Purification and characterization of a new cholesterol-binding protein from rat liver cytosol.

A new cholesterol-binding protein (CBP) has been purified from rat liver cytosol by ammonium sulfate precipitation, gel filtration and ion exchange chromatography. It appears to be homogeneous by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, and, using antiserum to purified CBP, by Ouchterlony double-immunodiffusion analysis, by immunoelectrophoresis, and by SDS gel electrophoresis of radiolabeled immunoprecipitates. Although not completely homogeneous by polyacrylamide gel electrophoresis in two different nondissociating systems, more than 95% of the stainable protein is present in the major band. Isoelectric focusing shows two bands with pH, values of 5.75 and 5.80. The molecular weight is estimated to be 26,500 by SDS gel electrophoresis. Lipids bound to purified CBP include both free and esterified cholesterol, free fatty acids, and trace amounts of phospholipid. The moles of lipid per mol of protein are: free cholesterol, 0.1; esterified cholesterol, 0.1; free fatty acids, 1.4; and phospholipids, ~0.01. After in vitro incubation with lecithinlcholesterol vesicles, purified CBP increases in free cholesterol content to 1.03 f 0.22 mol/mol of protein, but there is no detectable increase in phospholipid content. CBP differs from the two sterol carrier proteins previously described in the literature in almost all characteristics compared, including immunochemical reactivity and electrophoretic mobilities under both dissociating and nondissociating conditions. CBP differs from the sterol carrier proteins in containing small amounts of cholesterol even after extensive purification and also in being able to bind larger amounts in uitro; CBP also differs by not aggregating in the presence of lecithin/cholesterol dispersions. Anti-CBP antiserum gives a single precipitin line on double immunodiffusion with rat liver cytosol; it also reacts with solubilized membranes from both washed liver mitochondria and microsomes and with extracts from nuclei. In addition, the antiserum cross-reacts with cytosol preparations from several extrahepatic tissues but not with rat serum or purified serum lipoproteins, red blood cell extracts or intestinal lymph.