Identification and purification of a human liver cytosolic tocopherol binding protein.
We recently purified rat tocopherol binding protein (TBP), a 32-kDa cytosol protein which specifically binds alpha-tocopherol, exists as two charge isoforms, and is expressed exclusively in liver and only in hepatocytes. For the present work, we sought to identify the human hepatic tocopherol binding protein from normal human livers harvested from organ donors. Gel filtration of hepatic cytosol identified a peak of [alpha-3H]-tocopherol binding in the 30- to 40-kDa fractions displaceable by excess unlabeled alpha-tocopherol. The fractions exhibiting this binding were pooled and run on Affi-Gel Blue affinity chromatography eluted with a salt gradient. A single major peak of tocopherol binding activity eluted at 22 mS/cm. This peak was further purified by FPLC chromatofocusing. A single protein peak of specific alpha-tocopherol binding eluted at pH 5.9. Finally, the peak from chromatofocusing was purified to apparent homogeneity by reversed-phase microbore HPLC chromatography. Two closely eluting protein peaks were separated and each was homogeneous, had identical migration on a SDS-PAGE (36 kDa), and had the same amino acid composition. The purified human TBP exhibited displaceable, specific alpha-tocopherol binding in the gel filtration assay of tocopherol binding. Laser desorption time-of-flight mass spectroscopy revealed a molecular weight of 36.6 kDa. Both forms of human TBP reacted in Western blot with polyclonal rabbit anti-rat TBP. Identification of the human tocopherol binding protein will allow future studies on its physiological function in human alpha-tocopherol metabolism.
Kuhlenkamp
J
J
Department of Medicine, USC School of Medicine, Los Angeles 90033.
Ronk
M
M
Yusin
M
M
Stolz
A
A
Kaplowitz
N
N
D000818:Animals; D002352:Carrier Proteins / Q000302:isolation & purification* / Q000378:metabolism; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003600:Cytosol / Q000737:chemistry; D006801:Humans; D007525:Isoelectric Focusing; D008099:Liver / Q000737:chemistry*; D013058:Mass Spectrometry; D008970:Molecular Weight; D051381:Rats; D014810:Vitamin E / Q000378:metabolism*
D002352:Carrier Proteins; C017763:alpha-tocopherol transfer protein; D014810:Vitamin E