Figure 2.

Activities of StAR mutants. A, Activity of full-length StAR in whole cells. COS-1 cells were cotransfected with expression vectors for the cholesterol side-chain cleavage system (F2) and either wild-type (WT) or mutant StAR, and pregnenolone was measured 48 h later by immunoassay. The StAR-independent substrate 22(R)-hydroxycholesterol (22R-OH) was added to the cell culture media to determine the maximum steroidogenic capacity of the cells. Data are expressed as the mean ± sem from at least three independent experiments, each performed in triplicate. B, Activity of isolated N-62 StAR on mitochondria in vitro. Purified proteins were added to mitochondria from steroidogenic mouse MA-10 Leydig cells, and pregnenolone production from endogenous mitochondrial cholesterol was measured. Data are expressed as mean ± sem from four experiments, each performed in duplicate. C, Cholesterol binding assay. Binding of various concentrations of NBD-cholesterol by wild-type and mutant StAR was measured by fluorescence; control is buffer without protein. Data are expressed as mean ± sem for three experiments, each performed in triplicate.