Different forms of the oxysterol-binding protein. Binding kinetics and stability.
Based upon measurements of the sedimentation coefficient and the Stokes radii, three forms of the oxysterol-binding protein were identified. The unliganded binding protein was the largest (7.7 S, Stokes radius = 71.6 A, Mr = 236,000) was relatively asymmetric (f/f0 = 1.7), and was composed of at least three subunits. Binding of 25-hydroxycholesterol was associated with a reduction in the size of the protein (7.5 S, Stokes radius = 50 A, Mr approximately 169,000) and an increase in symmetry (f/f0 = 1.4), due to the loss of a subunit of Mr approximately 67,000. At pH 6 or lower, the Mr = 169,000 sterol-protein complex was altered so that reversible dissociation to give a smaller (4.2 S, Stokes radius = 53 A, Mr = 97,000) more asymmetric (f/f0 = 1.8) sterol-protein complex occurred when it was sedimented in a sucrose gradient buffered at pH 7.4 containing 0.3 M KCl and 2.5 M urea. Irreversible dissociation of the 7.5 S, Mr = 169,000 form to a 4.2 S form occurred spontaneously when the complex in whole cytosol buffered at pH 7.8 was allowed to stand overnight at 0 degree C, or when the partially purified complex was incubated at pH 5.5 at 0 degree C for several days. The partially purified, unliganded binding protein was unstable at 0 degree C (approximately 75% loss of binding activity in 24 h) whereas the liganded protein was stable for 7 days at 0 degree C although irreversible conversion to a 4.2 S form occurred under some conditions. Rates of sterol binding and dissociation were increased in the presence of 2.5 M urea at pH 7.4 or when the pH was lowered to 5.5 Kd values were not greatly altered under the various incubation conditions.
D000818:Animals; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D004355:Drug Stability; D006863:Hydrogen-Ion Concentration; D006888:Hydroxycholesterols / Q000378:metabolism*; D007700:Kinetics; D007739:L Cells / Q000378:metabolism; D046911:Macromolecular Substances; D051379:Mice; D008970:Molecular Weight; D011487:Protein Conformation; D013261:Sterols / Q000302:isolation & purification / Q000378:metabolism*; D014316:Tritium
D002352:Carrier Proteins; D006888:Hydroxycholesterols; D046911:Macromolecular Substances; D013261:Sterols; D014316:Tritium; C007997:25-hydroxycholesterol