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. 2010 Jun 3;285(32):24529–24537. doi: 10.1074/jbc.M110.127779

FIGURE 7.

FIGURE 7.

Detection of 5β-reductase activity of AKR1D1 and its natural mutants in transfected HEK 293 cells. Production of 5β-reduced products from 0.5 μm (A) or 5.0 μm (B) testosterone was followed for up to 48 h in cells expressing either wild type or mutant AKR1D1 or in cells that had been transfected with empty vector (negative control). 5β-Reductase activity for AKR1D1-L106F and AKR1D1-R261C was clearly detectable but close to the detection limit. Cells expressing vector control or AKR1D1-P198L did not exhibit measurable 5β-reduction activity (<0.1 pmol/mg at 60 h of incubation; data not shown). For wild type (C) and AKR1D1-P133R (D), estimates of Km and Vmax were obtained in the transfected cells using testosterone as substrate. All values are normalized to total amount of protein. For A and B, each experiment was performed twice and gave similar results; one representative experiment is shown. For C and D, each point was measured in triplicate with S.E. values less than 10%.