Fig. 3.

Time course of 27-HC biosynthesis and HMGR activity in response to PM cholesterol enrichment. A: 27-HC biosynthesis. Five 75 cm² flasks of cells were incubated overnight in medium A. The medium was replaced with 4 ml PBS, a pulse of 18 mg HPCD + 0.43 mg cholesterol added to each, and the flasks incubated for 7 min at 37°C. This enriched the cells by ∼50%. The cells were rinsed, layered with 4 ml medium A, and incubated at 37°C. For the zero-time point, the cells were processed immediately after the cholesterol pulse. After the indicated incubation times, total 27-HC and cell protein were determined. Values are the means of duplicate assays that agreed to within 3%. B: HMGR activity. Six replicate 25 cm² flasks were incubated overnight in medium A. The medium was replaced with 1.5 ml PBS, a pulse of 6 mg HPCD + 0.14 mg cholesterol added, and the flasks incubated for 7 min at 37°C. The buffer was replaced with 1.5 ml medium A and the flasks incubated at 37°C for the times indicated. HMGR activity and cell protein were then determined in duplicate. The enriched cells contained, on average, 43 μg cholesterol/mg cell protein, whereas unenriched cells normally contain ∼35 μg cholesterol/mg protein (23). The time course was fit to a first order expression with a half-time of 8.5 min. Data are from one of two similar experiments.