Characterization and primary structure of a fatty acid-binding protein andits isoforms from the liver of the amphibia, Rana catesbeiana

Fatty acid-binding protein (FABP) was purified from the liver of the Amphib ia, Rana catesbeiana, by gel filtration and ion-exchange chromatography. Th e complete primary structure of the frog liver FABP was determined by prote in analysis. Two isoforms, I and II, were separated by reversed phase HPLC, and found to differ by 10 atomic mass units as measured by ion-spray ioniz ation mass spectrometry, A detailed analysis of enzymatic peptides revealed a single Pro (isoform I)/Ser (isoform II) replacement at position 16. It s eems remarkable that a rather neutral point mutation results in the nearly complete separation of the two isoforms by reversed phase chromatography. H omology modeling suggests the location of this site on the first helix of t he helix-turn-helix domain and the presence of a single thiol group of cyst eine-91 at the inside of the ligand-binding cavity, Binding studies using a natural fluorescent fatty acid, cis-parinaric acid, showed a lower K-d val ue for the serine form and large enhancement of fluorescence intensity upon glutathione-thiolation at cysteine-91, Examination of phylogenetic relatio nships identified the frog liver protein as a mammalian liver type FABP, an d suggested a change in the vertebrate liver FABP gene expression at the bo ny fish/cartilagenous fish boundary.