| Abstract: | Epididymal 4-ene steroid 5 alpha-reductase converts testosterone to 5 alpha-dihydrotestosterone. The enzyme is localized to the nuclear and microsomal fractions, and the activity can be altered by modifying the phospholipids in the membrane environment. To investigate the membrane dependence of 4-ene steroid 5 alpha-reductase, we have treated nuclear and microsomal membranes with combinations of phospholipase A2 and phospholipase C, and examined the effects on 4-ene steroid 5 alpha-reductase activity. Sequential addition of phospholipase A2 and phospholipase C to the nuclear fraction, reduced the 4-ene steroid 5 alpha-reductase activity to approx 25% of the control level. Neither the nature of the phospholipase, nor the sequence of addition altered the inhibition. When both phospholipases were added simultaneously, nuclear 4-ene steroid 5 alpha-reductase activity was inhibited in a linear fashion, and in tests for cooperativity, the effects of phospholipase A2 and phospholipase C were clearly additive. The microsomal enzyme responded differently to sequential phospholipase treatments; if phospholipase A2 was followed by phospholipase C, or phospholipase C followed by phospholipase A2, the 4-ene steroid 5 alpha-reductase activity was, respectively, 13 and 27% of the control. In contrast, sequential addition of the same phospholipase reduced the activity of 4-ene steroid 5 alpha-reductase to approx 40% of the control level. Furthermore, simultaneous addition of phospholipase A2 and phospholipase C to the microsomal fraction, resulted in non-linearity of 4-ene steroid 5 alpha-reductase activity with time, whereas when added individually, linearity of 4-ene steroid 5 alpha-reductase was maintained. Consequently, it was not possible to test for cooperative effects of phospholipases on the microsomal 4-ene steroid 5 alpha-reductase. These findings suggest that for the nuclear 4-ene steroid 5 alpha-reductase, the polar and non-polar regions of the membrane environment have similar functions, which are most likely involved in the maintenance of the structural integrity of the enzyme. For the microsomal enzyme, the polar and non-polar regions of the membrane appear to have different functions, not only for the maintenance of enzyme integrity, but also in the mechanism at the active site. |
