Modulation of epididymal delta 4-steroid 5 alpha-reductase activity in vitro by the phospholipid environment

Epididymal 5 alpha-reductase converts testosterone to 5 alpha-dihydrotestosterone. The enzyme is localized to the nuclear and microsomal membranes, and using two approaches, we investigated the relationship between 5 alpha-reductase activity and the membrane environment. In the first, nuclear and microsomal membrane fractions were treated with phospholipases to modify specifically the structure of the phospholipid component of the membranes, and the effects of these treatments on the kinetic parameters of 5 alpha-reductase were examined. The second approach was to observe the effects of phospholipids of known structure on solubilized 5 alpha-reductase activity. Treatment of the membrane fractions with phospholipase C increased the Km(app) of both the nuclear and microsomal 5 alpha-reductases for testosterone. Phospholipase A2 treatment also increased the Km(app) of the microsomal enzyme, but in contrast, the Km(app) of the nuclear 5 alpha-reductase for testosterone was unaffected. This demonstrated a fundamental difference in the role of the membrane environment in the expression of 5 alpha-reductase activity in these subcellular compartments. The ability of phospholipids to enhance the activity of solubilized 5 alpha-reductase was highly specific and structure related. Only phosphatidylcholines containing either unsaturated acyl chains or saturated acyl chains of 12 carbon atoms were found to activate 5 alpha-reductase. The most potent activator was dilauroyl phosphatidylcholine, which reduced the Km(app) values of both nuclear and microsomal 5 alpha-reductases for testosterone, without affecting the concentration of active 5 alpha-reductase (Vmax(app) ). This is the first time that an activator of 5 alpha-reductase has been found. These findings suggest that epididymal 5 alpha-reductase activity may be regulated by changes in the phospholipid environment.     

Properties of 4-ene-5 alpha-reductase and studies on its solubilization from porcine testicular microsomes

The activity of 4-ene-5 alpha-reductase was assayed in porcine testis homogenates and subcellular fractions, using testosterone as substrate. 'Marker' enzyme activities were utilized to indicate the purity of the subcellular fractions. 4-Ene-5 alpha-reductase activity was associated with the microsomal fraction; there was no activity in the purified nuclear fraction. Enzyme activity was higher in the testes of 6 week old pigs than those of 3 and 17 week old animals, and a range of activity was found. The enzyme was unstable when stored at -20 degrees C but the addition of albumin (0.1%, w/v) or glycerol (20%, v/v) to the buffer and storage at -70 degrees C or in liquid nitrogen ensured that maximal activity was retained for at least 35 days. In addition to 5 alpha-DHT, other 5 alpha-reduced metabolites and 4-androstenedione were formed in this reaction; NADPH was the preferred cofactor, but 40% of the 4-ene-5 alpha-reductase activity was retained when NADH was used. Solubilization of the microsomal enzyme was achieved using sodium citrate (0.1 M); 4-ene-5 alpha-reductase activity was enhanced to greater than 120% and 60% of this activity was in the soluble fraction. The optimum pH and temperature for both soluble and membrane-bound 4-ene-5 alpha-reductase were 6.9 and 32 degrees C, respectively. The mean apparent Km and Vmax were 0.6 mumol/l and 158 pmol/min/mg microsomal protein for the microsomal enzyme and 1.42 mumol/l and 212.0 pmol/min/mg soluble protein for the solubilized 4-ene-5 alpha-reductase. The estimated sedimentation coefficient was 11.6.     

Subcellular distribution of steroid delta 4-5 alpha-reductase and 3 alpha-hydroxysteroid dehydrogenase in the rat epididymis during sexual maturation

The curve of the specific activity of rat epididymal nuclear delta 4-5 alpha-reductase is bell shaped as a function of age, whereas that of cytoplasmic 3 alpha-hydroxysteroid dehydrogenase does not change significantly with age. The present study examines the subcellular distribution of delta 4-5 alpha-reductase and 3 alpha-hydroxysteroid dehydrogenase in the caput-corpus and cauda epididymidis during development. A 5-step discontinuous sucrose gradient was developed for fractionation of epididymal homogenates. By using enzyme markers specific for different subcellular organelles, the five different subcellular fractions obtained were shown to be of cytoplasmic, microsomal, mitochondrial, nuclear and spermatozoal origin. 3 alpha-Hydroxysteroid dehydrogenase activity was associated only with the cytoplasmic fraction. The activity of the enzyme did not change significantly with age in either the caput-corpus or cauda epididymidis. delta 4-5 alpha-Reductase activity was found in fractions containing microsomal and nuclear markers. delta 4-5 alpha-Reductase activity in the nuclear fraction of the caput-corpus epididymidis was evident in the youngest age group (Day 25), increased 4-fold and peaked in the next age group (Day 35), and declined with each successive age group: Day 45 (60% of maximum), Day 60 (20% of maximum), Day 75 (15% of maximum) and Day 105 (10% of maximum). In contrast, microsomal delta 4-5 alpha-reductase activity increased successively from Day 25 to Day 105; enzyme activity doubled between these two ages. The ratio of nuclear to microsomal delta 4-5 alpha-reductase activity from the caput-corpus epididymidis thus changed markedly with age: Day 25:1.32; Day 35:3.76; Day 45:2.44; Day 60:1.03; Day 75:0.41; and Day 105:0.21. In the cauda epididymidis nuclear delta 4-5 alpha-reductase activity was only evident at Day 35 and Day 45; in microsomal fractions, activity was first found at Day 35 and did not subsequently change with age. These results demonstrate that: 1) epididymal 3 alpha-hydroxysteroid dehydrogenase activity is found only in the cytoplasmic fraction; 2) delta 4-5 alpha-reductase activity is found in nuclear and microsomal fractions; and 3) the subcellular distribution of delta 4-5 alpha-reductase activity changes markedly with age and epididymal section, suggesting differential regulation of nuclear and microsomal delta 4-5 alpha-reductase activities.     

Properties and subcellular distribution of delta4-steroid (progesterone) 5alpha-reductase in rat anterior pituitary.

The properties and subcellular distribution of anterior pituitary delta4-steroid (progesterone) 5alpha-reductase, which stimulates the conversion of progesterone to 5alpha-pregnane-3,20-dione, have been investigated utilizing 3H-substrate and a reverse isotopic dilution assay system. The enzymic activity was stimulated by NADPH but not NADH and exhibited a Km of 2.7+/-0.9 times 10(-7) M for progesterone. The substrate specificity of the enzyme for other delta4-3-ketosteroids and the effect of estradiol-17beta were also studied. 20alpha-hydroxy-4-pregnen-3-one was more reactive than progesterone, while testosterone was less reactive. Estradiol-17beta in vitro had an inhibitory effect on the 5alpha-reduction of progesterone. Studies on the subcellular distribution of the 5alpha-reductase activity indicate that the bulk of the activity was widely distributed amongst particulates sedimenting at 1,000, 15,000 and 100,000xg; with the 15,000xg pellet containing the most enzymic activity. The 100,000xg supernatant possessed only a small fraction of the total activity. After further fractionation of the 1,000xg pellet, the activity was distributed equally between the purified nuclear and cell debris-membranes fractions.     

Solubilization of nuclear steroid 5 alpha-reductase from rat ventral prostate

delta 4-Steroid-5 alpha-reductase (3-oxo-5 alpha-steroid:NADP+ delta 4-oxidoreductase, EC 1.3.1.22), is a membrane-bound enzyme. In the ventral prostate of the rat, its activity is found within the nuclear envelope. Solubilization of this enzyme can only be achieved in the presence of detergents. We studied the inhibitory effect of various detergents on 5 alpha-reductase activity as a function of detergent concentration, of pH, of incubation time, of salt concentration and of additives to the buffer system. Four detergents (Lubrol WX, CHAPS, L-alpha-lysophosphatidylcholine and octyl D-glucopyranoside) were selected for subsequent solubilization studies. The overall recovery of solubilized enzyme activity was about 30% when compared to 100% of 5 alpha-reductase activity found in freshly prepared nuclei. Up to 20-30% of the nuclear proteins were extracted during the solubilization procedure. Among the various treatments tested, a concentration of 3 mg/ml L-alpha-lysophosphatidylcholine per 10 mg/ml of nuclear protein in the presence of 5 mM MgCl2, 0.1 M KCl, 0.1 M sodium citrate and 5 mM NADPH yielded the maximal enzymic activity of 56%, 15% of the nuclear proteins being solubilized in an active and stable form. The activity in these extracts could be kept stable for 2 days at 4 degrees C with a recovery of 75% of enzymic activity. A 3-fold increase of specific 5 alpha-reductase activity was obtained during solubilization under optimal conditions.     

Pituitary progesterone 5 alpha-reductase: solubilization and partial characterization

The microsomal progesterone 5 alpha-reductase activity from female rat anterior pituitary has been solubilized and partially characterized with regard to some of its kinetic and physical properties. The solubilization of progesterone 5 alpha-reductase has been achieved through the use of either an n-octyl glucoside (OG)-KCl- or a digitonin-KCl-extraction. The total yield and specific activity of solubilized enzyme activity is greater using the OG-KCl method. Kinetic analyses of microsomal and OG-KCl-solubilized progesterone 5 alpha-reductase have indicated that both of these preparations exhibit a similar apparent Km for progesterone (microsomal Km = 117 +/- 12 nM; solubilized Km = 123 +/- 11 nM), suggesting that the solubilization procedure does not appreciably alter the kinetic behavior of this enzyme activity. The OG-KCl-extracted progesterone 5 alpha-reductase activity also appears quite stable, since essentially no enzyme activity is lost following dialysis at 4 degrees C for 22 h. In addition, the activity of the solubilized-dialyzed enzyme preparation can be slightly stimulated via the addition of phospholipids. Studies on the properties of the microsomal enzyme activity have indicated that this preparation is unaffected by metal chelators (EDTA or EGTA) but can be completely inhibited by the powerful sulfhydryl blocking agent p-chloromercuribenzoic acid. An evaluation of the possible role of flavins (as a hydride carrier between NADPH and the steroid) has shown that progesterone 5 alpha-reduction is inhibited by high levels of flavins and flavin analogs.     

Functional characteristics of nuclear 5 alpha-reductase from rat ventral prostate

The subcellular distribution and functional characteristics of 5 alpha-reductase (3-oxo-5 alpha-steroid: NADP+ 4-ene-oxidoreductase, EC 1.3.1.22) from rat ventral prostate were studied and compared to the 5 alpha-reductase from female rat liver. Tissue fractionation retained main enzymic activity in the microsomal fraction of rat liver, while 5 alpha-reductase from rat prostate was localized in the nuclear membrane with a specific activity 160 times that of the initial homogenate. The purity of nuclear envelopes was checked by electron microscopy. Solubilization experiments indicated that the hepatic 5 alpha-reductase is attached to the endoplasmic reticulum as a peripheral protein, while the nuclear prostatic enzyme is an integral membrane protein. Incubation experiments with phospholipases suggested a decisive role of the surrounding phospholipids for the prostatic enzyme activity. To elucidate the characteristics of hydrogen transfer of the enzyme, the effect of flavins and different other cofactors on 5 alpha-reductase activity in isolated prostatic nuclei were studied. Our findings indicate that in rat ventral prostate the conversion of testosterone to 5 alpha-dihydrotestosterone proceeds by a direct hydrogen transfer from NADPH to testosterone. Concerning these parameters the behaviour of hepatic 5 alpha-reductase is absolutely different from the prostatic enzyme. The localization of 5 alpha-reductase within the nuclear envelope of rat ventral prostate as an integral membrane protein seems to be of physiological significance with regard to the action of androgens.     

Solubilization of human prostatic 5 alpha-reductase

A sensitive assay for 5 alpha-reductase was introduced which is capable of detecting at least 0.2 U of activity per sample. The assay was used in developing a method for the solubilization of human prostatic 5 alpha-reductase. Homogenisation conditions were devised under which 95% of the total prostatic 5 alpha-reductase was released into the microsomal fraction. A combination of 0.1 M sodium citrate, 0.1 M KCl, 20% (v/v) glycerol, 0.5 mM NADPH and 1 microM testosterone was found to stabilise 5 alpha-reductase in the presence of detergents. The effect of the presence of low concentrations of detergents in the assay on the activity of 5 alpha-reductase was studied. Triton X-100, Lubrol PX and Nonidet P-40, caused a concentration-dependent inhibition of activity. The ability of several detergents (Triton X-100 MEGA-9, Tween 20, Tween 80, digitonin, Lubrol PX and Nonidet P-40) to solubilise 5 alpha-reductase was studied. All detergents caused a concentration-dependent solubilization of 5 alpha-reductase. Significant amounts of active solubilized enzyme were recovered only with Lubrol PX at concentrations less than 1.1 mg/ml. Seventy percent of the 5 alpha-reductase was solubilized in an active form by extracting the membranes 3 times with 0.8 mg/ml Lubrol PX.     

Photoaffinity labelling of nuclear steroid 5 alpha-reductase of rat ventral prostate

In order to get more information on the molecular structure of the rat prostatic 5 alpha-reductase (3-oxo-5 alpha-steroid: NADP+ 4-ene-oxidoreductase, EC 1.3:1.22) a systematic photoaffinity labelling study has been performed. To irreversibly freeze the status quo of interaction, either testosterone, the physiological ligand, or diazo-MAPD (21-diazo-4-methyl-4-aza-5 alpha-pregnane-3,20-dione), a specific 5 alpha-reductase inhibitor, was irradiated with isolated nuclei or with purified nuclear membranes or with solubilized nuclear membrane proteins and checked for optimal labelling conditions. The principal substances covalently labelled were phospholipids and at a minor ratio proteins. Analysis by SDS-PAGE and autoradiofluorography revealed two labelled polypeptides with molecular weights of 20 kDa and 26 kDa. The following evidence indicates that these polypeptides might be derived from the enzyme 5 alpha-reductase: both proteins are labelled only when specific ligands for 5 alpha-reductase are used; binding can be reduced by the addition of an excess of unlabelled ligand; enzyme activity is irreversibly suppressed when irradiated in the presence of these ligands; only subcellular fractions containing 5 alpha-reductase reveal the labelled proteins; in all 5 alpha-reductase containing preparations with increasing specific activity, independent of the polypeptide pattern, the same proteins are labelled.     

Influence of rete testis fluid deprivation on the kinetic parameters of goat epididymal 5 alpha-reductase

A surgical technique to cannulate the rete testis of the goat was utilized to examine the effects of rete testis fluid (RTF) deprivation on the enzymatic activity of epididymal 5 alpha-reductase. Kinetic techniques were used to determine whether the regional enzymatic effect of RTF deprivation is to decrease the apparent number of 5 alpha-reductase active sites or the catalytic activity of each active site within the epididymal epithelium. Paired comparisons of (Vmax)app and (Km)app values between control and RTF-deprived epididymides indicated that RTF deprivation affected the value of (Vmax)app with no apparent change in the values of (Km)app in caput, corpus, and cauda epididymal regions. We conclude that RTF deprivation in the goat epididymis for 7 days results in a decreased number of apparent 5 alpha-reductase active sites within the epididymal epithelium.     

Differential effects of combinations of phospholipase A2 and phospholipase C on the activity of rat epididymal nuclear and microsomal 4-ene steroid 5 alpha-reductase

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.     

Effects of unilateral orchidectomy on rat epididymal delta 4-5 alpha-reductase and 3 alpha-hydroxysteroid dehydrogenase

The effects of unilateral orchidectomy on the adult rat epidiymal testosterone metabolizing enzymes, delta 4-5 alpha-reductase and 3 alpha-hydroxysteroid dehydrogenase, are investigated. Five weeks following unilateral orchidectomy, it is found that the activity of 3 alpha-hydroxysteroid dehydrogenase per organ is not altered, whereas delta 4-5 alpha-reductase activity decreased by more than 80% on the side of the orchidectomy. Neither accessory sex tissue weights, ventral prostate and seminal vesicles, nor the concentration of circulating testosterone, luteinizing hormone, follicle-stimulating hormone, or prolactin is altered by unilateral orchidectomy. These data indicate that (1) epididymal 3 alpha-hydroxysteroid dehydrogenase activity can be maintained by circulating androgens and that (2) the major factor regulating delta 4-5 alpha-reductase activity is not a substance secreted by the testes into the peripheral circulation. It is suggested that a substance directly secreted into the epididymis by the testis regulates epididymal delta 4-5 alpha-reductase activity.     

Properties and biochemical characterization of NADH 5 alpha-reductase from rat liver microsomes

NADH 5 alpha-reductase is present in microsomes of various rat organs: heart and skeletal muscle, liver, adrenal glands, kidney, testes and prostate. The enzyme from rat liver microsomes utilizes B-hydrogen from the coenzyme NADH for steroid reduction. After solubilization of the enzyme with the nonionic detergent lubrol, phosphatidylcholine is necessary to restore the activity. This reactivation of the enzyme activity is paralleled by a corresponding increase of Vmax for testosterone (17 beta-hydroxy-4-androsten-3-one). Km and Vmax for testosterone change, Km and Vmax for the coenzyme NADH remain constant with an alteration of phosphate concentration in the incubation medium. The NADH 5 alpha-reductase is inhibited by numerous substances: amytal, phenobarbital, mepacrin, thenoyltrifluoracetone, gallic acid propyl ester, dicoumarol, pentachlorophenol, NADP and antibodies against rat liver NADPH ferrihemoprotein reductase. Antibodies against rat liver cytochrome-b5 reductase cause an activation of NADH 5 alpha-reductase.     

Hormonal regulation of rat liver microsomal enzymes. Role of gonadal steroids in programming, maintenance, and suppression of delta 4-steroid 5 alpha-reductase, flavin-containing monooxygenase, and sex-specific cytochromes P-450

Neonatal gonadectomy studies and hormonal replacement regimens were employed to characterize the regulation of delta 4-steroid 5 alpha-reductase, microsomal flavin-containing monooxygenase, and several forms of rat hepatic microsomal cytochrome P-450, including three that are sexually differentiated. Rats of both sexes that had been gonadectomized at birth were either untreated or were administered testosterone propionate or estradiol benzoate neonatally (subcutaneous injection on days 1 and 3 of life), postpubertally (an implant of a hormone-packed capsule at 5 weeks of age), or both neonatally and postpubertally. At the age of 10 weeks, all rats were killed, and several liver microsomal enzymes were assayed using immunochemical and catalytic techniques. Expression in the 10-week-old male and female rats of two male-specific cytochrome P-450 forms, termed P-4502c/UT-A and P-4502a/PCN-E, and their associated respective 16 alpha- and 6 beta-steroid hydroxylase activities could either be imprinted (programmed) by androgen exposure during the early neonatal period or, alternatively, could be stimulated by continuous hormone treatment after the age of 5 weeks. By contrast, hepatic expression of two female-specific enzymes, P-4502d/UT-1 and delta 4-steroid 5 alpha-reductase, was only partially dependent on estradiol; birth-gonadectomized rats expressed as much as 30-50% of the enzyme levels present in untreated adult females. Expression of both female-specific enzymes was fully suppressed upon postpubertal exposure to testosterone. In another study, birth sham-operated female rats were administered testosterone using the same regimens described above for the birth-gonadectomized rats. Although neonatal testosterone treatment alone did not affect the expression in these females of the four sex-specific enzymes examined in this study, it did enhance significantly the masculinization effected by postpubertal androgen exposure. This resulted in expression of the male-specific enzymes P-4502c/UT-A and P-4502a/PCN-E in these females at levels comparable to those found in adult males, while simultaneously suppressing the two female-specific enzymes, P-4502d/UT-I and delta 4-steroid 5 alpha-reductase, by approximately 70-75% to levels characteristic of prepubertal rats of either sex. The levels of another microsomal enzyme, flavin-containing monooxygenase, were also measured and found to be regulated by testosterone, but the ontogenic profiles and the effects of gonadectomy and hormone replacement indicated clear differences in its regulation when compared to the other male-specific enzymes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Sexual difference in properties of 5 alpha-reductase in microsome of rat submandibular glands

The properties of 5 alpha-reductase activity in the submandibular glands of rats were investigated using microsomal fraction in the presence of NADPH, and we found a sexual difference in these properties. The formation of 5 alpha-dihydrotestosterone and 5 alpha-androstane-3 alpha, 17 beta-diol from testosterone demonstrated 5 alpha-reductase in the rat microsomal fraction of this tissue sample. Apparent michaelis constants (Km) of the 5 alpha-reductase activity for testosterone was 1.02 x 10(-6) M for the female tissue. Microsomal fraction of submandibular glands of male rats had two Kms and were determined as 1.12 x 10(-6) M and 1.01 x 10(-5) M. The lower Km of 5 alpha-reductase for male rats was similar to for the females.     

Mechanistic studies with solubilized rat liver steroid 5 alpha-reductase: elucidation of the kinetic mechanism

A solubilized preparation of steroid 5 alpha-reductase (EC 1.3.1.30) from rat liver has been used in studies focused toward an understanding of the kinetic mechanism associated with enzyme catalysis. From the results of analyses with product and dead-end inhibitors, a preferentially ordered binding of substrates and release of products from the surface of the enzyme is proposed. The observations from these experiments were identical with those using the steroid 5 alpha-reductase activity associated with rat liver microsomes. The primary isotope effects on steady-state kinetic parameters when [4S-2H]NADPH was used also were consistent with an ordered kinetic mechanism. Normal isotope effects were observed for all three kinetic parameters (Vm/Km for both testosterone and NADPH and Vm) at all substrate concentrations used experimentally. Upon extrapolation to infinite concentration of testosterone, the isotope effect on Vm/Km for NADPH approached unity, indicating that the nicotinamide dinucleotide phosphate is the first substrate binding to and the second product released from the enzyme. The isotope effects on Vm/Km for testosterone at infinite concentration of cofactor and on Vm were 3.8 +/- 0.5 and 3.3 +/- 0.4, respectively. Data from the pH profiles of these three steady-state parameters and the inhibition constants (1/Ki) of competitive inhibitors versus both substrates indicate that the binding of nicotinamide dinucleotide phosphate involves coordination of its anionic 2'-phosphate to a protonated enzyme-associated base with an apparent pK near 8.0. From these results, relative limits have been placed on several of the internal rate constants used to describe the ordered mechanism of the rat liver steroid 5 alpha-reductase.     

The mechanism of rat epididymal 4-ene steroid 5 alpha-reductase

Epididymal nuclear 4-ene steroid 5 alpha-reductase catalyses the bisubstrate reaction between testosterone and NADPH to produce 5 alpha-dihydrotestosterone (DHT) and NADP+. Previous studies from this laboratory have demonstrated that the 4-ene steroid 5 alpha-reductase reaction proceeds through the direct transfer of protons from NADPH to testosterone, and that while the product DHT does not affect 4-ene steroid 5 alpha-reductase activity, NADP+ is a potent inhibitor of this enzyme. In the present studies we have investigated the mechanism of 4-ene steroid 5 alpha-reductase with respect to the binding of the substrates, testosterone and NADPH. Kinetic analyses revealed that testosterone does not alter the Kmapp for NADPH, and that NADPH does not alter the Kmapp for testosterone. These findings excluded the possibility that the mechanism of 4-ene steroid 5 alpha-reductase is of the ping-pong variety, and that the sequential addition of both substrates is required before any products are released. The lack of change in Kmapp, observed for either substrate, further suggests that both testosterone and NADPH are able to bind to the free enzyme, negating the possibility that substrate addition occurs in an ordered manner. Indeed the kinetic profiles are entirely consistent with the mechanism of 4-ene steroid 5 alpha-reductase being a rapid equilibrium random sequential process in which the binding of the first substrate has no affect on the binding of the second. Mean values for the dissociation constants, Ktestosterone and KNADPH, were 200 nmol/l and 50 nmol/l, respectively. These findings, coupled with those from earlier studies, suggest that the mechanism of epididymal nuclear 4-ene steroid 5 alpha-reductase is a rapid equilibrium random bireactant process, with the possible dead-end complex: testosterone-4-ene steroid 5 alpha-reductase-NADP+.     

Testosterone 5 alpha-reductase in rat brain

We describe a simple procedure for the microassay of testosterone 5 alpha-reductase in homogenates of rat brain. This enzyme converts testosterone to dihydrotestosterone. We have used this assay to characterize the enzymatic activity and to map its distribution. The apparent Km is 4.1 x 10(-6) M and the Vmax is 85.6 pmol/mg protein/h. The pH optimum is broad and extends from pH 6.0 to 8.0. For the brain regions surveyed, testosterone 5 alpha-reductase activity varied over a 10-fold range. The highest activities were observed in homogenates of the midbrain and pons (37-39 pmol/mg protein/h). The lowest were seen in homogenates of the thalamus, caudate nucleus, frontal cortex, hippocampus, hypothalamus, olfactory tubercle, and preoptic area (3-7 pmol/mg protein/h).     

Relationship of changing delta 4-steroid 5 alpha-reductase activity to [125I]iododeoxyuridine uptake during regeneration of involuted rat prostates

To elucidate the phenotypic expression of proliferating prostatic cells, rats were castrated, and the regenerating process of involuted ventral prostates during testosterone propionate (TP) administration was investigated by examining morphology, [5-125I]iododeoxyuridine (125I-UdR) uptake, DNA content, weight, acid phosphatase, and delta 4-steroid 5 alpha-reductase (5 alpha-reductase) activities. Morphologically, TP treatment initially increased the number of epithelial cells lining glandular lobules and subsequently restored the shape of epithelial cells. 125I-UdR uptake peaked on Day 3 of TP treatment and stayed at higher levels than for uncastrated controls until Day 14 of treatment. Prostatic weight, protein content, acid phosphatase, and DNA content returned to uncastrated control levels by Day 14 of TP treatment. TP administration markedly stimulated prostatic 5 alpha-reductase activity, which peaked on the Day 5 of treatment and decreased to uncastrated control levels by Day 14 of treatment. It is concluded that TP administration to castrated rats initially induced active mitotic division of the remaining stem cells, followed by formation of differentiated functional epithelial cells. Prostatic 5 alpha-reductase was highly active at the initial phase of active mitotic cell division. The major portion of the increased enzyme activity can be regarded as a phenotypic expression of stem or transient cells of prostatic epithelium.     

Studies on the mechanism of the antiandrogenic effect of a putative 5 alpha-reductase inhibitor

The mechanism of the antiandrogenic effect of 5,10-seco-19-norpregnane-4,5-diene-3,10,20-trione (secosteroid), reputedly an irreversible inhibitor of 5 alpha-reductase, was investigated. Its addition (10 microM) to culture media effectively suppressed the synthesis of rat epididymal proteins specifically induced by 0.1 microM testosterone (T) or dihydrotestosterone (DHT). Under the same conditions, secosteroid did not change the rate at which labeled T was metabolized to 5 alpha-reduced compounds. In a comparative study, secosteroid inhibited 5 alpha-reductase in an isolated microsomal fraction while not affecting the enzyme activity in minced tissue. Secosteroid was shown to be a competitor of the binding of [3H]T and [3H]DHT (both at 4 nM) to the epididymal cytosol androgen receptor, with ID50 of 1 microM for the former and 4 microM for the latter, thus explaining the mechanism involved in its antiandrogenic properties.