FIGURE 3.

[³H]Sterol binding to His₆-Scap(Loop1). Each reaction, in a final volume of 100 μl of buffer C with 0.004% Nonidet P-40 and 0.001% Fos-choline 13, contained 5 pmol of purified His₆-SCAP(Loop1), 1 μg of bovine serum albumin, and the indicated concentration of either [³H]cholesterol (222 dpm/fmol) or 25-[³H]hydroxycholesterol (165 dpm/fmol) in the absence (●) or presence (○) of 10 μm unlabeled sterol, as indicated. After 4 h at room temperature, the bound [³H]sterol was measured as described under “Experimental Procedures.” Each data point denotes the mean ± S.E. of triplicate assays. A, total [³H]cholesterol binding without subtraction of blank values. B, specific [³H]cholesterol or 25-[³H]hydroxycholesterol binding after subtraction of blank values determined in the presence of the respective unlabeled sterol at 10 μm (<127 fmol/tube for [³H]cholesterol binding; <30 fmol/tube for 25-[³H]hydroxycholesterol binding).