J. st er oid Biochem. Vol. 26, No. 5, pp. 581-588, 1987 0022-4731/ 87 53.00 + 0.00 Printed in Great Britain. All rights reserved Copyright 0 1987 Pergamon Journals Ltd DIFFERENTIAL EFFECTS OF COMBINATIONS OF PHOSPHOLIPASE A, AND PHOSPHOLIPASE C ON THE ACTIVITY OF RAT EPIDIDYMAL NUCLEAR AND MICROSOMAL 4-ENE STEROID 5a -REDUCTASE G. M. COOKEand B. ROBAIRE* Centre for the Study of Reproduction, Departments of Obstetrics and Gynecology, and Pharmacology and Therapeutics, McGill University, Montreal, Quebec, Canada (Received 4 August 1986) Summary-Epididymal Gene steroid Sa-reductase converts testosterone to Sa-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 Sa-reductase, we have treated nuclear and microsomal membranes with combinations of phospholipase A, and phospholipase C, and examined the effects on Cene steroid 5x-reductase activity. Sequential addition of phospholipase A, and phospholipase C to the nuclear fraction, reduced the 4-ene steroid 5a-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 Sa-reductase activity was inhibited in a linear fashion, and in tests for cooperativity, the effects of phospholipase A, and phospholipase C were clearly additive. The microsomal enzyme responded differently to sequential phospholipase treatments; if phospholipase A, was followed by phospholipase C, or phospholipase C followed by phospholipase A,, the 4-ene steroid Sa-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 5a-reductase to approx 40% of the control level. Furthermore, simultaneous addition of phospholipase A, and phospholipase C to the microsomal fraction, resulted in non-linearity of 4-ene steroid Sa-reductase activity with time, whereas when added individually, linearity of Gene steroid Sa-reductase was maintained. Consequently, it was not possible to test for cooperative effects of phospholipases on the microsomal Qene steroid Sa-reductase. These findings suggest that for the nuclear 4-ene steroid Sa-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. INTRODUCTION In the adult male rat, Sa-dihydrotestosterone (DHT) is important for the normal function of androgen target tissues such as the prostate [l] and seminal vesicles [2,3], and in the epididymis, DHT is essential for the maturation of spermatozoa [4-6]. The con- version of testosterone to DHT is catalysed by the enzyme 4-ene steroid Sa-reductase (3-0~0 Sa-steroid: NADP+ 4-ene-oxidoreductase, EC 1.3.1.22) [7]. which is found in the nuclear and microsomal sub- fractions of epididymal homogenates [8]. The dependence of several steroid transforming enzymes on the membrane environment has been demonstrated using a variety of approaches. Solu- bilization of liver 4-ene steroid Sa-reductase, from the phospholipid environment has been shown to cause a loss of specific activity, which can be *Address for correspondence: Department of Pharma- cology and Therapeutics, McGill University, 3655 Drummond Street, Montreal, Quebec, H3G lY6 Canada. partially restored by the addition of phosphatidyl- choline [9-l 11. Similarly the activities of adrenal cholesterol side-chain cleavage and 1l/I?-hydroxylase enzymes have also been shown to be dependent upon membrane phospholipids [12-161. The treat- ment of testicular microsomal membranes with phos- pholipases, has demonstrated that the enzymes re- lated to testosterone biosynthesis and metabolism are profoundly affected by modifications of their phos- pholipid environments [17-201. In the case of the adrenal cholesterol side-chain cleaving enzyme, the phospholipids are believed to enhance the binding of both the sterol to the active site and of the proteins in this complex to each other. However, in many other instances, the function of the membrane envi- ronment in the catalytic process has not been in- vestigated. Recently, we demonstrated that epididymal Cene steroid Sa-reductase is not only dependent upon the membrane environment for the maintenance of struc- tural integrity, but that certain phospholipids are capable of modulating the specific activity [21]. Fur- thermore, treatment of the nuclear and microsomal 581