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 liver-type fatty acid-binding protein (L-FABP). Charge fractions of L-FABP focusing at pH 6.0 and at pH 7.0/7.1 were first isolated from bovine 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 chromatography and analyzed by Edman degradation and plasma desorption mass spectrometry. The complete amino acid sequences of the isoforms were established; they consist of 127 amino acids and each is N-terminally blocked with an acetyl group. The difference between pI 6.0 L-FABP and 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 analyzed, discrepancies between sequence and mass data of the peptides containing at position 69 the sole cysteine of L-FABP led to the disclosure of a cysteinylation occurring at this position and giving rise to the slightly more basic pH 7.1 species. Moreover, chemical modification studies revealed that a part of the pH 6.0 fraction was pI 7.0 L-FABP that was glutathionylated at Cys69. Neither modification, however, prevented the binding of fatty acids. Together amino acid exchange and covalent modification of cysteine entirely explain the heterogeneity of L-FABP from bovine liver.
Dörmann
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P
Department of Biochemistry, University of Münster, Germany.
Börchers
T
T
Korf
U
U
Højrup
P
P
Roepstorff
P
P
Spener
F
F
D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000378:metabolism*; D050556:Fatty Acid-Binding Proteins; D005227:Fatty Acids / Q000378:metabolism*; D005978:Glutathione / Q000378:metabolism*; D007525:Isoelectric Focusing; D008099:Liver / Q000378:metabolism*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins; D017386:Sequence Homology, Amino Acid
D002352:Carrier Proteins; C493320:Fabp1 protein, mouse; D050556:Fatty Acid-Binding Proteins; D005227:Fatty Acids; D009363:Neoplasm Proteins; D005978:Glutathione; D003545:Cysteine