Cytosolic fatty acid binding protein enhances rat hepatocyte [3H]palmitate uptake

1999 ◽  
Vol 77 (11) ◽  
pp. 896-901 ◽  
Author(s):  
F J Burczynski ◽  
S Fandrey ◽  
G Wang ◽  
P A Pavletic ◽  
Y Gong
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Fatty Acid Binding Protein ◽  
Binding Protein ◽  
Long Chain Fatty Acids ◽  
Clearance Rates ◽  
Fatty Acid Binding ◽  
Acid Binding Protein ◽  
Palmitate Uptake ◽  
Long Chain

Liver cytosolic fatty acid binding protein (FABP) represents the intracellular equivalent to extracellular serum albumin, participating in the intracellular transport of long-chain fatty acids. In this study we observed the effect of increasing and decreasing FABP levels on hepatocyte [3H]palmitate uptake in male Sprague-Dawley rats. We also were interested to determine whether uptake, from either the unbound or unbound and protein-bound fractions, was fundamentally different at the different FABP levels. FABP levels were modified by hypophysectomy and clofibrate treatment (50 mg/100 g body weight for 10 days). Results showed that the [3H]palmitate clearance rates paralleled the 54% decrease and 73% increase in FABP levels in hypophysectomy and clofibrate-treated animals, respectively. In the presence of 2 and 20 µM albumin, hepatocyte clearance rates of unbound [3H]palmitate from hypophysectomized animals (0.16 ± 0.01 and 0.64 ± 0.01 mL·s-1·10-6 cells, respectively) were significantly lower (p < 0.01) than those of the sham group (0.30 ± 0.02 and 1.00 ± 0.06 mL·s-1·10-6 cells, respectively). However, the unbound [3H]palmitate clearance rates from the clofibrate-treated group (0.39 ± 0.04 and 1.18 ± 0.12 mL·s-1·10-6 cells) were significantly higher (p < 0.01) than the control group (0.29 ± 0.02 and 0.81 ± 0.05 mL·s-1·10-6 cells) for 2 and 20 µM albumin, respectively. To investigate whether uptake was fundamentally different between the hypophysectomized and clofibrate-treated groups, we expressed the clearance rates as enhancement factors, i.e., EF = CL20µM/CL2µM. No statistical difference was observed between EF of the hypophsectomized (3.8 ± 0.4) and EF of the clofibrate-treated (3.1 ± 0.3) groups, suggesting that the extracted ligand originated from similar fractions.Key words: palmitic acid, albumin, growth hormone, liver, fatty acid binding protein, uptake, hepatic, long-chain fatty acids, clofibrate, hypophysectomy.

scholarly journals Up-regulation of the expression of the gene for liver fatty acid-binding protein by long-chain fatty acids

1996 ◽  
Vol 319 (2) ◽  
pp. 483-487 ◽  
Author(s):  
Claire MEUNIER-DURMORT ◽  
Hélène POIRIER ◽  
Isabelle NIOT ◽  
Claude FOREST ◽  
Philippe BESNARD
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Fatty Acid Binding Protein ◽  
Fold Increase ◽  
Long Chain Fatty Acids ◽  
Liver Fatty Acid ◽  
Fatty Acid Binding ◽  
Acid Binding Protein ◽  
Fabp Gene ◽  
Long Chain

The role of fatty acids in the expression of the gene for liver fatty acid-binding protein (L-FABP) was investigated in the well-differentiated FAO rat hepatoma cell line. Cells were maintained in serum-free medium containing 40 µM BSA/320 µM oleate. Western blot analysis showed that oleate triggered an approx. 4-fold increase in the cytosolic L-FABP level in 16 h. Oleate specifically stimulated L-FABP mRNA in time-dependent and dose-dependent manners with a maximum 7-fold increase at 16 h in FAO cells. Preincubation of FAO cells with cycloheximide prevented the oleate-mediated induction of L-FABP mRNA, showing that protein synthesis was required for the action of fatty acids. Run-on transcription assays demonstrated that the control of L-FABP gene expression by oleate was, at least in part, transcriptional. Palmitic acid, oleic acid, linoleic acid, linolenic acid and arachidonic acid were similarly potent whereas octanoic acid was inefficient. This regulation was also found in normal hepatocytes. Therefore long-chain fatty acids are strong inducers of L-FABP gene expression. FAO cells constitute a useful tool for studying the underlying mechanism of fatty acid action.

Inhibition of binding to fatty acid binding protein reduces the intracellular transport of fatty acids

1996 ◽  
Vol 271 (1) ◽  
pp. G113-G120 ◽  
Author(s):  
B. A. Luxon
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Intracellular Transport ◽  
Fatty Acid Binding Protein ◽  
Binding Protein ◽  
Female Rats ◽  
Dependent Manner ◽  
Fatty Acid Binding ◽  
Acid Binding Protein ◽  
Cytoplasmic Transport

Male livers, containing lesser amounts of fatty acid binding protein (FABP), utilize fatty acids more slowly than female livers. Conventional wisdom dictates that FABP stimulates fatty acid use by increasing cytoplasmic transport rates. Previously, we showed that the cytoplasmic diffusion of a fatty acid analogue [12-N-methyl-7-nitrobenzo-2-oxa-1,3-diazol-amino stearate (NBD-stearate)] is faster in female hepatocytes, paralleling the larger amounts of FABP. Sex differences in other cytoplasmic factors could also lead to faster diffusion, independent of FABP levels. The aim of this study was to determine the effect of inhibition of fatty acid binding to FABP on the directly measured intracellular transport rate of NBD-stearate. The binding of NBD-stearate to FABP was reduced by incubating hepatocytes isolated from male and female rats with alpha-bromo-palmitate (0-1,500 microM), a modified long-chain fatty acid that binds to FABP. The inhibition by alpha-bromo-palmitate on NBD-stearate binding to FABP was measured with the use of centrifugation to separate cytosol from cytoplasmic membranes. Laser photobleaching (fluorescence recovery after photobleaching) was used to measure the cytoplasmic diffusion of NBD-stearate in hepatocytes. Alpha-Bromo-palmitate incubation reduced NBD-stearate binding to FABP in a dose-dependent manner. The measured diffusion rate was also reduced in proportion to the degree of binding inhibition. We conclude that cytoplasmic transport of NBD-stearate is modulated by binding to soluble proteins like FABP. FABP enhances diffusive transport by reducing binding to immobile cytosolic membranes.

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