Characterization of a novel alpha-tocopherol-binding protein from bovine heart cytosol.
We previously reported the identification of a new alpha-tocopherol-binding protein (approximately 15 kDa) in the cytosol of rat liver and heart and in rabbit heart (A.K. Dutta-Roy et al., J. Nutr. Biochem. 5, 562-570, 1994). This protein specifically binds alpha-tocopherol and enhances its transfer between separate membranes. In the present paper we have purified and characterized the alpha-tocopherol-binding protein from bovine heart cytosol and compared its various structural and functional properties with the similar size (approximately 15 kDa) cytosolic fatty acid-binding protein of this tissue. alpha-Tocopherol-binding protein was purified to electrophoretic homogeneity from bovine heart cytosol by a procedure involving precipitation with 70% ammonium sulfate, followed sequentially by gel filtration chromatography and chromatofocusing. The purified protein migrated as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular mass of 16 kDa. Isoelectric focusing of the purified protein showed that the pI value is around 4.5. The binding of alpha-tocopherol to the purified protein was rapid, reversible, and saturable. The alpha-tocopherol-binding protein did not bind oleate as assessed by direct radiolabeled fatty acid binding and fluorescence enhancement assay. Amino acid analysis showed the presence of a large number of Ala, Gly, Ser, Lys, and Pro residues and a lesser number of aromatic residues in this protein. Anti-bovine heart fatty acid-binding protein antibody did not recognize the alpha-tocopherol-binding protein in the Western blot. The Western blot, ligand affinity, molecular size, and amino acid analysis data suggest that the alpha-tocopherol-binding protein is different from the cytosolic fatty acid-binding protein and that it may be involved in intracellular transport and metabolism of alpha-tocopherol.
Gordon
M J
MJ
Division of Biochemical Sciences, Rowett Research Institute, Aberdeen, Scotland, United Kingdom.
Campbell
F M
FM
Duthie
G G
GG
Dutta-Roy
A K
AK
D000818:Animals; D001693:Biological Transport, Active; D002352:Carrier Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D002417:Cattle; D003600:Cytosol / Q000378:metabolism; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D005227:Fatty Acids / Q000378:metabolism; D066298:In Vitro Techniques; D007700:Kinetics; D015394:Molecular Structure; D008970:Molecular Weight; D009206:Myocardium / Q000378:metabolism*; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; D014810:Vitamin E / Q000378:metabolism*
D002352:Carrier Proteins; C490174:Fabp7 protein, rat; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D005227:Fatty Acids; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; C017763:alpha-tocopherol transfer protein; D014810:Vitamin E