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. 1998 Feb 17;95(4):1899–1902. doi: 10.1073/pnas.95.4.1899

Figure 2.

Figure 2

Heterologous expression of the 5′ truncated Δ7-sterol reductase cDNA (D7-ORF) and a myc-tagged derivative (mycD7-ORF) in S. cerevisiae strain JB811 (ade2–1 leu2–3,112 pep4–3 trp1–289 ura3–52). (A) For Western blotting, 5 μg of microsomal protein was separated on a 12% (wt/vol) SDS gel under reducing conditions (sample buffer containing 10 mM DTT), immunostained with 20 ng/ml 9E10 c-myc antibody as in ref. 15, and developed with CDP-Star chemiluminescent reagent. The arrow indicates the migration of a c-myc immunoreactive band with a migration corresponding to 40 kDa (lane 1, mock; lane 2, D7-ORF; lane 3, mycD7-ORF). (B) Time course of Δ7-sterol reductase activity. Microsomes (0.5 mg) prepared from strains transformed with the vector without cDNA (mock, ▪), D7-ORF (•), and mycD7-ORF (⧫) were incubated anaerobically for the indicated times with 7-dehydrocholesterol in the presence of 2 mM NADPH. Mean values from duplicate determinations are shown. (C) Lineweaver Burk plot from yeast microsomes expressing D7-ORF (•) and mycD7-ORF (⧫). From this experiment Km values of 30 μM and 50 μM, respectively, were determined for D7-ORF and mycD7-ORF (Vmax 0.85 and 1.65 nmol/min per mg of protein, respectively). (D) Inhibition of the Δ7-sterol reductase (D7-ORF) by AY9944 (•, IC50 = 0.013 μM), BM15766 (⧫, IC50 = 1.2 μM), triparanol (▴, IC50 14.2 μM), and tamoxifen (▾, IC50 > 100 μM). Data shown are mean ± SD (n = 3).