Binding of 25-hydroxycholesterol and cholesterol to different cytoplasmic proteins

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Abstract
Studies were carried out to determine whether or not oxygenated derivatives of cholesterol (e.g., 25-hydroxycholesterol) that specifically suppress the activity of 3-hydroxy-3-methylglutaryl-CoA reductase [mevalonate:NADP+ oxidoreductase (CoA-acylating), EC 1.1.1.34], bind to a soluble component of the cytoplasm different from that which binds the nonsuppressor, cholesterol. Density gradient fractionation of the cytosolic fraction isolated from L cell [mouse fibroblast] cultures that were incubated with low concentrations of 25-hydroxy[26,27-3H]cholesterol or [1,2-3H]cholesterol provided evidence for the existence of at least 2 different sterol-binding proteins. Bound cholesterol sedimented in a sucrose density gradient as 2 or more broad bands with coefficients of approximately 9 S and 21 S. Two relatively narrow bands of bound 25-hydroxycholesterol had sedimentation coefficients of 5 S and 8 S. Preincubation of the cells with a relatively high concentration of unlabeled 25-hydroxycholesterol altered the banding pattern of the 25-hydroxy[3H]cholesterol taken up during a subsequent incubation period by decreasing the size of the major (8S) band. Under these conditions, cholesterol did not affect the banding pattern of 25-hydroxy[3H]cholesterol. The density gradient banding pattern of bound [3H]cholesterol was only slightly affected by preincubating the cells with unlabeled cholesterol or 25-hydroxycholesterol. Both sterols appeared to be bound to proteins because the bound sterols were eliminated from cytosol that had been heated at 100.degree. C, and their sedimentation coefficients were altered by proteolysis.