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Effect of inhibitors on testicular microsomal steroid 17α-hydroxylase and 17α-hydroxypregnene C17–C20 lyase
BIOCHIMICA ET BIOPHYSICA ACTA BBA 6526I EFFECT OF INHIBITORS ON TESTICULAR MICROSOMAL STEROID
IJa-HYDROXYLASE AND 17a-HYDROXYPREGNENE C17-C20 LYASE MIKIO SHIKITA, TOMOKO OGISO
AND
BUN-IeBI TAMAOKI
Nation at Institute oj Radiological Sciences, Chiba-shi (Japan) (Received March 23rd, 1965)
SUMMARY 1. 3-(6-Chloro-3-methyl-2-indenyl)pyridine (SU 8000) and 3-(I,2,3.4-tetrahydro-4-oxo-J-chloro-2-naphthyl)pyridine (SU 10603) competitively inhibited IJahydroxylation of [4-14C]progesterone by rat-testicular microsomes. 2. The compounds also strongly but non-competitively inhibited the cleavage of the side chain of [4-14C]IJa-hydroxyprogesterone by the microsomes, 3. The apparent inhibitor constants of SU 8000 were determined as 1.35 pM and 1.00 pM against the IJa-hydroxylase and the IJa-hydroxypregnene C17-C20 lyase respectively. Corresponding K 1 values for SU 10603 were 0.86 pM and 0.54 pM. 4- IJf.J-Hydroxysteroid dehydrogenase and zcc-hydroxysteroid dehydrogenase of the rat testes were not influenced by the compounds.
INTRODUCTION
1Ja-Hydroxylation of adrenal corticosteroids is reported to be specifically inhibited by several chemical compounds-A, SHARMA et at. 3 , however, reported that 3(6-chloro-3-methyl-z-indenyl)pyridine (SU 8000), one of the inhibitors of the adrenocorticoid 17a-hydroxylase, also inhibited up-hydroxylation of II-deoxycorticosterone by adrenal enzyme preparation. HALL et ai», furthermore, reported that the compound also inhibited 17a-hydroxylation of progesterone by homogenates of rabbit testes. Moreover, they found with canine testes that not only 1Ja-hydroxylation of progesterone but also the cleavage of the side chain of IJa-hydroxyprogesterone was inhibited by the compound. The steroid hydroxylases and the IJ,20-desmolase (IJahydroxypregnene C17-C20 lyase) resemble each other, requiring NADPH and molecular oxygen for their action's". These reports led us to study further detailed kinetics of the influence of the inhibitors on the 1Ja-hydroxylase in comparison with that on the 17,20-desmolase. Both enzymes were reported to be contained in the microsomes obtained from rat testes", MATERIALS AND METHODS
Substrate steroids [4-14(lProgesterone (83 pC/mg), [4-l 4CJandrosteneclione ([4-14C]androst-4-eneBioohim. Biophys. Acta, I05 (1965) 5I6-522
INHIBITION OF TESTICULAR MICROSO:MAL
ENZY~IES
3,17-dione) (71 ftC/mg) and [4-14C]I7a-hydroxyprogesterone (44 ftCfmg) were purchased from New England Nuclear Corp. (Boston, Mass.), [r,z-3H 2]Progest erone (2 mC/mg) was supplied by Endocrinology Study Section, National Institutes of Health. Prior to use, their purity was checked by thin-layer chromatography. Tissue preparation
Testes of rats of the Wistar strain, 2 to 3 months old, were isolated, homogenized and fractionated by the differential centrifugation method as described in the previous report". The microsomal fraction was suspended in 0.25 M sucrose solution, and the suspension approximately equivalent to one testis (I.2 g) was added to each incubation flask. The 105 000 X g supernatant was transparent and equivalent two testes per flask.
to
I ncubation
Radioactive steroid precursors with the respective carrier steroid, the inhibitors and a drop per flask of propylene glycol were dissolved in dichloromethane and added to the incubation flask (50 ml centrifuge tube with round bottom). Dichloromethane was gently removed under reduced pressure. Shortly before incubation, 3 ml of 0.25 M sucrose solution containing 0.5 {lmoles of N"ADPH and SOD flmoles of Tris (pH 7-4) and then 3 ml of the testicular preparation were added to the flask. The mixtures were incubated for 30 min at 37° with continuous shaking under the atomosphere of 95 % O2 and 5 % CO2 , An incubation time of 30 min was chosen, as time studies ensured a linear rate of the enzymic reactions at least for 45 min. Extraction, separation, identification and quaniitation oj the products
The reaction was stopped by rapid extraction of the steroids from the incubation mixture- with dichloromethane. The products were separated and purified by silica gel G thin-layer chromatography with a solvent system of benzene-acetone (8:2, v/v) , and quantitatively determined by a liquid scintillation spectrometers. The efficiency of counting was 32% for tritium and 57% for [14CJcarbon. Total recovery of the radioactivity was between 85 and 95 %. The products were pooled and identified as described in detail in our previous papers. RESULTS
Specificity of the effect oj the inhibitors
As shown in Table I 17a-hydroxylase and 17,20-desmolase activities of rat testicular microsomes were both inhibited markedly by SU 8000 as well as by SU 10603. This was confirmed by another series of experiments in which case [1,2-SH2~ progesterone (35' r0 8 counts/min; 5.0 p,g) and [4-14C]I7a-hydroxyprogesterone (I4' lOS counts/min; 5.0 fig) were incubated together in the same flask. Without inhibitors, 55·3% of [I,2- 8H 2]progesterone was hydroxylated and 20.0% of [4-14C]17a-hydroxyprogesterone cleaved to the C19 steroids, while II.8% and 4.9% in the presence of SU 8000 [I flg/ml) and 8.5 % and 2.5% in the presence of SU 10603 (I fig/m]), respectively. These results show that the inhibition by the compounds is not specific on the 17a-hydroxylase. Besides the 17a-hydroxylase and the 17.20-desmolase, the rat-testicular microBiochim. Biophys, Acta, r05 (r965) 5 y6-5 2 2
M. SHIKITA,
518
T.
OGISO,
B-I.
TAMAOKI
TABLE I EFFECT OF SU 8000 AND SU I0603 ON" I7a-HYDROXYLASE, I7,20-DESMOLASE, I7fJ- AND 20a-HYDROXYSTEROID DEHYDROGEN"ASES OF RAT TESTES The enzyme activity is expressed in flg of the products obtained by the 30-min incubation: I7a-hydroxylase, sum of 17a-hydroxyprogesterone, androstenedione and testosterone produced from [4-l.4C]pro gesterons (62 . 103 counts/min; 6.2 fig) by the testicular microsomes; 7,zo-desmolase, sum of androstenedione and testosterone produced from r4-14C)I7a-hydroxyprogesterone (68 '10 8 counts/min; 5.0 p,g) by the microsomes ; 17fJ-hydroxysteroid dehydrogenase, testosterone produced from [:f- 14C]a n drost en ed ione (35' 10' counts/min; 5.0 flg) by the microsomes; zorz-hydroxysteroid dehydrogenase, 17a,20a-dihydroxypregn-4-en-3-one produced from ['I-14C]17a-hydroxyprogesterone (68 '10 3 counts/min; 5.0 ,ug) by the testicular 105 000 X g supernatant.
Inhibitov
I7a-H y drosylase
I7,2o-Desmolase I7 fJ-Hydroxysteroid eoa-Hydrosysteroid dehydrogenase dehydrogenase
None SU 8000, I p,g/m! SU 10603. I flg/m!
3·55 0.86 0·44
2.03 0.27 0.15
0.7 8 0·73 0.89
'-54 1.55 1.64
somes contain n,B-hydroxysteroid dehydrogenase activity as reported previously? The 105 000 X g supernatant, on the other hand, containes zoc-hydroxysteroid dehydrogenase activity (SHIKITA AND TAMAOKI9). Neither of these dehydrcgenases, however, was inhibited by the compounds (Table I). Double tracer experiments, using [I,2-3H 2 ]progest erone (95' 103 counts/min: 5.0 flg) and [4-14C]androstenedione (35' 103 counts/min; 5·0 flg) also demonstrated that the compounds inhibited the 17a-hydroxylase but not the 17,B-hydroxysteroid dehydrogenase. Furthermore, in the presence of SU 8000 (I /lg/ml), only 0.6% and 2.5% of [4-14C]I7ahydroxyprogesterone (68' 103 counts/min; 5.0 /lg) was converted to androstenedione and testosterone respectively, by the incubation with the recombined mixture of the microsome suspension and the 105 000 X g supernatant. 17a,20a-Dihydroxypregn-4en-g-one, however, was obtained in 37.8% yield by the incubation. This is comparable with the yield of the dihydroxy compound obtained by the incubation with the supernatant only. The result confirmed again that the zoc-hydrcxysteroid dehydrogenase was not influenced by the inhibitors. TABLE II APPARENT DISSOCIATION CONSTANT OF THE ENZYME-INHIBITOR COMPLEX The inhibitor constant was determined graphically as shown in Figs. I and 2.
Tnhibitor
I7a-Hydro:'.;ylase I7, go-Desmolase (flM) (~lM)
SU 8000 SU 10603
1.35 0.86
1.00 0·54
Mode of action oj the inhibitors on the I7a-hydroxylase 17a-Hydroxylation of [4-14C]progesterone by the testicular microsomes was measured with a series of inhibitor concentrations and with substrate concentrations Biochim, Biophys. Aota, 105 (1965) 5 16-5 22
INHIBITION OF TESTICULAR MICROSOMAL ENZYMES
519
of I p,g/ml and 4P,g/ml. The result was analyzed by the method of DIXONlO, and it was shown that both compounds inhibited the IJa-hydroxylase competitively (Fig. I). The inhibitor constant (K i ) estimated by the graphical method are presented in Table II. The K 1 value for SU 8000 is 1.6 times as large as that for SU 10603, indicating that the latter compound has larger affinity against the enzyme than has the former. When the amount of [4-14C]progesterone consumed during the incubation was used, instead of the sum of IJa-hydroxyprogesterone, androstenedione and testosterone produced, to represent the 17a-hydroxylase activity, straight lines similar to those
1.4 "i
1.2
'2u .g
1.0
o
a '00.8 01
3-
"'.... 0.6
~
S=4
-0.4 - 0.2
0.1
0.40.7
[rJ (fJ9/m l)
1.0
Fig. I. Competitive inhibition of 17a-hydroxylation of [4-14C]progesterone by SU 8000 C&.-.) and SU ro603 (0-0). Rat-testicular microsomes equivalent to one testis were incubated with [4-14C]progesterone (7.8' IDS countsjrninjrnl ; I or 4 pg/ml) for 30 min in the presence of 0.1-1.0 pg/ ml of SU 8000 or SU ID603. The reciprocal of the 17a-hydroxylase activity (the sum of 17ahydroxyprogesterone, androstenedione and testosterone produced) is plotted against the inhibitor concentration by the method of DIXON 1 " .
shown in Fig. I were obtained. In this case, also, the points of the intersection of the lines did not lie on the base line, showing that the compounds are competitive inhibitors against the IJa-hydroxylase. Mode of action of the inhibitors on the I7,2o-desmolase Similar experiment was performed to study the effect of the inhibitors on the side-chain cleavage of [4-14C]IJa-hydroxyprogesterone by the testicular microsomes. In this case, however, the point of the intersection of the two lines was on the base line with regard to both inhibitors (Fig. 2). The result suggests that the inhibition of the IJ,2o-desmolase by the compounds is of a non-competitive type. The same conclusion was obtained by plotting against inhibitor concentrations the reciprocal of the amount of [4-14C]IJa-hydroxyprogesterone which was consumed during the incubation. Biochim, Biophys. Acta, lOS (196S) SI6-S22
M. SHIKITA, T. OGISO, B-1. TAMAOKI
520
3.0
2.5
,
"'..... u
:J
2.0
"0
0 c, o,
...0
1.5
01
~
:>'
-;s-
'8=4
-0.2
0.4
0.1
0.7
1.0
[1J (}J9 /mll
Fig. 2. Non-competitive inhibition of the cleavage of the side chain of [4-14C]I7u-hydroxyprogesterone by SU 8000 CA.-A) and SU ra603 (0-0). r4-14C]I7u-Hydroxyprogesterone (1.3' 10 3 countsjrnin/ml: lor 4 flg/ml) was incubated with the rat-testicular microsomes as in the experiment shown in Fig. I. The reciprocal of the I7,:20-desmolase activity (the sum of androstenedione and testosterone produced) is plotted ag-ainst the inhibitor concentration.
The inhibitor constants obtained graphically are shown in Table II. SU I0603 is a stronger inhibitor against the desmolase than is SU I0603 as observed in the case of the IJa-hydroxylase. DISCUSSION
From the measurements shown in Fig. I, the apparent substrate constant of the 17a-hydroxylase was calculated (Table III). Mean K s value is I.29 fig/ml or 4.I TABLE III APPARENT SUBSTRATE CONSTANT OF THE 1.·ESTICULAR MICROSOMAL I7U-HYDROXYLASE
Apparent substrate constant (1(.) was calculated from the intercept (-i) on the base line (Fig. I), by the equation; K. = [S]/(i/KI- I)' where [5] is the substrate concentration and KI the inhibitor constant. Inhibitor
Substrate Intercept on concentration. the base line (5) (flg/ml) (--i) (ltg/ml)
SU 8000 SU 10603
4 I
4
0.60 L3 I 0.38 0.9 0
Substrate constant (K.) (flg/ml)
LIS L37 1.3 0 1.3 2
Biochim, Biophys. Acta, 105 (1965) 516-522
INJ:lIBITION OF TESTICULAR MICROSOMAL ENZYMES
521
I'M. In a separate experiment, the testicular microsomes were incubated with varying concentrations of [4-14C]progesterone without addition of the inhibitors. The apparent substrate constant was graphically obtained (Fig. 3) to be 1.3 1 ttg/rol or 4.2 I'M which is almost same as obtained by the method using inhibitors. The microsome preparation used in these incubations contained many components other than IJa-hydroxylase. The amount of the substrate progesterone and the inhibitor compounds bound with these components was, however, neglected for the estimation of the K a and K I; values, assuming that the substrate and the inhibitors added were sufficiently in excess over the amount of the the compounds bound On the microsomes. This may be justified by the fact that almost similar K s values were obtained by the two different methods, one using the inhibitors and the other using the substrate only. It is, however, possible that the K a of such IJa-hydroxylase as is bound on the microsomes 0.4
,.
0.3
3u :J "0
o
a. 0.2
....o
C1
2$0.1
0.1250.25
0.5
1.0
l/[SJ (pg Iml,-l Fig. 3. Determination of the substrate constant (Kg) of the testicular microsomal 17a-hydroxylase by a Lineweaver-Burk plot. The rat-testicular microsornes were incubated for 30 min as in the preceding experiment with [4- 14C]progesterone (39.ro S counts/flask). The concentration of the carrier progesterone was varied as indicated in the figure.
differs from that of the enzyme which is solubilized from the particles. It may be, furthermore, possible that the solubilized enzyme is inhibited by the compounds noncompetitively, although the enzyme in a bound form is inhibited competitively. Notwithstanding these possibilities, the result presented in this paper suggests that the SU compounds competitively inhibit the IJa-hydroxylase activity of the testicular micro somes and that both compounds have larger affinity than the substrate progesterone against the microsomal IJa-hydroxylase. It is of much interest, on the other hand, that the IJ,2o-desmolase activity of the testicular micro somes was inhibited by the compounds non-competitively. This result suggests that there is some important difference in the active centers of these two enzymes, although the substrate steroids of these enzymes have the same chemical structure except for the hydroxy group at C17• Neither thiamine hydrochloride (Vitamin B l ) nor pyridoxine hydrochloride (Vitamin B o) even at 100 I'M had significant effect on the testicular microsomal enzyme Biochim, Biophys. Acta. 105 (1965) 5 16-5 22
522
M. SHIKITA, T. OGISO, B-I. TAMAOKI
activities. Purine and pyrimidine bases and their ribosides added in 5 ,uM also had no influence on the microsomal enzyme activities. It was reported that the addition of the 105 000 X g supernatant to the microsomal suspension increased the microsomal 17a-hydroxylase and the 17,2o-desmolase activities". The present experiment showed, however, that the inhibition of the enzyme activities by SU 8000 was not prevented by the addition of the supernatant to the incubation mixture. These results may suggest that the inhibition by the SU compounds is a specific one. The observation that neither of the 17,B-hydroxysteroid dehydrogenase nor zoc-hydroxysteroid dehydrogenase was influenced by the compounds supports the view on the specificity of the inhibition of the 17a-hydroxylase and the 17,2o-desmolase by the compounds.
ACKNOWLEDGEMENTS The authors are indebted to Dr. J. J. CHART of Ciba Pharmaceutical Co. (Summit, N.].) for kindly supplying SU 8000 and SU 10603. This work was supported in part by Grants (RH-oOOI4 and CA-07083) from U.S. Public Health Service. REFERENCES I 2
F. W. KAHN'! AND R. NEHER, Esperientia, 18 (1962) 499.
J. J. CHART, H. SHEPPARD, T. MOWLES AND N. HOWIE, Endocrinology, 71 (19 62) 479· 3 D. C. SHARMA, E. FORCHIELLI AND R. 1. DORFMAN, ]. Biol, Chem., 238 (1963) 57 2 . 4 P. F. HALL, K. B. EIK-NES AND L. T. SAMUELS, Endocrinology, 73 (19 63) 547. .5 W. S. LYNN AND R. H. BROWN, J. BioI. Chem., 232 (1958) 1 0 1 5. 6 M. HAYANO, N. SABA, R. L DORFMAN AND O. HECHTER, Recent Progr. Hormone Res., 12 (19.55) 79· M. SHIKITA AND B. TAMAOKI, Endocrinology, M. SHIKITA, H. KAKIZAKI AND B. TAMAOKI, 9 M. SHIKITA AND B. TAMAOKI, Biochemislry, 10 M. DIXON, Biochem, 55 (1953) 170.
7 8
J.,
Biocbim, Biophys, Acta, 105 (1965) 516-522
76 (196 5) 5 63 . Steroids, 4 (1964) 52!. 4 (1965) 118 9 .
IJa-HYDROXYLASE AND 17a-HYDROXYPREGNENE C17-C20 LYASE MIKIO SHIKITA, TOMOKO OGISO
AND
BUN-IeBI TAMAOKI
Nation at Institute oj Radiological Sciences, Chiba-shi (Japan) (Received March 23rd, 1965)
SUMMARY 1. 3-(6-Chloro-3-methyl-2-indenyl)pyridine (SU 8000) and 3-(I,2,3.4-tetrahydro-4-oxo-J-chloro-2-naphthyl)pyridine (SU 10603) competitively inhibited IJahydroxylation of [4-14C]progesterone by rat-testicular microsomes. 2. The compounds also strongly but non-competitively inhibited the cleavage of the side chain of [4-14C]IJa-hydroxyprogesterone by the microsomes, 3. The apparent inhibitor constants of SU 8000 were determined as 1.35 pM and 1.00 pM against the IJa-hydroxylase and the IJa-hydroxypregnene C17-C20 lyase respectively. Corresponding K 1 values for SU 10603 were 0.86 pM and 0.54 pM. 4- IJf.J-Hydroxysteroid dehydrogenase and zcc-hydroxysteroid dehydrogenase of the rat testes were not influenced by the compounds.
INTRODUCTION
1Ja-Hydroxylation of adrenal corticosteroids is reported to be specifically inhibited by several chemical compounds-A, SHARMA et at. 3 , however, reported that 3(6-chloro-3-methyl-z-indenyl)pyridine (SU 8000), one of the inhibitors of the adrenocorticoid 17a-hydroxylase, also inhibited up-hydroxylation of II-deoxycorticosterone by adrenal enzyme preparation. HALL et ai», furthermore, reported that the compound also inhibited 17a-hydroxylation of progesterone by homogenates of rabbit testes. Moreover, they found with canine testes that not only 1Ja-hydroxylation of progesterone but also the cleavage of the side chain of IJa-hydroxyprogesterone was inhibited by the compound. The steroid hydroxylases and the IJ,20-desmolase (IJahydroxypregnene C17-C20 lyase) resemble each other, requiring NADPH and molecular oxygen for their action's". These reports led us to study further detailed kinetics of the influence of the inhibitors on the 1Ja-hydroxylase in comparison with that on the 17,20-desmolase. Both enzymes were reported to be contained in the microsomes obtained from rat testes", MATERIALS AND METHODS
Substrate steroids [4-14(lProgesterone (83 pC/mg), [4-l 4CJandrosteneclione ([4-14C]androst-4-eneBioohim. Biophys. Acta, I05 (1965) 5I6-522
INHIBITION OF TESTICULAR MICROSO:MAL
ENZY~IES
3,17-dione) (71 ftC/mg) and [4-14C]I7a-hydroxyprogesterone (44 ftCfmg) were purchased from New England Nuclear Corp. (Boston, Mass.), [r,z-3H 2]Progest erone (2 mC/mg) was supplied by Endocrinology Study Section, National Institutes of Health. Prior to use, their purity was checked by thin-layer chromatography. Tissue preparation
Testes of rats of the Wistar strain, 2 to 3 months old, were isolated, homogenized and fractionated by the differential centrifugation method as described in the previous report". The microsomal fraction was suspended in 0.25 M sucrose solution, and the suspension approximately equivalent to one testis (I.2 g) was added to each incubation flask. The 105 000 X g supernatant was transparent and equivalent two testes per flask.
to
I ncubation
Radioactive steroid precursors with the respective carrier steroid, the inhibitors and a drop per flask of propylene glycol were dissolved in dichloromethane and added to the incubation flask (50 ml centrifuge tube with round bottom). Dichloromethane was gently removed under reduced pressure. Shortly before incubation, 3 ml of 0.25 M sucrose solution containing 0.5 {lmoles of N"ADPH and SOD flmoles of Tris (pH 7-4) and then 3 ml of the testicular preparation were added to the flask. The mixtures were incubated for 30 min at 37° with continuous shaking under the atomosphere of 95 % O2 and 5 % CO2 , An incubation time of 30 min was chosen, as time studies ensured a linear rate of the enzymic reactions at least for 45 min. Extraction, separation, identification and quaniitation oj the products
The reaction was stopped by rapid extraction of the steroids from the incubation mixture- with dichloromethane. The products were separated and purified by silica gel G thin-layer chromatography with a solvent system of benzene-acetone (8:2, v/v) , and quantitatively determined by a liquid scintillation spectrometers. The efficiency of counting was 32% for tritium and 57% for [14CJcarbon. Total recovery of the radioactivity was between 85 and 95 %. The products were pooled and identified as described in detail in our previous papers. RESULTS
Specificity of the effect oj the inhibitors
As shown in Table I 17a-hydroxylase and 17,20-desmolase activities of rat testicular microsomes were both inhibited markedly by SU 8000 as well as by SU 10603. This was confirmed by another series of experiments in which case [1,2-SH2~ progesterone (35' r0 8 counts/min; 5.0 p,g) and [4-14C]I7a-hydroxyprogesterone (I4' lOS counts/min; 5.0 fig) were incubated together in the same flask. Without inhibitors, 55·3% of [I,2- 8H 2]progesterone was hydroxylated and 20.0% of [4-14C]17a-hydroxyprogesterone cleaved to the C19 steroids, while II.8% and 4.9% in the presence of SU 8000 [I flg/ml) and 8.5 % and 2.5% in the presence of SU 10603 (I fig/m]), respectively. These results show that the inhibition by the compounds is not specific on the 17a-hydroxylase. Besides the 17a-hydroxylase and the 17.20-desmolase, the rat-testicular microBiochim. Biophys, Acta, r05 (r965) 5 y6-5 2 2
M. SHIKITA,
518
T.
OGISO,
B-I.
TAMAOKI
TABLE I EFFECT OF SU 8000 AND SU I0603 ON" I7a-HYDROXYLASE, I7,20-DESMOLASE, I7fJ- AND 20a-HYDROXYSTEROID DEHYDROGEN"ASES OF RAT TESTES The enzyme activity is expressed in flg of the products obtained by the 30-min incubation: I7a-hydroxylase, sum of 17a-hydroxyprogesterone, androstenedione and testosterone produced from [4-l.4C]pro gesterons (62 . 103 counts/min; 6.2 fig) by the testicular microsomes; 7,zo-desmolase, sum of androstenedione and testosterone produced from r4-14C)I7a-hydroxyprogesterone (68 '10 8 counts/min; 5.0 p,g) by the microsomes ; 17fJ-hydroxysteroid dehydrogenase, testosterone produced from [:f- 14C]a n drost en ed ione (35' 10' counts/min; 5.0 flg) by the microsomes; zorz-hydroxysteroid dehydrogenase, 17a,20a-dihydroxypregn-4-en-3-one produced from ['I-14C]17a-hydroxyprogesterone (68 '10 3 counts/min; 5.0 ,ug) by the testicular 105 000 X g supernatant.
Inhibitov
I7a-H y drosylase
I7,2o-Desmolase I7 fJ-Hydroxysteroid eoa-Hydrosysteroid dehydrogenase dehydrogenase
None SU 8000, I p,g/m! SU 10603. I flg/m!
3·55 0.86 0·44
2.03 0.27 0.15
0.7 8 0·73 0.89
'-54 1.55 1.64
somes contain n,B-hydroxysteroid dehydrogenase activity as reported previously? The 105 000 X g supernatant, on the other hand, containes zoc-hydroxysteroid dehydrogenase activity (SHIKITA AND TAMAOKI9). Neither of these dehydrcgenases, however, was inhibited by the compounds (Table I). Double tracer experiments, using [I,2-3H 2 ]progest erone (95' 103 counts/min: 5.0 flg) and [4-14C]androstenedione (35' 103 counts/min; 5·0 flg) also demonstrated that the compounds inhibited the 17a-hydroxylase but not the 17,B-hydroxysteroid dehydrogenase. Furthermore, in the presence of SU 8000 (I /lg/ml), only 0.6% and 2.5% of [4-14C]I7ahydroxyprogesterone (68' 103 counts/min; 5.0 /lg) was converted to androstenedione and testosterone respectively, by the incubation with the recombined mixture of the microsome suspension and the 105 000 X g supernatant. 17a,20a-Dihydroxypregn-4en-g-one, however, was obtained in 37.8% yield by the incubation. This is comparable with the yield of the dihydroxy compound obtained by the incubation with the supernatant only. The result confirmed again that the zoc-hydrcxysteroid dehydrogenase was not influenced by the inhibitors. TABLE II APPARENT DISSOCIATION CONSTANT OF THE ENZYME-INHIBITOR COMPLEX The inhibitor constant was determined graphically as shown in Figs. I and 2.
Tnhibitor
I7a-Hydro:'.;ylase I7, go-Desmolase (flM) (~lM)
SU 8000 SU 10603
1.35 0.86
1.00 0·54
Mode of action oj the inhibitors on the I7a-hydroxylase 17a-Hydroxylation of [4-14C]progesterone by the testicular microsomes was measured with a series of inhibitor concentrations and with substrate concentrations Biochim, Biophys. Aota, 105 (1965) 5 16-5 22
INHIBITION OF TESTICULAR MICROSOMAL ENZYMES
519
of I p,g/ml and 4P,g/ml. The result was analyzed by the method of DIXONlO, and it was shown that both compounds inhibited the IJa-hydroxylase competitively (Fig. I). The inhibitor constant (K i ) estimated by the graphical method are presented in Table II. The K 1 value for SU 8000 is 1.6 times as large as that for SU 10603, indicating that the latter compound has larger affinity against the enzyme than has the former. When the amount of [4-14C]progesterone consumed during the incubation was used, instead of the sum of IJa-hydroxyprogesterone, androstenedione and testosterone produced, to represent the 17a-hydroxylase activity, straight lines similar to those
1.4 "i
1.2
'2u .g
1.0
o
a '00.8 01
3-
"'.... 0.6
~
S=4
-0.4 - 0.2
0.1
0.40.7
[rJ (fJ9/m l)
1.0
Fig. I. Competitive inhibition of 17a-hydroxylation of [4-14C]progesterone by SU 8000 C&.-.) and SU ro603 (0-0). Rat-testicular microsomes equivalent to one testis were incubated with [4-14C]progesterone (7.8' IDS countsjrninjrnl ; I or 4 pg/ml) for 30 min in the presence of 0.1-1.0 pg/ ml of SU 8000 or SU ID603. The reciprocal of the 17a-hydroxylase activity (the sum of 17ahydroxyprogesterone, androstenedione and testosterone produced) is plotted against the inhibitor concentration by the method of DIXON 1 " .
shown in Fig. I were obtained. In this case, also, the points of the intersection of the lines did not lie on the base line, showing that the compounds are competitive inhibitors against the IJa-hydroxylase. Mode of action of the inhibitors on the I7,2o-desmolase Similar experiment was performed to study the effect of the inhibitors on the side-chain cleavage of [4-14C]IJa-hydroxyprogesterone by the testicular microsomes. In this case, however, the point of the intersection of the two lines was on the base line with regard to both inhibitors (Fig. 2). The result suggests that the inhibition of the IJ,2o-desmolase by the compounds is of a non-competitive type. The same conclusion was obtained by plotting against inhibitor concentrations the reciprocal of the amount of [4-14C]IJa-hydroxyprogesterone which was consumed during the incubation. Biochim, Biophys. Acta, lOS (196S) SI6-S22
M. SHIKITA, T. OGISO, B-1. TAMAOKI
520
3.0
2.5
,
"'..... u
:J
2.0
"0
0 c, o,
...0
1.5
01
~
:>'
-;s-
'8=4
-0.2
0.4
0.1
0.7
1.0
[1J (}J9 /mll
Fig. 2. Non-competitive inhibition of the cleavage of the side chain of [4-14C]I7u-hydroxyprogesterone by SU 8000 CA.-A) and SU ra603 (0-0). r4-14C]I7u-Hydroxyprogesterone (1.3' 10 3 countsjrnin/ml: lor 4 flg/ml) was incubated with the rat-testicular microsomes as in the experiment shown in Fig. I. The reciprocal of the I7,:20-desmolase activity (the sum of androstenedione and testosterone produced) is plotted ag-ainst the inhibitor concentration.
The inhibitor constants obtained graphically are shown in Table II. SU I0603 is a stronger inhibitor against the desmolase than is SU I0603 as observed in the case of the IJa-hydroxylase. DISCUSSION
From the measurements shown in Fig. I, the apparent substrate constant of the 17a-hydroxylase was calculated (Table III). Mean K s value is I.29 fig/ml or 4.I TABLE III APPARENT SUBSTRATE CONSTANT OF THE 1.·ESTICULAR MICROSOMAL I7U-HYDROXYLASE
Apparent substrate constant (1(.) was calculated from the intercept (-i) on the base line (Fig. I), by the equation; K. = [S]/(i/KI- I)' where [5] is the substrate concentration and KI the inhibitor constant. Inhibitor
Substrate Intercept on concentration. the base line (5) (flg/ml) (--i) (ltg/ml)
SU 8000 SU 10603
4 I
4
0.60 L3 I 0.38 0.9 0
Substrate constant (K.) (flg/ml)
LIS L37 1.3 0 1.3 2
Biochim, Biophys. Acta, 105 (1965) 516-522
INJ:lIBITION OF TESTICULAR MICROSOMAL ENZYMES
521
I'M. In a separate experiment, the testicular microsomes were incubated with varying concentrations of [4-14C]progesterone without addition of the inhibitors. The apparent substrate constant was graphically obtained (Fig. 3) to be 1.3 1 ttg/rol or 4.2 I'M which is almost same as obtained by the method using inhibitors. The microsome preparation used in these incubations contained many components other than IJa-hydroxylase. The amount of the substrate progesterone and the inhibitor compounds bound with these components was, however, neglected for the estimation of the K a and K I; values, assuming that the substrate and the inhibitors added were sufficiently in excess over the amount of the the compounds bound On the microsomes. This may be justified by the fact that almost similar K s values were obtained by the two different methods, one using the inhibitors and the other using the substrate only. It is, however, possible that the K a of such IJa-hydroxylase as is bound on the microsomes 0.4
,.
0.3
3u :J "0
o
a. 0.2
....o
C1
2$0.1
0.1250.25
0.5
1.0
l/[SJ (pg Iml,-l Fig. 3. Determination of the substrate constant (Kg) of the testicular microsomal 17a-hydroxylase by a Lineweaver-Burk plot. The rat-testicular microsornes were incubated for 30 min as in the preceding experiment with [4- 14C]progesterone (39.ro S counts/flask). The concentration of the carrier progesterone was varied as indicated in the figure.
differs from that of the enzyme which is solubilized from the particles. It may be, furthermore, possible that the solubilized enzyme is inhibited by the compounds noncompetitively, although the enzyme in a bound form is inhibited competitively. Notwithstanding these possibilities, the result presented in this paper suggests that the SU compounds competitively inhibit the IJa-hydroxylase activity of the testicular micro somes and that both compounds have larger affinity than the substrate progesterone against the microsomal IJa-hydroxylase. It is of much interest, on the other hand, that the IJ,2o-desmolase activity of the testicular micro somes was inhibited by the compounds non-competitively. This result suggests that there is some important difference in the active centers of these two enzymes, although the substrate steroids of these enzymes have the same chemical structure except for the hydroxy group at C17• Neither thiamine hydrochloride (Vitamin B l ) nor pyridoxine hydrochloride (Vitamin B o) even at 100 I'M had significant effect on the testicular microsomal enzyme Biochim, Biophys. Acta. 105 (1965) 5 16-5 22
522
M. SHIKITA, T. OGISO, B-I. TAMAOKI
activities. Purine and pyrimidine bases and their ribosides added in 5 ,uM also had no influence on the microsomal enzyme activities. It was reported that the addition of the 105 000 X g supernatant to the microsomal suspension increased the microsomal 17a-hydroxylase and the 17,2o-desmolase activities". The present experiment showed, however, that the inhibition of the enzyme activities by SU 8000 was not prevented by the addition of the supernatant to the incubation mixture. These results may suggest that the inhibition by the SU compounds is a specific one. The observation that neither of the 17,B-hydroxysteroid dehydrogenase nor zoc-hydroxysteroid dehydrogenase was influenced by the compounds supports the view on the specificity of the inhibition of the 17a-hydroxylase and the 17,2o-desmolase by the compounds.
ACKNOWLEDGEMENTS The authors are indebted to Dr. J. J. CHART of Ciba Pharmaceutical Co. (Summit, N.].) for kindly supplying SU 8000 and SU 10603. This work was supported in part by Grants (RH-oOOI4 and CA-07083) from U.S. Public Health Service. REFERENCES I 2
F. W. KAHN'! AND R. NEHER, Esperientia, 18 (1962) 499.
J. J. CHART, H. SHEPPARD, T. MOWLES AND N. HOWIE, Endocrinology, 71 (19 62) 479· 3 D. C. SHARMA, E. FORCHIELLI AND R. 1. DORFMAN, ]. Biol, Chem., 238 (1963) 57 2 . 4 P. F. HALL, K. B. EIK-NES AND L. T. SAMUELS, Endocrinology, 73 (19 63) 547. .5 W. S. LYNN AND R. H. BROWN, J. BioI. Chem., 232 (1958) 1 0 1 5. 6 M. HAYANO, N. SABA, R. L DORFMAN AND O. HECHTER, Recent Progr. Hormone Res., 12 (19.55) 79· M. SHIKITA AND B. TAMAOKI, Endocrinology, M. SHIKITA, H. KAKIZAKI AND B. TAMAOKI, 9 M. SHIKITA AND B. TAMAOKI, Biochemislry, 10 M. DIXON, Biochem, 55 (1953) 170.
7 8
J.,
Biocbim, Biophys, Acta, 105 (1965) 516-522
76 (196 5) 5 63 . Steroids, 4 (1964) 52!. 4 (1965) 118 9 .