Isolation and characterization of two distinct forms of liver fatty acid binding protein from the rat.
Liver fatty acid binding protein (L-FABP) appears to contain several different forms that may result from post-translational modification or bound ligand. To further assess this possibility, L-FABP was purified from rat liver homogenate and two putative isoforms separated using a sulfonyl column, a strong cation exchange resin. Fraction I eluted at 0.2 M NaCl, had a pI of 7.59, and following a final size exclusion step contained > 98% L-FABP. Fraction II eluted at 1.0 M NaCl, had a pI of 7.59, and following a final size exclusion step contained > 99% L-FABP. Both fractions contained approx. 0.15 moles of endogenous bound fatty acid per mole of protein, while L-FABP not subjected to the cation exchange step contained 0.75 moles of fatty acid per mole of protein. Fractions I and II had a greater proportion of saturated and monounsaturated fatty acids with a large reduction in polyunsaturated fatty acids compared to L-FABP not fractionated by cation exchange. Mass spectral analysis indicated the molecular mass of Fraction I was 14,315.02 +/- 0.35 Da and Fraction II was 14,315.86 +/- 0.34 Da. The peptide map for each fraction was determined by limited digestion of each fraction with either trypsin, Asp-N, or chymotrypsin to yield overlapping peptide fragments. Mass spectral analysis of these digests indicated the two proteins had identical amino acid fragments and that Cys69 was reduced and there were no Asn to Asp exchanges. Hence, these two forms of L-FABP were not isoforms and were not the result of differences in bound fatty acid. It is proposed that these two distinct forms of rat L-FABP were structural conformers based on two alternative folding pathways.
Murphy
E J
EJ
Department of Physiology and Pharmacology, Texas A&M University, College Station 77843, USA. murphye@lns.nia.nih.gov
Edmondson
R D
RD
Russell
D H
DH
Colles
S
S
Schroeder
F
F
D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D002852:Chromatography, Ion Exchange; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D005227:Fatty Acids / Q000737:chemistry / Q000378:metabolism*; D007526:Isoelectric Point; D008099:Liver / Q000378:metabolism*; D008297:Male; D008969:Molecular Sequence Data; D008970:Molecular Weight; D018996:Myelin P2 Protein / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D051381:Rats; D017207:Rats, Sprague-Dawley; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization