K. Baba et al., Characterization and primary structure of a fatty acid-binding protein andits isoforms from the liver of the amphibia, Rana catesbeiana, J BIOCHEM, 125(1), 1999, pp. 115-122
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.