Purification of oxysterol binding protein from hamster liver cytosol.
We have purified to apparent homogeneity an oxysterol binding protein from cytosol of hamster livers. This protein, which corresponds to the protein described by Taylor and Kandutsch (Taylor, F. R., and Kandutsch, A. (1985) Chem. Phys. Lipids 38, 187-194), binds oxysterols such as 25-hydroxycholesterol but does not bind cholesterol or steroid hormones in vitro. It may participate in the feedback repression of enzymes of cholesterol biosynthesis and the low density lipoprotein receptor. The protein was purified more than 40,000-fold with a series of ion exchange chromatography steps. The final preparation contained a doublet of peptides with molecular weights (Mr) of 101,000 and 96,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These components formed a complex that migrated on gel filtration with an apparent Mr of 280,000 in the absence or presence of 25-hydroxycholesterol. The amino acid sequence of a tryptic peptide from this protein complex was obtained, and a monoclonal antipeptide antibody was prepared. The antibody stained both the 101,000- and 96,000-Da proteins on immunoblots, suggesting that these two components are closely related and that one may be a modified or proteolyzed form of the other. With the purified protein now available, it should become possible to determine the role, if any, that this protein plays in the regulation of intracellular cholesterol metabolism.
Dawson
P A
PA
Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas 75235.
Van der Westhuyzen
D R
DR
Goldstein
J L
JL
Brown
M S
MS
D000818:Animals; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D006224:Cricetinae; D003600:Cytosol / Q000378:metabolism; D006888:Hydroxycholesterols / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000378:metabolism*; D008297:Male; D008647:Mesocricetus; D008970:Molecular Weight; D011987:Receptors, Steroid / Q000302:isolation & purification* / Q000378:metabolism; D013261:Sterols / Q000378:metabolism*; D000098825:Oxysterol Binding Proteins
D006888:Hydroxycholesterols; D011987:Receptors, Steroid; D013261:Sterols; D000098825:Oxysterol Binding Proteins; C007997:25-hydroxycholesterol