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Affinity labeling of rat liver microsomal NADH-5α-reductase with a nucleoside analogue
1
2920
AFFINITY LABELING OF RAT LIVER MICROSOMAL NADH-5x-RHDUCTASE WITH A NUCLEOSIDE ANALOGUE S. W. Golf and V. Graef Institute of Clinical Chemistry and Pathbbiochemistry, University Medical School, 63 Giessen, Germany Received
1-26-82
ABSTRACT A time dependent irreversible loss of rat liver microsomal NADH-5d-reductase activity is caused by incubation of microsomes with the nucleoside 5'-E-fluorosulfonylbenzoyladenosine (FSA). The decrease of activity is dependent on FSA concentration and shows first order kinetics. Presence of NADH partially stabilizes the NADH-5a-reductase. Thioglycerol present before incubation prevents loss of activity, and stops decrease of activity when added during incubation. NADPH5d-reductase (E.C. 1.3.1.4) and NADPH-cytochrome c reductase (E.C. 1.6.2.4) are not influenced while NADH-cytochrome c reductase (E.C. 1.6.99.3) is inhibited by FSA. Evidently FSA causes inactivation of the enzymes by binding to the NADH-binding site. Affinity labeling by FSA thus clearly distinguishes between NADH- and NADPH-dependent 5d-reductases from rat liver microsomes. INTRODUCTION In rat liver microsomes two 5d-reductases
are present
with different affinity toward the hydrogen donors NADH and NADPH for reduction of the 4,5-double bond of 4-ene-3-oxosteroids (1). The NADPH-5U-reductase
from rat liver micro-
somes is an enzyme-complex consisting of two proteins: NADPHcytochrome c reductase and 5d-reductase the NADPH-dependent 5d-reductase
(2, 3, 4). While
has received much attention
(5), not much data are available concerning the NADH-5 ti-reductase. Differentiation between both enzymes is possible by solubilization
(6), inhibitors (3) and by phosphate in the
incubation medium Volwne40, Nwnber 1
(1). In this paper FSA is shown to inhibit
u
TEEOXDI
July,
1982
S
2
TDPROIDIP
NADH-5al-reductase selectively while keeping the NADPH5d-reductase
unaltered.
FSA has been mostly used for affinity labeling of NADHor ATP-specific enzymes unrelated to steroid metabolism: pyruvate kinase (7), Fl-ATPase
(8), glutamate dehydrogenase
(9) and CAMP-dependent protein kinase
(10). 3al,20R-Hydroxy-
steroid dehydrogenase loses both 3a-
and 20B-dehydrogenase
activity when labeled with FSA, suggesting a common active center (11) for both reactions. MATERIALS AND METHODS Materials: FSA was obtained from Sigma Chemical Co., D-8000 Miinchen, Germany: steroids, inorganic and organic chemicals were purchased from Merck, D-6100 Darmstadt, Germany, NADH and NADPH from Boehringer, D-6800 Mannheim, Germany. Liver microsomes: The preparation of liver microsomes from female rats (Wistar) has been described elsewhere (12). Enzyme-assays: The activities of 5d-reductases in microsomes were determined with testosterone as substrate and NADH and NADPH as cosubstrates. The reaction mixture contained in a final volume of 3.0 mL 0.2 M potassium phosphate buffer pH 6.5, 2.4 poles NADH or NADPH, 300 nmoles testosterone and 0.07 mg microsomal protein. Preincubation (varying time) was carried out with buffer, microsomes, 1.67% (v/v) ethyleneglycolmonomethylether and 10 poles or less FSA (dissolved in ethyleneglycolmonomethylether) and NADH or NADPH as indicated in the results section. After preincubation testosterone with or without NADH or NADPH was added. The reaction was stopped by ether extraction after 10 minutes (37OC). Steroids were determined after Cr03-oxidation (13) by gas-chromatography using a PerkinElmer 3920 GC with a 2 m column (3% OV-17 W/AW-DMCS)or a Perkin-Elmer F-20 equipped with a fused silica column (OV-101). NADPH- and NADH-cytochrome c reductase were determined spectrophotometrically (548 nm): 1.1 mL final volume, 50 mM sodium phosphate buffer pH 7.7, 100 nmoles NADPH or NADH, 40 nmoles cytochrome c, 0.24 mg microsomal protein )NADPH) or 0.015 mg microsomal protein (NADH), 33 nmoles FSA (dissolved in ethyleneglycolmonomethylether or 1.67 % (v/v) ethyleneglycolmonomethylether.
The protein concentration was determined by the Biuretmethod (14). The values presented here are the mean of two simultaneous determinations with at least one corresponding repetition. RESULTS Several enzymes present in rat liver microsomes were tested for reaction with the nucleoside analogue FSA. In particular NADPH- and NADH-dependent reductions of testosterone and cytochrome c were carried out. When NADH-5dreductase was preincubated for varying time, a decrease of
20
O
IO 5 Minutes preincubation
15 time
Fig. 1. Inactivation kinetics of NADH-5d-reductase activity by varying FSA concentration. The preincubation mixtures contained 1.67% ethyleneglycolmonomethylether. (x-x) control, (d-u) 5 poles FSA, (o-e) 2.5 poles FSA, (o---o) 0.5 poles FSA. After preincubation NADH and testosterone were added to initiate the NADH-5Oi -reductase reaction.
S
4
?3?1JEOXDI
activity was observed both in absence and presence of FSA. The nucleoside analogue, however, strongly accelerates the inactivation of microsomal NADH-5CX -reductase (Fig. 1). Increasing FSA concentration results in an accelerated decrease of enzymatic activity, which, however, declines only to about 50% of its original value. The semilogarithmic plot shows a linear slope of inactivation, suggesting first order
I
5 Minutes
IO preincubation
15 time
20
Fig. 2. Inactivation kinetics of NADH-5oL-reductase activity by FSA in presence of NADH. The preincubation mixtures contained 1.67% ethyleneglycolmonomethylether and 2.4 poles NADH. (x-x1 control, (m-•) 0.5 moles FSA, (o--o) 2.5 poles FSA,(tl--&I) 5 poles FSA. After preincubation time testosterone was added to initiate the NADH-5ti-reductase reaction.
kinetics of inactivation (Fig. 1). NADH presence during preincubation partially prevents inactivation (Fig. 2) with the FSA concentration being crucial for the rate of inactivation. Addition of thioglycerol during incubation immediately stops decrease of NADH-5d-reductase
activity caused by FSA
(Fig. 3). When thioglycerol is added 10 min after the be-
a g
.-
I,8
“u 0
2 I,6 ‘j 2 7 1,4 ‘d “: 6
Q Z
12
s
0”
-
I,0 ,
5 10 Minutes preincubation
20 time
Fig. 3. Inactivation of NADH-5d-reductase by FSA in presence of thioglycerol. The preincubation mixtures contained 1.67% ethyleneglycolmonomethylether. (x-x) Control, 5 poles FSA, (o-1 5 poles FSA and 9 poles (0-0) thioglycerol.
ginning of the preincubation, it prevents the further loss of activity. NADPH-Lid-reductase
from rat liver microsomes, in con-
trast, is not modified by the nucleoside analoyue (Table I.). Its activity remains unchanged when incubated in presence of FSA if compared to the control. The same is true for microsomal NADPH-cytochrome
c reductase, which is not modified by
preincubation of microsomes with FSA (substrate cytochrome c). NADH-dependent reduction of cytochrome c by microsomes, however, is decreased after preincubation with the nucleoside analogue (Table 1).
Table 1. Relative activity of different microsomal enzymes after preincubation with FSA. Activity is expressed as percent of control activity (absence of FSAf. Preincubation was carried out with 1.65% ethyleneglycolmonomethylether (control) or FSA (0.5; 0.033; 0.033 poles in above sequence) in ethyleneglycolmonomethylether. NADPH and testosterone (NADPH-5d -reductase) and NAD(P)H and cytochrome c (cytochrome c reductases) were added after preincubation to start the reaction. Time of preincubation (min)
NADPH-5rYreductase
NADPH-cytochrome c reductase
0
98.8%
90.6%
88.9%
5
89.0%
94.2%
77.5%
10
106.0%
98.3%
81.5%
15 20
99.0%
98.3%
66.7%
90.8%
61.9%
25
95.6
90.9% 87.0%
NADH-cytochrome c reductase
60.0%
S
WREOX
DI
DISCUSSION FSA was found to be an affinity label for several enzymes (7,8,9). Similarly, FSA is bound to the active center of microsomal NADH-5aC-reductase with a resulting irreversible loss of activity, producing first order inactivation kinetics. The rate of inactivation by FSA proceeds until 50% of microsomal NADH-5 d-reductase
activity is reached. There is no
ready explanation for this observation, except that there exist two different 5ti-reductases in rat liver microsomes which accept NADH as cofactor. One of these enzymes remains unchanged by FSA. Rat liver microsomes contain a second 5ci-reductase which requires NADPH as hydrogen donor. This NADPH-5d-reductase is well characterized (4,5). It consists of an enzyme complex with two different proteins. The NADPH-cytochrome c reductase transfers the electrons from NADPH to coenzyme Ql, (3) which then reduces a 4-ene-3-oxosteroid via the 5aL-reductase. The NADPH-cytochrome c reductase is able to use NADH for various hydroxylation reactions (19). The KM value for these hydroxylations are very high and range from 6.7 x 10m4M (20) or 1 x 10-3M (24) to 5 x 10-3M (19). In contrast, the KM value for the NADH-dependent 5d-reduction
of testosterone is much
lower (10s5M) (15). The NADH-concentration used in our experiments was 800 p
so that one should expect a 5cC-reduc-
tion of testosterone by means of NADH via NADPH-cytochrome c reductase and this reaction is not influenced by FSA.
A differentiation between NADH- and NADPH-5 d-reductase is possible by various means. Solubilization of both enzymes is achieved only by different systems (6), where the NADHdependent enzyme needs phosphatidylcholine micelles for reactivation (15). Different inhibitor characteristics phosphate dependency
(9,15),
(l), circadian rhythm of activity (16),
different inducibility by ethanol (15,17) point to different enzyme activities. There are, however, several similarities between both enzymes. Both activities are inhibited by gallic acid propylester
(2,15), a substance characterized as a spe-
cific inhibitor for NADPH-cytochrome c reductase
(18) from rat
liver microsomes. Both enzyme activities are suppressed by antibody (rabbit) against purified NADPH-cytochrome c reductase (4,15), and are inhibited by NADP+, where the inhibition is competitive in the case of NADH-5&-reductase
(15).
The presented results indicate that FSA is a useful tool in resolving whether microsomal NADPH-cytochrome c reductase is proton donor for the NADH-dependent 5a!-reductase or not. It was clearly shown that the corresponding active sites accepting NADH or NADPH for reduction of 4-ene-3-oxosteroids by rat liver microsomes are different. ACKNOWLEDGMENT This work was supported by Dsutsche Forschungsgemeinschaft. ABBREVIATION 5 CY-DHT: 5X-dihydrotestosterone 3-one).
(17!3-hydroxy-5oC-androstan-
SC
WEEOIDI
9
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9.
10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.
Leybold, K. and Staudinger, Hj., Arch. Biochem. Biophys. 96, 626-628 (1962,). Golf, S.W., Graef, V. and Staudinger, Hj., 2. Physiol. Chem. 355, 1499-1507 (1974). Graef,T and Golf, S.W., Z. Klin. Chem. Klin. Biochem. 2, 333-339 (1975). Golf, S.W. and Graef, V., J. Steroid Biochem. 2, 10871092 (1978). Clark, A.F., "Steroid Biochemistry", Chem. Rubber Comp. Cleveland, Ohio, USA, l-27 (1980). Graef, V., Golf, S.W. and Rempeters, G., J. Steroid Biochem. 2, 369-371 (1978). Wyatt, J.L. and Colman, R.F., Biochem. -16, 1333-1342 (1977). Esch, F.S. and Allison, W.S., J. Biol. Chem. 253, 61006106 (1978). Pal, K., Wechter, W.J. and Colman, R.F., J. Biol. Chem. 3, 8140-8147 (1975). Zoller, M.J. and Taylor, S.S., J. Biol. Chem. 254, 83638368 (1979). Sweet, F. and Samant, B., Steroids 36, 373-381 (1980). Golf, S.W., Graef, V. and Nowotny, E., Z. Physiol. Chem. 357, 35-40 (1976). Graef, V., Golf, S.W. and Tiischen, M., J. Steroid Biochem.14, 883-887 (1981). Gornan, A.G., Bardawill, C.J. and David, M.M., J. Biol. Chem. 177, 751-766 (1949). Rempet=, G., Dissertation, University Giessen, l-109 (1978). Graef, V. and Golf, S.W., J. Steroid Biochem. ll_, 12991303 (1979). Rubin, E. and Lieber, Ch. S., Science 191, 563-564 (1976). Torielli, N.F. and Slater, T.F., Biochem. Pharmacol. 20, 2027-2032 (1971). Prough, R.A. and Burke, M.D., Arch. Biochem. Biophys. 170, 160-168 (1975). Noshiro, M. and Omura, T., J. Biochem. 83, 61-77 (1978). Ichikawa, Y. and Yamano, T., J. Biochem. 66, 351-360 (1969).
2920
AFFINITY LABELING OF RAT LIVER MICROSOMAL NADH-5x-RHDUCTASE WITH A NUCLEOSIDE ANALOGUE S. W. Golf and V. Graef Institute of Clinical Chemistry and Pathbbiochemistry, University Medical School, 63 Giessen, Germany Received
1-26-82
ABSTRACT A time dependent irreversible loss of rat liver microsomal NADH-5d-reductase activity is caused by incubation of microsomes with the nucleoside 5'-E-fluorosulfonylbenzoyladenosine (FSA). The decrease of activity is dependent on FSA concentration and shows first order kinetics. Presence of NADH partially stabilizes the NADH-5a-reductase. Thioglycerol present before incubation prevents loss of activity, and stops decrease of activity when added during incubation. NADPH5d-reductase (E.C. 1.3.1.4) and NADPH-cytochrome c reductase (E.C. 1.6.2.4) are not influenced while NADH-cytochrome c reductase (E.C. 1.6.99.3) is inhibited by FSA. Evidently FSA causes inactivation of the enzymes by binding to the NADH-binding site. Affinity labeling by FSA thus clearly distinguishes between NADH- and NADPH-dependent 5d-reductases from rat liver microsomes. INTRODUCTION In rat liver microsomes two 5d-reductases
are present
with different affinity toward the hydrogen donors NADH and NADPH for reduction of the 4,5-double bond of 4-ene-3-oxosteroids (1). The NADPH-5U-reductase
from rat liver micro-
somes is an enzyme-complex consisting of two proteins: NADPHcytochrome c reductase and 5d-reductase the NADPH-dependent 5d-reductase
(2, 3, 4). While
has received much attention
(5), not much data are available concerning the NADH-5 ti-reductase. Differentiation between both enzymes is possible by solubilization
(6), inhibitors (3) and by phosphate in the
incubation medium Volwne40, Nwnber 1
(1). In this paper FSA is shown to inhibit
u
TEEOXDI
July,
1982
S
2
TDPROIDIP
NADH-5al-reductase selectively while keeping the NADPH5d-reductase
unaltered.
FSA has been mostly used for affinity labeling of NADHor ATP-specific enzymes unrelated to steroid metabolism: pyruvate kinase (7), Fl-ATPase
(8), glutamate dehydrogenase
(9) and CAMP-dependent protein kinase
(10). 3al,20R-Hydroxy-
steroid dehydrogenase loses both 3a-
and 20B-dehydrogenase
activity when labeled with FSA, suggesting a common active center (11) for both reactions. MATERIALS AND METHODS Materials: FSA was obtained from Sigma Chemical Co., D-8000 Miinchen, Germany: steroids, inorganic and organic chemicals were purchased from Merck, D-6100 Darmstadt, Germany, NADH and NADPH from Boehringer, D-6800 Mannheim, Germany. Liver microsomes: The preparation of liver microsomes from female rats (Wistar) has been described elsewhere (12). Enzyme-assays: The activities of 5d-reductases in microsomes were determined with testosterone as substrate and NADH and NADPH as cosubstrates. The reaction mixture contained in a final volume of 3.0 mL 0.2 M potassium phosphate buffer pH 6.5, 2.4 poles NADH or NADPH, 300 nmoles testosterone and 0.07 mg microsomal protein. Preincubation (varying time) was carried out with buffer, microsomes, 1.67% (v/v) ethyleneglycolmonomethylether and 10 poles or less FSA (dissolved in ethyleneglycolmonomethylether) and NADH or NADPH as indicated in the results section. After preincubation testosterone with or without NADH or NADPH was added. The reaction was stopped by ether extraction after 10 minutes (37OC). Steroids were determined after Cr03-oxidation (13) by gas-chromatography using a PerkinElmer 3920 GC with a 2 m column (3% OV-17 W/AW-DMCS)or a Perkin-Elmer F-20 equipped with a fused silica column (OV-101). NADPH- and NADH-cytochrome c reductase were determined spectrophotometrically (548 nm): 1.1 mL final volume, 50 mM sodium phosphate buffer pH 7.7, 100 nmoles NADPH or NADH, 40 nmoles cytochrome c, 0.24 mg microsomal protein )NADPH) or 0.015 mg microsomal protein (NADH), 33 nmoles FSA (dissolved in ethyleneglycolmonomethylether or 1.67 % (v/v) ethyleneglycolmonomethylether.
The protein concentration was determined by the Biuretmethod (14). The values presented here are the mean of two simultaneous determinations with at least one corresponding repetition. RESULTS Several enzymes present in rat liver microsomes were tested for reaction with the nucleoside analogue FSA. In particular NADPH- and NADH-dependent reductions of testosterone and cytochrome c were carried out. When NADH-5dreductase was preincubated for varying time, a decrease of
20
O
IO 5 Minutes preincubation
15 time
Fig. 1. Inactivation kinetics of NADH-5d-reductase activity by varying FSA concentration. The preincubation mixtures contained 1.67% ethyleneglycolmonomethylether. (x-x) control, (d-u) 5 poles FSA, (o-e) 2.5 poles FSA, (o---o) 0.5 poles FSA. After preincubation NADH and testosterone were added to initiate the NADH-5Oi -reductase reaction.
S
4
?3?1JEOXDI
activity was observed both in absence and presence of FSA. The nucleoside analogue, however, strongly accelerates the inactivation of microsomal NADH-5CX -reductase (Fig. 1). Increasing FSA concentration results in an accelerated decrease of enzymatic activity, which, however, declines only to about 50% of its original value. The semilogarithmic plot shows a linear slope of inactivation, suggesting first order
I
5 Minutes
IO preincubation
15 time
20
Fig. 2. Inactivation kinetics of NADH-5oL-reductase activity by FSA in presence of NADH. The preincubation mixtures contained 1.67% ethyleneglycolmonomethylether and 2.4 poles NADH. (x-x1 control, (m-•) 0.5 moles FSA, (o--o) 2.5 poles FSA,(tl--&I) 5 poles FSA. After preincubation time testosterone was added to initiate the NADH-5ti-reductase reaction.
kinetics of inactivation (Fig. 1). NADH presence during preincubation partially prevents inactivation (Fig. 2) with the FSA concentration being crucial for the rate of inactivation. Addition of thioglycerol during incubation immediately stops decrease of NADH-5d-reductase
activity caused by FSA
(Fig. 3). When thioglycerol is added 10 min after the be-
a g
.-
I,8
“u 0
2 I,6 ‘j 2 7 1,4 ‘d “: 6
Q Z
12
s
0”
-
I,0 ,
5 10 Minutes preincubation
20 time
Fig. 3. Inactivation of NADH-5d-reductase by FSA in presence of thioglycerol. The preincubation mixtures contained 1.67% ethyleneglycolmonomethylether. (x-x) Control, 5 poles FSA, (o-1 5 poles FSA and 9 poles (0-0) thioglycerol.
ginning of the preincubation, it prevents the further loss of activity. NADPH-Lid-reductase
from rat liver microsomes, in con-
trast, is not modified by the nucleoside analoyue (Table I.). Its activity remains unchanged when incubated in presence of FSA if compared to the control. The same is true for microsomal NADPH-cytochrome
c reductase, which is not modified by
preincubation of microsomes with FSA (substrate cytochrome c). NADH-dependent reduction of cytochrome c by microsomes, however, is decreased after preincubation with the nucleoside analogue (Table 1).
Table 1. Relative activity of different microsomal enzymes after preincubation with FSA. Activity is expressed as percent of control activity (absence of FSAf. Preincubation was carried out with 1.65% ethyleneglycolmonomethylether (control) or FSA (0.5; 0.033; 0.033 poles in above sequence) in ethyleneglycolmonomethylether. NADPH and testosterone (NADPH-5d -reductase) and NAD(P)H and cytochrome c (cytochrome c reductases) were added after preincubation to start the reaction. Time of preincubation (min)
NADPH-5rYreductase
NADPH-cytochrome c reductase
0
98.8%
90.6%
88.9%
5
89.0%
94.2%
77.5%
10
106.0%
98.3%
81.5%
15 20
99.0%
98.3%
66.7%
90.8%
61.9%
25
95.6
90.9% 87.0%
NADH-cytochrome c reductase
60.0%
S
WREOX
DI
DISCUSSION FSA was found to be an affinity label for several enzymes (7,8,9). Similarly, FSA is bound to the active center of microsomal NADH-5aC-reductase with a resulting irreversible loss of activity, producing first order inactivation kinetics. The rate of inactivation by FSA proceeds until 50% of microsomal NADH-5 d-reductase
activity is reached. There is no
ready explanation for this observation, except that there exist two different 5ti-reductases in rat liver microsomes which accept NADH as cofactor. One of these enzymes remains unchanged by FSA. Rat liver microsomes contain a second 5ci-reductase which requires NADPH as hydrogen donor. This NADPH-5d-reductase is well characterized (4,5). It consists of an enzyme complex with two different proteins. The NADPH-cytochrome c reductase transfers the electrons from NADPH to coenzyme Ql, (3) which then reduces a 4-ene-3-oxosteroid via the 5aL-reductase. The NADPH-cytochrome c reductase is able to use NADH for various hydroxylation reactions (19). The KM value for these hydroxylations are very high and range from 6.7 x 10m4M (20) or 1 x 10-3M (24) to 5 x 10-3M (19). In contrast, the KM value for the NADH-dependent 5d-reduction
of testosterone is much
lower (10s5M) (15). The NADH-concentration used in our experiments was 800 p
so that one should expect a 5cC-reduc-
tion of testosterone by means of NADH via NADPH-cytochrome c reductase and this reaction is not influenced by FSA.
A differentiation between NADH- and NADPH-5 d-reductase is possible by various means. Solubilization of both enzymes is achieved only by different systems (6), where the NADHdependent enzyme needs phosphatidylcholine micelles for reactivation (15). Different inhibitor characteristics phosphate dependency
(9,15),
(l), circadian rhythm of activity (16),
different inducibility by ethanol (15,17) point to different enzyme activities. There are, however, several similarities between both enzymes. Both activities are inhibited by gallic acid propylester
(2,15), a substance characterized as a spe-
cific inhibitor for NADPH-cytochrome c reductase
(18) from rat
liver microsomes. Both enzyme activities are suppressed by antibody (rabbit) against purified NADPH-cytochrome c reductase (4,15), and are inhibited by NADP+, where the inhibition is competitive in the case of NADH-5&-reductase
(15).
The presented results indicate that FSA is a useful tool in resolving whether microsomal NADPH-cytochrome c reductase is proton donor for the NADH-dependent 5a!-reductase or not. It was clearly shown that the corresponding active sites accepting NADH or NADPH for reduction of 4-ene-3-oxosteroids by rat liver microsomes are different. ACKNOWLEDGMENT This work was supported by Dsutsche Forschungsgemeinschaft. ABBREVIATION 5 CY-DHT: 5X-dihydrotestosterone 3-one).
(17!3-hydroxy-5oC-androstan-
SC
WEEOIDI
9
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9.
10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.
Leybold, K. and Staudinger, Hj., Arch. Biochem. Biophys. 96, 626-628 (1962,). Golf, S.W., Graef, V. and Staudinger, Hj., 2. Physiol. Chem. 355, 1499-1507 (1974). Graef,T and Golf, S.W., Z. Klin. Chem. Klin. Biochem. 2, 333-339 (1975). Golf, S.W. and Graef, V., J. Steroid Biochem. 2, 10871092 (1978). Clark, A.F., "Steroid Biochemistry", Chem. Rubber Comp. Cleveland, Ohio, USA, l-27 (1980). Graef, V., Golf, S.W. and Rempeters, G., J. Steroid Biochem. 2, 369-371 (1978). Wyatt, J.L. and Colman, R.F., Biochem. -16, 1333-1342 (1977). Esch, F.S. and Allison, W.S., J. Biol. Chem. 253, 61006106 (1978). Pal, K., Wechter, W.J. and Colman, R.F., J. Biol. Chem. 3, 8140-8147 (1975). Zoller, M.J. and Taylor, S.S., J. Biol. Chem. 254, 83638368 (1979). Sweet, F. and Samant, B., Steroids 36, 373-381 (1980). Golf, S.W., Graef, V. and Nowotny, E., Z. Physiol. Chem. 357, 35-40 (1976). Graef, V., Golf, S.W. and Tiischen, M., J. Steroid Biochem.14, 883-887 (1981). Gornan, A.G., Bardawill, C.J. and David, M.M., J. Biol. Chem. 177, 751-766 (1949). Rempet=, G., Dissertation, University Giessen, l-109 (1978). Graef, V. and Golf, S.W., J. Steroid Biochem. ll_, 12991303 (1979). Rubin, E. and Lieber, Ch. S., Science 191, 563-564 (1976). Torielli, N.F. and Slater, T.F., Biochem. Pharmacol. 20, 2027-2032 (1971). Prough, R.A. and Burke, M.D., Arch. Biochem. Biophys. 170, 160-168 (1975). Noshiro, M. and Omura, T., J. Biochem. 83, 61-77 (1978). Ichikawa, Y. and Yamano, T., J. Biochem. 66, 351-360 (1969).