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Characterisation of 5α-reductase in human benign prostatic hyperplasia (BPH)
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CBAUCTEBISATION OF Sr-REDUCTbsE IN EIJMANBENIGN PROSTATIC EYPERPLASIA (BPR) Husson J.M. and Raynaud J.P. Laboratoire de Canc6rologie Le Goff J.M., Martin P.M., 75007 Paris, FRANCE. Experimentale, 13015 Marseille ; Rousael-Uclaf,
Widely different affinity constant (Km) values (0.1 to 15 p) have been reported in the 11 terature for Sk-reducrase activity. These differences could be due to species, organ or tissue heterogeneity. Nevertheless this variation has been observed for a single tissue : human benign / prostatic hyperplasla (BPH). We decided therefore to determine the optimum conditions for a 5*- i reductase assay with the aim of perfecting a test to monitor enzyme actlvlty in prostate patho- 1 logy. Aliquots from a single microsomal preparatlon from a pool of 10 BPH samples incubated at 37°C were used to study enzyme-concentration and enzyme-affinity. The optimum pH was 5.5 ; at this pH and if the product formed represents less than 10 % of the substrate, the measurement of; of Michaelis-Menten kinetics gives a valid estimation of the enzyme concentrs4 ‘“ma, on the basis 1 depends little on the dissociation rate of the substrate-enzyme complex. On ,tion since V,, j ‘calculating the V and ‘h, of the enzyme for 3 ranges of substrate concentration (0.1, 1 and rhe steady-state, similar V ‘10 fl testosterone Tat / values were obtained but highly slgnifi/ icant differences In Km were recorded (0.48 F to S”$f). An In-depth study of the Influence of 1 ionic strength, pH, buffer, the presence of stabilizers (DTT), protease Inhibitors (PMFS) and ,ion-chelating agents, during tissue pulverization, microsome preparation and/or incubation indi-i /cated that affinity varied primarily according to incubation time. A short lag period was noted / [in the in vitro formation of dihydrocestosterone In these BPH samples suggesting that the con1 formation of the enzyme does not adapt itself Immediately to the imposed in vitro conditions. This could have important implications in the interpretation of the true physiological activity 1 /of the enzyme. _
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183 PRELIMINARY STUDIES ON THE NATURE OF THE AROMATASE ENZYME SYSTEM OF SHEEP PLACENTAL MICROSOMES: France, J.T., Mason, J. I., Magness, R.R., Rosenfeld, C.R. and Cecil H. and Ida Green Center for Reproductive Biology Sciences, Departments Murry B.A. of Obstetrics and Gynecology, Biochemistry, and Pediatics, University of Texas Health Science Center, Dallas, Texas, U.S.A. Apart from the last 3-4 days preceeding parturition, pregnancy in the sheep is associated with relatively low placental production of estrogen compared with human pregnancy. It is uncertain whether this lower production is due to low tissue concentrations of enzymes or limited availability of substrate. In associated with estrogen synthesis, inactive enzymes, our preliminary studies have utilized investigations of seeking to resolve this uncertainly, microsomal aromatase activity in the fetal tissue component of pre-term ovine placentomes. Aromatase activity was determined by an assay based on the measurement of the [ 3H] water The assay reaction was carried by-product when [ 1 -3H landrostenedione is used as substrate. out in the presence of a NADPH generating system at 37OC and pH 7.4. Specific activities, as of fresh microsomal preparations in four animals studies were 8.2 pm01 /min. mg protein, (gestational period: 105 days), 2.9 (125 days), 7.6 (127 days), and 4.0 (130 days). For human term placental microsome preparations had a mean (fSD) aromatase activity comparison, Under the assay conditions, the apparent Km for of 60.1 ? 27.4 pmol/min.mg protein (n=6). Despite the sheep placental aromatase was 45 nM and the Vmax 10.5 pmol/min.mg protein. aromatase is still unlikely to be rate depressed activity compared with human placenta, Our results would be consistent with the view! limiting in ovine placental estrogen synthesis. that the major factor influencing the low production rate is a limited availability of substrate.
184
COMPARATIVEAROMATIZATION OF C-19 and 19-NORANDROGENSBY STALLION TESTIS MICROSOMES ; Gaillard, J-L. ; Dintinger, T.; Al-Timimi, I., Silberzahn, P.; Laboratoire de Biochimie, C.N.R.S. UA 609 UniversitiZ; 14032 Caen, FRANCE.
;
19-norandrogens (NA) are aromatized less efficiently than the correspondant methylandrogens (A) by human placental microsomes. Stallion testes are known to produce high quantities of estrc gens. The present work compares A and NA aromatization by stallion testis microsomes. Aromatase activity was estimated by 3H20 release and by RIA of El and E2. 3 PM androgens were incubated with equine testicular microsomes (1 mg protein) and 0.3 mM NADPH in I ml Tris maleate buffer pH 7.4 for 20 min at 37’ C. Rates of aromatization were 73.4 * 11.3, 100 * 11.5, 42.4 * 4, 47.6 * 6.8 pmol/min/mg for A T, NA and NT. T had an apparent Km of 0.6 uM. Inhibition of 3H 0 release from 1.5 UM (16 4;83H)% by A T,NA andNT in a 1 : 1 molar ratio was 51.7, 38.3, 22,7 and 14.9 X. At 30°‘C, the ar$matiz.%ion rate (in pmol/min/mg) of T and NT together (3 + 3 PM) was 21.5. The aromatization rates of individual su1 were 17.9 and 20.7 for 3 MMT or NT, 19.9 and 25.1 for 6 )IM T or NT. Equine testicular microsomes converted NA with a slightly higher efficiency than A. If there distinct A and NA aromatases, the expected reaction velocity of an equimolar mixture of sul would be the sum (38.6 pmol/min/mg) of velocities of individual substrates. Therefore ou Istrates enzyme system for aromatization of A and NA. A! the apparent affinity for the enzyme system was in this agrees well with the obtained El/E2 ratio equal to 10
67s
CBAUCTEBISATION OF Sr-REDUCTbsE IN EIJMANBENIGN PROSTATIC EYPERPLASIA (BPR) Husson J.M. and Raynaud J.P. Laboratoire de Canc6rologie Le Goff J.M., Martin P.M., 75007 Paris, FRANCE. Experimentale, 13015 Marseille ; Rousael-Uclaf,
Widely different affinity constant (Km) values (0.1 to 15 p) have been reported in the 11 terature for Sk-reducrase activity. These differences could be due to species, organ or tissue heterogeneity. Nevertheless this variation has been observed for a single tissue : human benign / prostatic hyperplasla (BPH). We decided therefore to determine the optimum conditions for a 5*- i reductase assay with the aim of perfecting a test to monitor enzyme actlvlty in prostate patho- 1 logy. Aliquots from a single microsomal preparatlon from a pool of 10 BPH samples incubated at 37°C were used to study enzyme-concentration and enzyme-affinity. The optimum pH was 5.5 ; at this pH and if the product formed represents less than 10 % of the substrate, the measurement of; of Michaelis-Menten kinetics gives a valid estimation of the enzyme concentrs4 ‘“ma, on the basis 1 depends little on the dissociation rate of the substrate-enzyme complex. On ,tion since V,, j ‘calculating the V and ‘h, of the enzyme for 3 ranges of substrate concentration (0.1, 1 and rhe steady-state, similar V ‘10 fl testosterone Tat / values were obtained but highly slgnifi/ icant differences In Km were recorded (0.48 F to S”$f). An In-depth study of the Influence of 1 ionic strength, pH, buffer, the presence of stabilizers (DTT), protease Inhibitors (PMFS) and ,ion-chelating agents, during tissue pulverization, microsome preparation and/or incubation indi-i /cated that affinity varied primarily according to incubation time. A short lag period was noted / [in the in vitro formation of dihydrocestosterone In these BPH samples suggesting that the con1 formation of the enzyme does not adapt itself Immediately to the imposed in vitro conditions. This could have important implications in the interpretation of the true physiological activity 1 /of the enzyme. _
_
183 PRELIMINARY STUDIES ON THE NATURE OF THE AROMATASE ENZYME SYSTEM OF SHEEP PLACENTAL MICROSOMES: France, J.T., Mason, J. I., Magness, R.R., Rosenfeld, C.R. and Cecil H. and Ida Green Center for Reproductive Biology Sciences, Departments Murry B.A. of Obstetrics and Gynecology, Biochemistry, and Pediatics, University of Texas Health Science Center, Dallas, Texas, U.S.A. Apart from the last 3-4 days preceeding parturition, pregnancy in the sheep is associated with relatively low placental production of estrogen compared with human pregnancy. It is uncertain whether this lower production is due to low tissue concentrations of enzymes or limited availability of substrate. In associated with estrogen synthesis, inactive enzymes, our preliminary studies have utilized investigations of seeking to resolve this uncertainly, microsomal aromatase activity in the fetal tissue component of pre-term ovine placentomes. Aromatase activity was determined by an assay based on the measurement of the [ 3H] water The assay reaction was carried by-product when [ 1 -3H landrostenedione is used as substrate. out in the presence of a NADPH generating system at 37OC and pH 7.4. Specific activities, as of fresh microsomal preparations in four animals studies were 8.2 pm01 /min. mg protein, (gestational period: 105 days), 2.9 (125 days), 7.6 (127 days), and 4.0 (130 days). For human term placental microsome preparations had a mean (fSD) aromatase activity comparison, Under the assay conditions, the apparent Km for of 60.1 ? 27.4 pmol/min.mg protein (n=6). Despite the sheep placental aromatase was 45 nM and the Vmax 10.5 pmol/min.mg protein. aromatase is still unlikely to be rate depressed activity compared with human placenta, Our results would be consistent with the view! limiting in ovine placental estrogen synthesis. that the major factor influencing the low production rate is a limited availability of substrate.
184
COMPARATIVEAROMATIZATION OF C-19 and 19-NORANDROGENSBY STALLION TESTIS MICROSOMES ; Gaillard, J-L. ; Dintinger, T.; Al-Timimi, I., Silberzahn, P.; Laboratoire de Biochimie, C.N.R.S. UA 609 UniversitiZ; 14032 Caen, FRANCE.
;
19-norandrogens (NA) are aromatized less efficiently than the correspondant methylandrogens (A) by human placental microsomes. Stallion testes are known to produce high quantities of estrc gens. The present work compares A and NA aromatization by stallion testis microsomes. Aromatase activity was estimated by 3H20 release and by RIA of El and E2. 3 PM androgens were incubated with equine testicular microsomes (1 mg protein) and 0.3 mM NADPH in I ml Tris maleate buffer pH 7.4 for 20 min at 37’ C. Rates of aromatization were 73.4 * 11.3, 100 * 11.5, 42.4 * 4, 47.6 * 6.8 pmol/min/mg for A T, NA and NT. T had an apparent Km of 0.6 uM. Inhibition of 3H 0 release from 1.5 UM (16 4;83H)% by A T,NA andNT in a 1 : 1 molar ratio was 51.7, 38.3, 22,7 and 14.9 X. At 30°‘C, the ar$matiz.%ion rate (in pmol/min/mg) of T and NT together (3 + 3 PM) was 21.5. The aromatization rates of individual su1 were 17.9 and 20.7 for 3 MMT or NT, 19.9 and 25.1 for 6 )IM T or NT. Equine testicular microsomes converted NA with a slightly higher efficiency than A. If there distinct A and NA aromatases, the expected reaction velocity of an equimolar mixture of sul would be the sum (38.6 pmol/min/mg) of velocities of individual substrates. Therefore ou Istrates enzyme system for aromatization of A and NA. A! the apparent affinity for the enzyme system was in this agrees well with the obtained El/E2 ratio equal to 10
67s