Molecular mechanism of recombinant liver fatty acid binding protein's antioxidant activity.
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 approximately 80 Da mass shift compared with native L-FABP. Protection against lipid peroxidation of L-FABP after binding with palmitate or alpha-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.
Yan
Jing
J
Faculty of Pharmacy, University of Manitoba, Winnipeg, Canada.
Gong
Yuewen
Y
She
Yi-Min
YM
Wang
Guqi
G
Roberts
Michael S
MS
Burczynski
Frank J
FJ
D000578:Amidines / Q000737:chemistry / Q000494:pharmacology; D000818:Animals; D000975:Antioxidants / Q000737:chemistry / Q000378:metabolism*; D001205:Ascorbic Acid / Q000494:pharmacology; D001391:Azo Compounds / Q000737:chemistry / Q000494:pharmacology; D004926:Escherichia coli / Q000737:chemistry / Q000378:metabolism; D050556:Fatty Acid-Binding Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D005452:Fluoresceins / Q000737:chemistry; D005453:Fluorescence; D005609:Free Radicals / Q000737:chemistry / Q000494:pharmacology; D005982:Glutathione Transferase / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D015227:Lipid Peroxidation / Q000187:drug effects; D009570:Nitriles / Q000737:chemistry / Q000494:pharmacology; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D024502:alpha-Tocopherol / Q000494:pharmacology
D000578:Amidines; D000975:Antioxidants; D001391:Azo Compounds; D050556:Fatty Acid-Binding Proteins; D005452:Fluoresceins; D005609:Free Radicals; D009570:Nitriles; D011994:Recombinant Proteins; C055792:2,2'-azobis(2,4-dimethylvaleronitrile); C029569:diacetyldichlorofluorescein; C046728:2,2'-azobis(2-amidinopropane); D005982:Glutathione Transferase; D024502:alpha-Tocopherol; D001205:Ascorbic Acid