Research Article

Cysteine-10 on 17β-Hydroxysteroid Dehydrogenase 1 Has Stabilizing Interactions in the Cofactor Binding Region and Renders Sensitivity to Sulfhydryl Modifying Chemicals

Figure 5

Stability of 17β-HSD1 and Cys10Ser mutant. C-terminal histidine-tagged wild-type 17β-HSD1 and Cys10Ser mutant enzymes were expressed in HEK-293 cells. A, 24 h posttransfection cells were incubated with 50 μg/mL cycloheximide to inhibit de novo protein synthesis. After 0, 12, 24, and 48 h cells were harvested and the amount of expressed protein was analyzed semiquantitatively using anti-histidine antibody. β-actin served as control. B, the protein half-life of 17β-HSD1 and Cys10Ser mutant was estimated by pulse-chase experiments, labeling cellular proteins with tritiated L-leucine, followed by washing and incubation in leucine-free medium for different times. After purification of histidine-tagged proteins by Ni-NTA agarose, proteins were separated by SDS-PAGE and gels were dried and exposed to tritium-sensitive screens for detection of radioactivity by phosphor-imaging. Representative experiments are shown.