AMINO-ACID EXCHANGE AND COVALENT MODIFICATION BY CYSTEINE AND GLUTATHIONE EXPLAIN ISOFORMS OF FATTY-ACID-BINDING PROTEIN OCCURRING IN BOVINE LIVER

A unique property of the liver-type member of the family of fatty acid -binding proteins is the heterogeneic pattern observed upon isolation, which can only partly be ascribed to the state of lipidation. Here we unraveled the structural basis of the heterogeneity of delipidated li ver-type fatty acid-binding protein (L-FABP). Charge fractions of L-FA BP focusing at pH 6.0 and at pH 7.0/7.1 were first isolated from bovin e liver. Upon reduction, however, two distinct isoforms, namely pI 6.0 L-FABP and pI 7.0 L-FABP, were observed. From these isoforms peptides were generated enzymically and chemically by four independent methods . Peptides were separated by reverse phase high performance liquid chr omatography and analyzed by Edman degradation and plasma desorption ma ss spectrometry. The complete amino acid sequences of the isoforms wer e established; they consist of 127 amino acids and each is N-terminall y blocked with an acetyl group. The difference between pI 6.0 L-FABP a nd pI 7.0 L-FABP was attributed to an asparagine-aspartate exchange at position 105. When tryptic peptides of the pH 7.0/7.1 fraction were a nalyzed, discrepancies between sequence and mass data of the peptides containing at position 69 the sole cysteine of L-FABP led to the discl osure of a cysteinylation occurring at this position and giving rise t o the slightly more basic pH 7.1 species. Moreover, chemical modificat ion studies revealed that a part of the pH 6.0 fraction was pl 7.0 L-F ABP that was glutathionylated at Cys69. Neither modification, however, prevented the binding of fatty acids. Together amino acid exchange an d covalent modification of cysteine entirely explain the heterogeneity of L-FABP from bovine liver.