Distribution of 3α-hydroxysteroid dehydrogenase in rat brain and molecular cloning of multiple cDNAs encoding structurally related proteins in humans

J. Steroid Biochem. Molec. Biol. Vol. 53, No. 1-6, pp. 41-46, 1995 Copyright © 1995 Elsevier Science Ltd 0960-0760(95)00019-4 Printed in Great Britain. All rights reserved 0960-0760/95 $9.50 + 0.00

Pergamon

Distribution of 3 -Hydroxysteroid D e h y d r o g e n a s e in Rat Brain and Molecular Cloning of Multiple cDNAs Encoding Structurally Related Proteins in H u m a n s Marilyn Khanna, Ke-Nan Qin and K-C. Cheng* Department of Pediatrics, Cornell University Medical College, New York, N Y 10021, U.S.A. 3 ~ - H y d r o x y s t e r o i d d e h y d r o g e n a s e in t he b r a i n is r e s p o n s i b l e f o r p r o d u c t i o n o f n e u r o a c t i v e t e t r a h y d r o s t e r o i d s t h a t i n t e r a c t with t h e m a j o r i n h i b i t o r y g a m m a - a m i n o b u t y r i c aci d r e c e p t o r c o m p l e x e s . D i s t r i b u t i o n o f 3 ~ - h y d r o x y s t e r o i d d e h y d r o g e n a s e in d i f f e r e n t r e g i o n s o f t he b r a i n in r a t s was e v a l u a t e d by a c t i v i t y assay a n d by W e s t e r n i m m u n o b l o t t i n g using a m o n o c l o n a l a n t i b o d y a g a i n s t l i v er 3 ~ - h y d r o x y s t e r o i d d e h y d r o g e n a s e as t h e p r o b e . T h e o l f a c t o r y b u l b was f o u n d to c o n t a i n th e h i g h e s t level o f 3 ~ - h y d r o x y s t e r o i d d e h y d r o g e n a s e act i vi t y, while m o d e r a t e levels o f t h e e n z y m e a c t i v i t y w e r e f o u n d in o t h e r r e g i o n s such as c e r e b e l l u m , c e r e b r a l c o r t e x , h y p o t h a l a m u s a n d p i t u i t a r y . S o m e a c t i v i t y was f o u n d in t h e r e s t o f t he b r a i n such as a m y g d a l a , b r a i n s t e m , c a u d a t e p u t a m e n , c i n g u l a t e c o r t e x , h i p p o c a m p u s , m i d b r a i n , a n d t h a l a m u s . T h e p r o t e i n levels o f 3~h y d r o x y s t e r o i d d e h y d r o g e n a s e in d i f f e r e n t r e g i o n s o f t he b r a i n as d e t e c t e d by W e s t e r n i m m u n o b l o t t i n g a r e c o m p a r a b l e to t hos e o f t he e n z y m e activity. We used t he r a t cDNA as t he p r o b e to s c r e e n a h u m a n liver 2 gt11 cDNA l i b r a r y . A t o t a l o f f o u r d i f f e r e n t cDNAs w e r e i d e n t i f i e d a n d s e q u e n c e d . O n e o f t h e cDNAs is i d e n t i c a l to t h a t o f t h e h u m a n c h l o r d e c o n e r e d u c t a s e cDNA e x c e p t t h a t o u r clone c o n t a i n s a m u c h l o n g e r 5'- c odi ng s e q u e n c e t h a n p r e v i o u s l y r e p o r t e d . T h e o t h e r t h r e e cDNAs d i s p l a y high d e g r e e s o f s e q u e n c e h o m o l o g y to t h ose o f b o t h r a t 3 ~ - h y d r o x y s t e r o i d d e h y d r o g e n a s e a n d h u m a n c h l o r d e c o n e r e d u c t a s e . We a r e c u r r e n t l y i n v e s t i g a t i n g t he f u n c t i o n a l r e l a t i o n s h i p b e t w e e n t h e e n z y m e s e n c o d e d by these h u m a n cDNAs a n d 3 ~ - h y d r o x y s t e r o i d d e h y d r o g e n a s e .

J. Steroid Biochem. Molec. Biol., Vol. 53, No. 1-6, pp. 41-46, 1995

INTRODUCTION

adrenocortical axis (HPA), and, finally, by exhaustion. Two hypothalamic peptides which appear to play an important role in stress-related HPA activation are the corticotropin-release factor (CRF) and vasopressin (AVP) [2]. T he secretion of both CRF and AVP to the portal system of the anterior pituitary during stress prompts an increase in the release of adrenocorticotropin (ACTH) into circulation [3, 4]. T he elevated A C T H levels stimulate the release of glucocorticoids and progestins from the adrenal gland [5]. As these steroids reach the brain, they are quickly converted to tetrahydrosteroids by sequential reactions involving two brain enzymes: steroid 5~-reductase and 3~t-hydroxysteroid dehydrogenase. T he production of the tetrahydrosteroid metabolites is rapid and robust, but transient, and may last only an hour or so [6]. Recent studies have demonstrated that the steroid hormone metabolites, both tetrahydrodeoxycorticosterone

3~-Hydroxysteroid dehydrogenase (3ct-HSD) was first identified by its activity in converting dihydrocortisone to 3~-tetrahydrocortisone. It was subsequently found to reduce dihydrotestosterone and dihydroprogesterone to their respective tetrahydro metabolites. In addition to its role in the metabolism of steroid hormones, 3~-HSD has been shown to metabolize a wide range of other endogenous substrates as well as xenobiotics. Stress, as defined by Selye [1], develops in three stages: an initial alarm reaction, characterized by an immediate sympathoadrenomedullary discharge, a subsequent "stage of resistance" characterized by the activation of the hypothalamic-pituitaryProceedings of the I X International Congress on Hormonal Steroids, Dallas, Texas, U.S.A., 24-29 September 1994. *Correspondence to K-C. Cheng. 41

Marilyn Khanna et al.

42

( T H D O C ) and tetrahydroprogesterone ( T H P ) , serve as agonist-ligands for the major inhibitory GABA A receptor complex [7, 8]. Interaction between these tetrahydrosteroids and the GABAA receptor complex induces an anxiolytic response that may protect the neurons from overstimulation and therefore preserve the homeostasis of the C N S [9]. Because the neuroactive tetrahydro-steroid metabolites induce anxiolytic, analgesic, anaesthetic, and anticonflict behavior in animals and humans, it has been postulated that the lack of these steroid metabolites may cause adverse effects in brain functions [10]. As 3~-hydroxysteroid dehydrogenase is the key brain enzyme involved in the production of neuroactive tetrahydro-steroid hormones, its distribution in brain may have significant implications with regard to the physiology and pathology of brain functions. In order to characterize the enzyme we have previously made several monoclonal antibodies and used these monoclonal antibodies as probes to isolate a full-length c D N A encoding the rat liver 3~-hydroxysteroid dehydrogenase and several human c D N A s encoding enzymes structurally related to the rat 3~H S D [11-14]. In this communication we studied the distribution of 3~-hydroxysteroid dehydrogenase by activity assays and by Western immunoblotting. We also present results regarding the isolation of several c D N A s encoding structurally related proteins in humans.

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MATERIALS AND METHODS

Preparation of cytosolic proteins Rats (200-250 g) were sacrificed by carbon dioxide euthanasia. Brains were removed and homogenized in a solution containing 0 . 2 5 M sucrose and 1 0 m M Tris-HC1, p H 7.2. T h e homogenate was centrifuged at 10,000g for 10 min. Supernatant containing cytosols and microsomes was recovered and subjected to a second centrifugation at 100,000g for 30min. T h e supernatant containing cytosolic proteins was recovered for protein assay as described by Lowry [15].

3~-hydroxysteroid dehydrogenase activity assays 3 ~ - H S D activities were measured by a radioactive procedure [16]. T h e reaction mixture contained 300/~g of brain cytosolic protein, 20 nmol of dihydrotestosterone, 20 nCi of 14C-5~-dihydrotestosterone in 900/~1 of 1 0 0 m M sodium phosphate buffer, p H 7 . 2 . T h e reaction mixture was preincubated at 37°C for 3 min prior to the addition of 100/~1 of 10 m M N A D P H to start the reaction. T h e reaction was allowed to continue for 10 min and was stopped by the addition of 5 ml methylene chloride. Dihydrotestosterone and androsterone were extracted into methylene chloride using three successive 5 ml aliquot of the solvent. T h e solvent was dried under a stream of nitrogen. T h e dry residue was dissolved in 100/~1 of methylene chloride and applied to a T L C plate. T h e plate was developed using

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cross-reactivity between the liver and the brain 3a-HSD. Two pg of the liver cytosols w e r e u s e d f o r t h e i m m u n o b l o t , w h e r e a s 300/~g o f t h e o l f a c t o r y b u l b a n d t h e w h o l e b r a i n c y t o s o l s w e r e u s e d . The positions of the protein size markers are shown on the left side of the blots.

Distribution of 3e-HSD in Rat Brain

Amygdala Brain stem Caudate putamen Cerebellum Cerebral cortex

43

prepared against 3 ~ - H S D [18]. Fusion proteins adsorbed to nitrocellulose filters were detected by sequential incubation with p r i m a r y monoclonal antibodies, goat anti-mouse IgG-peroxidase, and 4-chloronaphthol plus hydrogen peroxide. Positive plaques were replated at lower titers and rescreened with monoclonal antibodies until all plaques reacted positively. Phage were isolated by the plate lysate method [18], and D N A was extracted after treatment with phenol/chloroform.

Cingulate cortex

Library screening with cDNA probes under low stringency washing conditions

Liver

T h e library was plated out and lifts were taken on nylon m e m b r a n e s . Phage D N A attached to the m e m branes was denatured, neutralized, and cross-linked to the m e m b r a n e by U V light. Plaque hybridization was performed at 5 5 C in a solution containing 6 x SSC (1 x SSC = 0.15 M NaC1 and 0.015 M sodium citrate, p H 7 . 5 ) , 0.1°i, SDS, and 5 x D e n h a r d t ' s solution. Washing was p e r f o r m e d at 55°C in solution containing 2 x SSC and 0.1% SDS. Positive plaques were isolated and rescreened after dilution.

Hippocampus Hypothalamus M idbrain

Subcloning and sequence analysis

Olfactory bulb

Pituitary Thalamus /

Liver

Fig. 2. W e s t e r n i m m u n o b l o t t i n g of 3 ~ - H S D in d i f f e r e n t r e g i o n s o f t h e b r a i n u s i n g m o n o c l o n a l a n t i b o d y M A b 7D3. E a c h l a n e c o n t a i n s 300 Augo f t h e cytosolic p r o t e i n s . T h e p o s i t i o n s o f t h e p r o t e i n size m a r k e r s a r e s h o w n on t h e left side o f t h e blot.

a mixed solvent system of c h l o r o f o r m : e t h y l acetate : ethanol/4:1 : 0.6. Radioactive spots were localized by autoradiography.

Immunoblotting Proteins were subjected to electrophoresis in a 8°/; p o l y a c r y l a m i d e - 0 . 1 % S D S gel and then electrotransferred to a nitrocellulose filter [17]. T h e filter was sequentially treated with 3 % BSA in PBS, h y b r i d o m a supernatant and goat anti-mouse I g G - h o r s e r a d i s h peroxidase conjugate. T h e proteins which interacted with monoclonal antibody were visualized by incubating the filter with a solution containing 4-chloronaphthol and hydrogen peroxide.

Library screening using monoclonal antibody as the probe A h u m a n liver c D N A library in the expression vector Z g t l l was screened using monoclonal antibody 3G6

Phage D N A was digested with Eco RI, subjected to electrophoresis in a low-melting agarose gel and isolated by binding to glass beads. Recovered D N A fragments were subcloned into the Eco R I site of pUC19. Chain termination sequencing was performed on denatured supercoiled plasmid D N A using T 7 D N A polymerase [19]. RESULTS

Immuno-cross reactivity between the rat liver and brain enzyme Figure 1 shows the Western immunoblotting of the 3 ~ - H S D in liver as well as in brain using two m o n o clonal antibodies, M A b s 7D3 and 3G6, made against the liver enzyme. T h e s e two monoclonal antibodies have been shown to interact with the liver 3 ~ - H S D at different epitopes [12]. Both M A b s 7D3 and 3G6 recognized a single protein in the liver, the olfactory bulb and the brain. T h e molecular size of the protein in these different tissues appears to be the same. T h e intensities of the protein bands detected by M A b 3G6 are m u c h lighter than those detected by M A b 7D3, which is likely due to a lower binding affinity of M A b 3G6.

Distribution of 3ot-HSD in different regions of the brain T h e regional expression of the 3 ~ - H S D in the brain was first analyzed by Western immunoblotting using M A b 7D3 as the probe. As shown in Fig. 2, expression of 3ct-HSD protein was detectable in every part of the brain that was analyzed. Nonetheless, the levels of

Marilyn Khanna et al.

44

3 a - H S D protein as reflected by the intensities of the protein bands on the blots vary in different areas of the brain. W e have p e r f o r m e d at least five blots which showed reproducible band patterns. F o r example, the protein band in the olfactory bulb was the strongest. Other regions, such as cerebellum, cerebral cortex, hypothalamus, and pituitary, contained less but m o d e r ate amounts of the 3 a - H S D protein. M u c h less 3~H S D was found in amygdala, brain stem, caudate putamen, cingulate cortex, hippocampus, midbrain and thalamus. A similar pattern of 3 a - H S D activities was also seen in various regions of the brain. T h e brain tissues from four animals were used for the activity assays. As shown in Fig. 3, the olfactory bulb also contains the highest level of 3 ~ - H S D activity. Moderate levels of 3 a - H S D activity were also found in cerebellum, cerebral cortex, hypothalamus, and pituitary. T h i s suggests that the differences in activities of 3 a - H S D in various regions of the brain are due to different levels of the 3 a - H S D enzyme.

Isolation of multiple human c D N A clones As reported previously, a monoclonal antibody ( M A b 3G6) raised against rat liver 3 a - H S D also crossreacted with a h u m a n protein of molecular weight similar to that of the rat enzyme [12]. Therefore, in our initial screening of the h u m a n c D N A library we used this monoclonal antibody as the probe. F r o m approx.

1 million phages we isolated 50 positive clones. Sequencing and comparison of the c D N A inserts in these positive clones indicated that we had isolated four distinct cDNAs. Subsequent use of these c D N A s as probes to re-screen the c D N A library produced several longer clones that contained three full-length and one near full-length cDNAs. Sequence comparison reveals a high degree of similarity (approx. 70%) between the h u m a n c D N A s and the rat 3 a - H S D . Search of the G e n e b a n k revealed that one of the h u m a n c D N A s ( H A K R a ) isolated in this study had the same D N A sequence as h u m a n chlordecone reductase [20] except the H A K R a contains a m u c h longer 5'-coding sequence than that of h u m a n chlordecone reductase. T h e start codons that were assigned are located within the potential eucaryotic translation initiation consensus sequences [21]. T h e same start codon was also shown to be used by several other rat enzymes belonging to the aldo-keto reductase superfamily [13]. Because these h u m a n c D N A s resemble both 3 a - H S D and h u m a n chlordecone reductase, and both of them belong to the rat aldo-keto reductase superfamily, we tentatively named them h u m a n aldo-keto reductase ( H A K R ) . In contrast to the 3 a - H S D c D N A , the h u m a n c D N A s contain m u c h shorter 3'-non-coding sequences. In two of the c D N A s , H A K R a and H A K R b , a polyadenylation signal ( A A T A A A ) was found, and in H A K R d a polyadenylation tail was located.

amygdala brain stem caudate putamen cerebellum cerebral cortex

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pmoles/mg/min Fig. 3. 3a-HSD activities in v a r i o u s r e g i o n s of the rat brain. A s s a y o f 3=-HSD activity is d e s c r i b e d in the M a t e r i a l s and M e t h o d s .

Distribution of 3~-HSD in Rat Brain

3a-HSD HAKRa HAKRb HAKRc HAKRd

1 MDSISLRDALNDGNFIPVLGFGTTVPEKVAKDEFIKATKIAIDNGFFHFDSAYLYELEEDVGQSI M PKYQ VE H M YA PE PRNRAVEV L EA R I NN Q M PKHKCVK H M YA PE PRSKALEV L EA R I H NN Q AKYQCVK H M YA AE PKSKALEA L EA R I H NN Q M SKYQCVK H M YA AE PKSKALEAV L EA H I HV NN Q

3g-HSD HAKRa HAKRb HAKRc HAKRd

66 RSKIEGKSGKREDIFYTSKLWSTFHRPELVRPCLEKTLEKHQQHYVDLYIIHFPMALQPGDIFFP ADG V C FQ QM Q A SS K L LD LL K ADG VE HR EL R A NS K A LD L S S K ADG V CNSHR ELDR A RS KNL LD LI F V S V K ADG V NSHR EL A ERS KNL LD LI F V S V K

3 ~-HS D HAKRa HAKRb HAKRc HAKRd

131 RDEHGKLLFETVDI K N VI D T N VI DI K N IL D K N ILD

3~-HSD HAKRa HAKRb HAKRc HAKRd

196 YLNQSKMLDYCKSKDIILVSYCTLGSSRGKTWVDQKSPVLLDDPVLCAIAKKYKQTPALVALRYQ L F V AHSA TQ H L PN E L H R I FR L F V ASA QDR PN E L HR I F R L F V A SA L EEP PN E L H R I F R L F V A SA H EEP PN E L H R I

3 ~-HSD HAKRa HAKRb HAKRc HAKRd

261 LQSGVEPLIRSFKPKRI R VV AK YNEQ R VV AK YNEQ R VV AK YNEQ R VV AK YNEQ

CDTWEAMEKCKDAGLAS LSA V L T L A V E LA

45 65 LA LA LA LA 130 ETPL EELS EEVI EEVI

195 S IGGSNFNCRRLERI LNKPGLKYKPVCNQVECHL K V Q M P K V R Q I P K V RST M QV P K V H L M E E P

322 KEPTQVFEFQLASEDMKALDGLNRNFRYNNAKS FHDHPNHPFTDE R NI T V Y VVMDFLM DY S RQNV TA AI D LH F SD AS Y YS RQNV T E AI V LTLDI AGP Y IS RQNV T E AI V LTLDI AGP Y IS

260

• Y. Y. Y. Y.

Fig. 4. Comparison of deduced a m i n o acid sequences of 3~t-HSD, HARKa, HARKb, HARKc, and HARKd. Only differences from the 3~-HSD sequence are displayed. Boldface one-letter codes denote the b e g i n n i n g of the sequences derived from the cDNA sequences. The amino acid residues are numbered in boldface letters in the amino terminus to craboxyl terminus direction. Figure 4 shows the alignment of the deduced sequences of the rat 3 e - H S D and the h u m a n proteins. T h e 3 ~ - H S D c D N A encodes a protein of 322 amino acids, whereas the open reading frames of the h u m a n c D N A s encode proteins of 323 amino acids. A high degree of sequence homology (about 6 5 % ) was found between the rat 3 ~ - H S D and each of the h u m a n proteins. A m u c h greater degree of homology ( > 8 5 % ) was found a m o n g the h u m a n proteins. O f the four h u m a n proteins, H A K R c and H A K R d showed the highest degree of homology (95%).

DISCUSSION

Over the last 50 years since Selye demonstrated that 3~-tetrahydrosteroids induced anxiolytic, analgesic and anticonvulsive effects in animals [10], the m e c h a n i s m by which these neuroactive steroids interact with neurons at molecular levels has been well delineated. As demonstrated by competitive ligand binding, these 3~-tetrahydrosteroids interact with the major inhibitory GABAA receptor complex near the binding site for SBMB 53, I ~ I )

barbiturates [22]. U p o n binding to the G A B A A receptor, tetrahydrosteroids induce a prolonged opening of the chloride channel leading to the transport of chloride ion and hyperpolarization of the neurons. As a result of the modulating function of 3~-tetrahydrosteroids on G A B A A receptor complex, it has been speculated that some psychiatric disorders, such as depression, premenstrual syndrome, and anxiety, m a y be due to abnormality in the metabolism of neuroactive steroid metabolites [17, 18]. Since 3ct-hydroxysteroid dehydrogenase is one of the key enzymes responsible for the production of the neuroactive tetrahydrosteroids, its expression and regulation may have direct implication to various functions of the tetrahydrosteroids in the brain. It is rather interesting that olfactory bulb contains a m u c h higher concentration of the 3 ~ - H S D than any other area of the rat brain. Since G A B A A receptors in the olfactory bulb is involved in the odor processing, it is possible that the neuroactive steroid metabolites may play a role in the odor coding pathway [23]. Deprivation of steroid hormones by adrenalectomy did not affect the levels of 3 e - H S D in the brain; neither did administration of various steroid hormones affect

46

Marilyn Khanna et al.

the e n z y m e activity in the brain. N o sex difference was found in the brain 3 a - H S D activity. In contrast, the liver 3 ~ - H S D has been s h o w n to exhibit sexual dimorphism. T h e s e results suggest that the regulation of brain enzyme, which is steroid hormone-independent, is different from that of the liver enzyme. Several groups of researchers have suggested the existence of multiple 3 ~ - H S D in the liver [24,25]. Penning has suggested that the brain e n z y m e is m u c h less active than the liver e n z y m e [26]. H o w e v e r , in this study we found that the liver and the brain e n z y m e are immunologically identical. U s i n g m o n o c l o n a l antibodies made against the liver e n z y m e to screen a 2 gtl 1 c D N A library derived from the rat brain we have isolated a c D N A clone that showed identical sequence to that of the liver e n z y m e (data not shown). Our studies, therefore, suggest the e n z y m e s expressed in the liver and the brain are identical. Multiple h u m a n 3ct-HSD have been purified from the cytosolic fraction of h u m a n liver. Since a m o n o clonal antibody previously raised against rat 3 ~ - H S D in our laboratory recognizes a h u m a n protein with a molecular weight similar to the rat enzyme, we used it as the probe to isolate c D N A s in a h u m a n liver library. As s h o w n in this study, h u m a n liver expresses multiple forms of e n z y m e s structurally related to rat 3 ~ - H S D . Our recent studies suggest that two of the proteins encoded by the c D N A s we isolated have 3 a - H S D activity.

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7. Kraulis I., Foldes G., Traikov H., Dubrosky B. and Birmingham M. K.: Distribution, metabolism and biological activity of deoxycorticosterone in the ventral nervous system. Brain Res. 88 (1975) 1-14. 8. Mendelson W. B., Martin J. V., Perlis M., Wagner R., Majewska M. D. and Paul S. M.: Sleep induction by adrenal steroid in the rat. Psychopharmacology 93 (1987) 226-229. 9. Majewska M. D.: Neurosteroids: endogenous bimodal modulators of the GABA A receptor. Mechanism of action and physiological significance. Prog. Neurobiol. 38 (1992) 379-395. 10. Selye H.: Correlation between the chemical structure and the pharmacological actions of the steroids. Endocrinology 30 (1992) 437-452. 11. Cheng K-C., White P. C. and Qin K.: Molecular cloning and expression of rat 3~-hydroxysteroid dehydrogenase. Molec. Endocr. 5 (1991) 823-828. 12. Cheng K-C.: Detection of multiple antigenetically related enzymes from various rat tissues by monoclonal antibodies against 3~-hydroxysteroid dehydrogenase. Arch. Biochem. Biophys. 291 (1991) 258-262. 13. Cheng K-C.: Molecular cloning of rat liver 3~-hydroxysteroid dehydrogenase and related enzymes from rat liver, kidney and lung. J. Steroid Biochem. Molec. Biol. 43 (1992) 1083-1088. 14. Qin K-N., New M. I. and Cheng K-C.: Molecular cloning of multiple cDNAs encoding human enzymes structurally related to 3~-hydroxysteroid dehydrogenase. J. Steroid Biochem. Molec. Biol. 46 (1994) 673-679. 15. Lowry O. H., Rosebrough N. J., Farr A. L. and Randall R. J.: Protein measurements with the folin-phenol reagent. J. Biol. Chem. 193 (1951) 265-275. 16. Cheng K-C. and Schenkam J. B.: Testosterone metabolism by cytochrome P-450 isozymes RLM3 and RLM5 and by microsomes. J. Biol. Chem. 258 (1983) 11,738-11,744. 17. Young R. A. and Davis R. W.: Efficient isolation of genes by using antibody probes. Proc. Nam. Acad. Sci. U.S.A. 80 (1983) 1194-1198.

18. Snyder M., Elledge S., Sweetser D., Young R. A. and Davis R . W.: Lambda gt 11: gene isolation with antibody probes and other applications. Meth. Enzymol. 154 (1987) 107-128. 19. Chen E. Y. and Seeburg P. H.: Supercoil sequencing: a fast and simple method for sequencing plasmid DNA. D N A 4 (1985) 165-170. 20. Winters C. J., Molowa D. T. and Guzelian P. S.: Isolation and characterization of cloned cDNAs encoding human liver chlordecone reductase. Biochemistry 29 (1990) 1080-1087. 21. Kozak M.: The scanning model for translation: an update. J. Cell. Biol. 108 (1989) 229-241. 22. Majewska M. D., Harrison N. L., Schwartz R. D., Barker J. L. and Paul S. M.: Steroid hormone metabolites are barbiturate-like modulators of the GABA receptor. Science 232 (1986) 1004-1007. 23. Duchamp-Viret P., Duchamp A. and Chaput M.: GABAnergic control of odor-induced activity in the frog olfactory bulb: electrophysiological study with picrotoxin and bicuculline. Neuroscience 53 (1993) 111-120. 24. Smithgall T. E. and Penning T. M.: Electrophoretic and immunochemical characterization of 3~-hydroxysteroid dihydrodiol dehydrogenase of rat tissues. Biochem. J. 254 (1988) 715-721. 25. Boutin J. A.: Camphoroquinone reduction: another reaction catalyzed by rat liver 3~-hydroxysteroid dehydrogenase. Biochim. Biophys. Acta 870 (1986) 463-472. 26. Penning T. M., Sharp R. B. and Krieger N. R.: Purification and properties of 3~-hydroxysteroid dehydrogenase from rat brain cytosol. J. Biol. Chem. 260 (1985) 15,266-15,272.