Testicular and adrenal 3 beta-hydroxy-5-ene-steroid dehydrogenase and 5-ene-4-ene isomerase

The purified multifunctional enzyme, 3 beta-hydroxysteroid dehydrogenase with steroid 5-ene-4-ene isomerase from rat testes and adrenals showed similar catalytic properties. They exhibited the same molecular weight of 46,500. Either NAD+ or NADH was required for steroid isomerizing activity, probably as an allosteric effector. It was clearly demonstrated by using the purified enzyme that without NAD(H) no isomerizing activity was detected. In the presence of NADH, or its analogue, 3 beta-hydroxysteroid dehydrogenase obtained from both tissues was inhibited; however, steroid isomerizing activity remained due to the allosteric effect. The results suggest that in these endocrine organs, both enzyme activities reside within the same protein.     

Evidence for distinct dehydrogenase and isomerase sites within a single 3 beta-hydroxysteroid dehydrogenase/5-ene-4-ene isomerase protein.

Complementary DNA encoding human 3 beta-hydroxysteroid dehydrogenase/5-ene-4-ene isomerase (3 beta-HSD) has been expressed in transfected GH4C1 with use of the cytomegalovirus promoter. The activity of the expressed protein clearly shows that both dehydrogenase and isomerase enzymatic activities are present within a single protein. However, such findings do not indicate whether the two activities reside within one or two closely related catalytic sites. With use of [3H]-5-androstenedione, the intermediate compound in dehydroepiandrosterone (DHEA) transformation into 4-androstenedione by 3 beta-HSD, the present study shows that 4MA (N,N-diethyl-4-methyl-3-oxo-4-aza-5 alpha-androstane-17 beta-carboxamide) and its analogues inhibit DHEA oxidation competitively while they exert a noncompetitive inhibition of the isomerization of 5-androstenedione to 4-androstenedione with an approximately 1000-fold higher Ki value. The present results thus strongly suggest that dehydrogenase and isomerase activities are present at separate sites on the 3 beta-HSD protein. In addition, using 5 alpha-dihydrotestosterone (DHT) and 5 alpha-androstane-3 beta, 17 beta-diol as substrates for dehydrogenase activity only, we have found that dehydrogenase activity is reversibly and competitively inhibited by 4MA. Such data suggest that the irreversible step in the transformation of DHEA to 4-androstenedione is due to a separate site possessing isomerase activity that converts the 5-ene-3-keto to a much more stable 4-ene-3-keto configuration.     

Affinity alkylation of human placental 3 beta-hydroxy-5-ene-steroid dehydrogenase and steroid 5----4-ene-isomerase by 2 alpha-bromoacetoxyprogesterone: evidence for separate dehydrogenase and isomerase sites on one protein

We have copurified human placental 3 beta-hydroxy-5-ene-steroid dehydrogenase and steroid 5----4-ene-isomerase, which synthesize progesterone from pregnenolone and androstenedione from fetal dehydroepiandrosterone sulfate, from microsomes as a homogeneous protein based on electrophoretic and NH2-terminal sequencing data. The affinity alkylator, 2 alpha-bromoacetoxyprogesterone, simultaneously inactivates the pregnene and androstene dehydrogenase activities as well as the C21 and C19 isomerase activities in a time-dependent, irreversible manner following first order kinetics. At four concentrations (50/1-20/1 steroid/enzyme M ratios), the alkylator inactivates the dehydrogenase activity (t1/2 = 1.5-3.7 min) 2-fold faster than the isomerase activity. Pregnenolone and dehydroepiandrosterone protect the dehydrogenase activity, while 5-pregnene-3,20-dione, progesterone, and androstenedione protect isomerase activity from inactivation. The protection studies and competitive kinetics of inhibition demonstrate that the affinity alkylator is active site-directed. Kitz and Wilson analyses show that 2 alpha-bromoacetoxyprogesterone inactivates the dehydrogenase activity by a bimolecular mechanism (k3' = 160.9 l/mol.s), while the alkylator inactivates isomerase by a unimolecular mechanism (Ki = 0.14 mM, k3 = 0.013 s-1). Pregnenolone completely protects the dehydrogenase activity but does not slow the rate of isomerase inactivation by 2 alpha-bromoacetoxyprogesterone at all. NADH completely protects both activities from inactivation by the alkylator, while NAD+ protects neither. From Dixon analysis, NADH competitively inhibits NAD+ reduction by dehydrogenase activity. Mixed cofactor studies show that isomerase binds NAD+ and NADH at a common site. Therefore, NADH must not protect either activity by simply binding at the cofactor site. We postulate that NADH binding as an allosteric activator of isomerase protects both the dehydrogenase and isomerase activities from affinity alkylation by inducing a conformational change in the enzyme protein. The human placental enzyme appears to express the pregnene and androstene dehydrogenase activities at one site and the C21 and C19 isomerase activities at a second site on the same protein.     

Molecular cloning, cDNA structure and predicted amino acid sequence of bovine 3 beta-hydroxy-5-ene steroid dehydrogenase/delta 5-delta 4 isomerase

We have used our recently characterized human 3 beta-hydroxy-5-ene steroid dehydrogenase/delta 5-delta 4-isomerase (3 beta-HSD) cDNA as probe to isolate cDNAs encoding bovine 3 beta-HSD from a bovine ovary lambda gtll cDNA library. Nucleotide sequence analysis of two overlapping cDNA clones of 1362 bp and 1536 bp in length predicts a protein of 372 amino acids with a calculated molecular mass of 42,093 (excluding the first Met). The deduced amino acid sequence of bovine 3 beta-HSD displays 79% homology with human 3 beta-HSD while the nucleotide sequence of the coding region shares 82% interspecies similarity. Hybridization of cloned cDNAs to bovine ovary poly(A)+ RNA shows the presence of an approximately 1.7 kb mRNA species.     

3beta-Hydroxysteroid dehydrogenase/5-ene-4-ene isomerase mRNA expression in rat brain: effect of pseudopregnancy and traumatic brain injury

Evidence that endogenous progesterone (PROG) is neuroprotective after traumatic brain injury (TBI) is supported by the findings that pseudopregnant female rats present less edema and achieve better functional recovery than do male rats. PROG in the nervous system may originate from steroidogenic glands or can be locally synthesized. 3β-Hydroxysteroid dehydrogenase/5-ene-4-ene isomerase (3β-HSD) is the key enzyme in the biosynthesis of PROG. In the present study, we investigated the effects of pseudopregnancy and TBI on brain 3β-HSD mRNA expression and on PROG levels. Twenty-four hours after bilateral contusion of the medial prefrontal cortex of rats, 3β-HSD mRNA expression was analyzed by in situ hybridization while PROG levels were measured by gas chromatography/mass spectrometry. Similar levels of 3β-HSD mRNA expression were observed in males and pseudopregnant females in the non-injured groups. At this time point, there was a significant decrease in the 3β-HSD mRNA expression in the contusion site within the frontal cortex in both males and pseudopregnant females. In all other regions analyzed, 3β-HSD mRNA expression was not affected by TBI and there was no difference between males and pseudopregnant females. The high decrease in the expression of the 3β-HSD mRNA in the lesion site 24 h after TBI suggests a possible decrease in locally synthesized PROG in lesion site without change in the other brain regions. This decrease has less impact in pseudopregnant females since they have high plasmatic and brain levels of PROG compared to males.     

Structure and tissue-specific expression of 3ß-hydroxysteroid dehydrogenase/5-ene-4-ene isomerase genes in human and rat classical and peripheral steroidogenic tissues

The enzyme 3ß-hydroxysteroid dehydrogenase/5-ene-4-ene isomerase (3ß-HSD) catalyzes the oxidation and isomerization of 5-ene-3ß-hydroxypregnene and 5-ene-hydroxyandrostene steroid precursors into the corresponding 4-ene-ketosteroids necessary for the formation of all classes of steroid hormones. We have recently characterized two types of human 3ß-HSD cDNA clones and the corresponding genes which encode deduced proteins of 371 and 372 amino acids, respectively, and share 93.5% homology. The human 3ß-HSD genes containing 4 exons were assigned by in situ hybridization to the p11–p13 region of the short arm of chromosome 1. We have also recently elucidated the structure of three types of rat 3ß-HSD cDNAs as well as that of one type of 3ß-HSD from bovine and macaque ovary λgt11 cDNA libraries which all encode 372 amino acid proteins. The human type I 3ß-HSD is the almost exclusive mRNA species detected in the placenta and skin, while the human type II is the predominant mRNA species in the adrenals, ovaries and testes. The predicted rat type I and type II 3ß-HSD proteins expressed in adrenals, gonads and adipose tissue share 94% homology while they share 80% similarity with the liver-specific type III 3ß-HSD. Transient expression of human type I and type II as well as rat type I and type II 3ß-HSD cDNAs in Hela human cervical carcinoma cells reveals that 3ß-ol dehydrogenase and 5-ene-4-ene isomerase activities reside within a single protein and these cDNAs encode functional 3ß-HSD proteins that are capable of converting 3ß-hydroxy-5-ene-steroids into 3-keto-4-ene derivatives as well as the interconversion of 3ß-hydroxy and 3-keto-5-androstane steroids. We have found that the rat type III mRNA species was below the detection limit in intact female liver while, following hypophysectomy, its accumulation increased to 55% of the levels measured in intact or HYPOX male rats, an increase which can be blocked by administration of ovine prolactin (oPRL). In addition, in female rats, treatment with oPRL for 10 days starting 15 days after HYPOX, markedly decreased ovarian 3ß-HSD mRNA accumulation accompanied by a similar decrease in 3ß-HSD activity and protein levels. Treatment with the gonadotropin hCG reversed the potent inhibitory effect of oPRL on these parameters and stimulated 3ß-HSD mRNA levels in ovarian interstitial cells. In intact females, hCG exerted marked trophic effects on rat corpora lutea with an increase in total ovarian 3ß-HSD expression and activity. We have also shown that treatment with hCG for 15 days in intact male rats caused a marked increase in testicular 3ß-HSD expression and activity while glucocorticoids exerted inhibitory effects on these parameters. We have also observed that the ontogeny of 3ß-HSD expression in human and rat adrenal gland, testis and ovary is closely correlated with steroid hormone biosynthesis, thus suggesting that regulation of the expression of 3ß-HSD is a limiting step in the biosynthesis of steroids in these tissues.     

Purification and characterization of rat adrenal 3 beta-hydroxysteroid dehydrogenase with steroid 5-ene-4-ene-isomerase

After solubilization of rat adrenal microsomes with sodium cholate, 3 beta-hydroxysteroid dehydrogenase with steroid 5-ene-4-ene isomerase (abbreviated as steroid isomerase) activity was purified to a homogeneous state. The following characteristics of the enzyme were obtained: 3 beta-Hydroxysteroid dehydrogenase together with steroid isomerase was detected as a single protein band in SDS-polyacrylamide gel electrophoresis, where its mol. wt was estimated as 46,500. Either NAD+ or NADH was required for demonstration of steroid isomerase activity. Treatment of the enzyme with 5'-p-fluorosulfonylbenzoyladenosine, an affinity labeling reagent for NAD+-dependent enzyme, diminished both the enzyme activities.     

Human placental 3 beta-hydroxy-5-ene-steroid dehydrogenase and steroid 5----4-ene-isomerase: purification from microsomes, substrate kinetics, and inhibition by product steroids

In human pregnancy, placental 3 beta-hydroxy-5-ene-steroid dehydrogenase and steroid 5----4-ene-isomerase produce progesterone from pregnenolone and metabolize fetal dehydroepiandrosterone sulfate to androstenedione, an estrogen precursor. The enzyme complex was solubilized from human placental microsomes using the anionic detergent, sodium cholate. Purification (500-fold, 3.9% yield) was achieved by ion exchange chromatography (Fractogel-TSK DEAE 650-S) followed by hydroxylapatite chromatography (Bio-Gel HT). The purified enzyme was detected as a single protein band in sodium dodecylsulfate-polyacrylamide gel electrophoresis (monomeric Mr = 19,000). Fractionation by gel filtration chromatography at constant specific enzyme activity supported enzyme homogeneity and determined the molecular mass (Mr = 76,000). The dehydrogenase and isomerase activities copurified. Kinetic constants were determined at pH 7.4, 37 degrees C for the oxidation of pregnenolone (Km = 1.9 microM, Vmax = 32.6 nmol/min/mg) and dehydroepiandrosterone (Km = 2.8 microM, Vmax = 32.0 nmol/min/mg) and for the isomerization of 5-pregnene-3,20-dione (Km = 9.7 microM, Vmax = 618.3 nmol/min/mg) and 5-androstene-3,17-dione (Km = 23.7 microM, Vmax = 625.7 nmol/min/mg). Mixed substrate analyses showed that the dehydrogenase and isomerase reactions use the appropriate pregnene and androstene steroids as alternative, competitive substrates. Dixon analyses demonstrated competitive inhibition of the oxidation of pregnenolone and dehydroepiandrosterone by both product steroids, progesterone and androstenedione. The enzyme has a 3-fold higher affinity for androstenedione than for progesterone as an inhibitor of dehydrogenase activity. Based on these competitive patterns of substrate utilization and product inhibition, the pregnene and androstene activities of 3 beta-hydroxy-5-ene-steroid dehydrogenase and steroid 5----4-ene-isomerase may be expressed at a single catalytic site on one protein in human placenta.     

Full length cDNA structure and deduced amino acid sequence of human 3 beta-hydroxy-5-ene steroid dehydrogenase

Polyclonal antibodies raised against 3 beta-hydroxysteroid dehydrogenase isolated from human placenta were used to screen a lambda gt11 expression cDNA library from the same tissue. The protein deduced from cDNA sequences contains 372 amino acids with a calculated mol wt of 42,216. Since 3 beta-hydroxysteroid dehydrogenase is the enzyme catalyzing the formation of all classes of hormonal steroids, the availability of the cDNA encoding this enzyme opens new possibilities for a detailed investigation of the factors regulating the expression and activity of this crucial enzyme in adrenal, gonadal as well as peripheral tissues.     

Human placental 3 beta-hydroxy-5-ene-steroid dehydrogenase and steroid 5----4-ene-isomerase: purification from mitochondria and kinetic profiles, biophysical characterization of the purified mitochondrial and microsomal enzymes

In human placenta, 3 beta-hydroxy-5-ene-steroid dehydrogenase and steroid 5----4-ene-isomerase, an enzyme complex found in microsomes and mitochondria, synthesizes progesterone from pregnenolone and androstenedione from fetal dehydroepiandrosterone sulfate. The dehydrogenase and isomerase activities of the mitochondrial enzyme were copurified (733-fold) using sequential cholate solubilization, ion exchange chromatography (DEAE-Toyopearl 650S), and hydroxylapatite chromatography (Bio-Gel HT). Enzyme homogeneity was demonstrated by a single protein band in SDS-polyacrylamide gel electrophoresis (monomeric Mr = 41,000), gel filtration at constant specific enzyme activity (Mr = 77,000), and a single NH2-terminal sequence. Kinetic constants were determined for the oxidation of pregnenolone (Km = 1.6 microM, Vmax = 48.6 nmol/min/mg) and dehydroepiandrosterone (Km = 2.4 microM, Vmax = 48.5 nmol/min/mg) and for the isomerization of 5-pregnene-3,20-dione (Km = 9.3 microM, Vmax = 914.2 nmol/min/mg) and 5-androstene-3,17-dione (Km = 27.6 microM, Vmax = 888.4 nmol/min/mg. Mixed substrate studies showed that the dehydrogenase and isomerase activities utilize their respective pregnene and androstene substrates competitively. Dixon analysis demonstrated that the product steroids, progesterone and androstenedione, are competitive inhibitors of the C-21 and C-19 dehydrogenase activities. Enzyme purified from mitochondria and microsomes had similar kinetic profiles with respect to substrate utilization, product inhibition, and cofactor (NAD+) reduction (mean Km +/- SD using C-19 and C-21 dehydrogenase substrates = 26.4 +/- 0.8 microM, mean Vmax = 73.2 +/- 1.3 nmol/min/mg). Pure enzyme from both organelles exhibited identical biophysical properties in terms of molecular weight and subunit composition, pH optima (pH 9.8, dehydrogenase; pH 7.5, isomerase), temperature optimum (37 degrees C), stability in storage and solution, effects of divalent cations, and the single NH2-terminal sequence of 27 amino acids. These results suggest that the mitochondrial and microsomal enzymes are the same protein localized in different organelles.     

Molecular cloning and expression of human trophoblast antigen FDO161G and its identification as 3 beta-hydroxy-5-ene steroid dehydrogenase

The monoclonal antibody FDO161G reacts with a 43-kDa protein found in human extravillous trophoblast, syncytiotrophoblast, adrenal cortex, interstitial cells of the testis and ovarian follicle cumulus cells. cDNAs for this protein have been isolated from the lambda gt11 library, sequenced, and expressed in COS-7 cells. The protein was identified as 3 beta-hydroxy-5-ene steroid dehydrogenase (HSD). The sequence of the HSD protein raises questions about its association with cell membrane systems. The lack of reactivity of FDO161G with other tissues suggests that HSD has a limited tissue distribution and that other enzymes may exist in peripheral tissues, which can convert delta 5 3-hydroxysteroids to delta 4 3-ketosteroids.     

Bovine adrenal 3beta-hydroxysteroid dehydrogenase (E.C. 1.1.1. 145)/5-ene-4-ene isomerase (E.C. 5.3.3.1): characterization and its inhibition by isoflavones

The isoflavones daidzein, genistein, biochanin A and formononetin inhibit potently and preferentially the γ-isozymes of mammalian alcohol dehydrogenase (γγ-ADH), the only ADH isozyme that catalyzes the oxidation of 3β-hydroxysteroids. Based on these results, we proposed that these isoflavones might also act on other enzymes involved in 3β-hydroxysteroid metabolism. Recently, we showed that they indeed are potent inhibitors of a bacterial β-hydroxysteroid dehydrogenase (β-HSD). To extend this finding to the mammalian systems, we hereby purified, characterized and studied the effects of isoflavones and structurally related compounds on, a bovine adrenal 3β-hydroxysteroid dehydrogenase (3β-HSD). This enzyme catalyzes the oxidation of 3β-hydroxysteroids but not 3-, 11β- or 17β-hydroxysteroids. The same enzyme also catalyzes 5-ene-4-ene isomerization, converting 5-pregnen 3, 20-dione to progesterone. The K_m values of its dehydrogenase activity determined for a list of 3β-hydroxysteroid substrates are similar (1 to 2 μM) and that of its isomerase activity, determined with 5-pregnen 3, 20-dione as a substrate, is 10 μM. The k_cat value determined for its isomerase activity (18.2 min⁻¹) is also higher than that for its dehydrogenase activity (1.4–2.4 min⁻¹). A survey of more than 30 isoflavones and structurally related compounds revealed that daidzein, genistein, biochanin A and formononetin inhibit both the dehydrogenase and isomerase activity of this enzyme. Inhibition is potent and concentration dependent. IC₅₀ values determined for these compounds range from 0.4 to 11 μM, within the plasma and urine concentration ranges of daidzein and genistein of individuals on vegetarian diet or semi-vegetarian diet. These results suggest that dietary isoflavones may exert their biological effects by inhibiting the action of 3β-HSD, a key enzyme of neurosteroid and/or steroid hormone biosynthesis.     

Differential inhibition of dehydrogenase and 5-ene→4-ene isomerase activities of purified 3β-hydroxysteroid dehydrogenase. Evidence for two distinct sites

The success in synthesis of [³H]5-androstene-3,17-dione, the intermediate product in the transformation of DHEA to 4-androstenedione by 3β-hydroxysteroid dehydrogenase/ 5-ene→4-ene isomerase (3β-HSD) offers the opportunity to determine whether or not the two activities reside in one active site or in two closely related active sites. The finding that N,N-dimethyl-4-methyl-3-oxo-4-aza-5-androstane-17β-carboxamide (4-MA) inhibits competitively and specifically the dehydrogenase activity whereas a non-competitive inhibition type with a K_i value 1000 fold higher was observed for the isomerase activity, indicated that dehydrogenase and isomerase activities belong to separate sites. Using 5-dihydro-testosterone and 5-androstane-3β,17β-diol, exclusive substrates for dehydrogenase activity, it was shown that dehydrogenase is reversible and strongly inhibited by 4-MA and that thus the irreversible step in the transformation of DHEA to 4-androstenedione is due to the isomerase activity.     

Conversion of androstenedione to 3 beta-hydroxy-5-androsten-17-one and 3 beta-hydroxy-4-androsten-17-one by the testicular microsomal fraction of Sprague-Dawley rats

When androstenedione was incubated with testicular microsomes of Sprague-Dawley rats in the presence of reduced nicotinamide-adenine dinucleotide (NADH), unknown metabolites were produced, in addition to testosterone and 7 alpha-hydroxyandrostenedione. The metabolites were identified as 3 beta-hydroxy-4-androsten-17-one and 3 beta-hydroxy-5-androsten-17-one (3:1) by biochemical and radiochemical methods. These results confirmed the occurrence of the reverse reactions from androstenedione to 3 beta-hydroxy-4-androsten-17-one and 3 beta-hydroxy-5-androsten-17-one catalyzed by the 3 beta-hydroxysteroid dehydrogenase and 5-ene-4-ene isomerase in the microsomal fraction of Sprague-Dawley rat testes.     

Synthesis of 19-hydroxyaldosterone and the 3 beta-hydroxy-5-ene analog of aldosterone, active mineralocorticoids

19-Hydroxyaldosterone (20) and the 3 beta-hydroxy-5-ene analog of aldosterone (HAA) (8) were synthesized from 21-acetoxy-4-pregnene-3,20-dion-20-ethylene ketal-18, 11 beta-lactone (2) as follows: the double bond was transposed from the 4,5 to the 5,6-position by enol acetylation to 3, followed by sodium borohydride reduction. Further reduction of the resulting lactone 4a with diisobutylaluminum hydride (DIBAH) furnished the 20-ketal of HAA 6, from which free HAA (8) and the 18,21-anhydro compound 7 were obtained by acid treatment. The [1H]NMR spectrum of 8 in CDCl3 showed it to be a mixture of two isomeric forms. Correlation with the known aldosterone-gamma-etiolactone (10) was established by periodate oxidation of HAA to the corresponding etiolactone 9 followed by chromic acid oxidation. The preparation of 20 was next effected in the following manner: the diacetate 4b was converted into the 6 beta, 19-oxido compound 13b by addition of hypobromous acid followed by the hypoiodite reaction of the bromohydrin 11. Mild saponification of 13b lead to the corresponding diol 13a, and was followed by selective oxidation to the 3-one 14, readily dehydrobrominated to 15a. Reductive ring opening furnished a mixture of the 19,21-diol 16a and its 5-ene isomer 16b, which was directly converted to the diketal 17. Reduction with DIBAH gave the hemiacetal 18, and hydrolysis of the latter 19-hydroxyaldosterone (20) as a water-soluble solid, accompanied by the 18,21-anhydro compound 19. 19-Hydroxyaldosterone exists in CHCl3 and water as a mixture of mainly two isomers. Periodate oxidation furnished the etiolactone 21. Preliminary results indicate that HAA and 19-hydroxyaldosterone are active mineralocorticoids in the Kagawa bioassay and short-circuit current measurements.     

Over-expression of human type I (placental) 3β-hydroxy-5-ene-steroid dehydrogenase/isomerase in insect cells infected with recombinant baculovirus

Human type I placental 3β-hydroxy-5-ene-steroid dehydrogenase/steroid 5→4-ene-isomerase (3β-HSD/isomerase) synthesizes androstenedione from fetal dehydroepiandrosterone and progesterone from pregnenolone. The full length cDNA that encodes type I 3β-HSD/isomerase was inserted into the baculovirus, Autographa californica multiple nucleocapsid polyhedrosis virus, and expressed in Spodoptera fungiperda (Sf-9) insect cells. Western blots showed that the baculovirus-infected Sf-9 cells produced an immunoreactive protein that co-migrated with purified placental 3β-HSD/isomerase. Ultracentrifugation localized the expressed enzyme activities in all the membrane-associated organelles of the Sf-9 cell (nuclear, mitochondrial and microsomal). Kinetic studies showed that the expressed enzyme has 3β-HSD and isomerase activities. The Michaelis-Menton constant is very similar for the 3β-HSD substrate, 5-androstan-3β-o1-17-one, in the Sf-9 cell homogenate (K_m = 17.9 μM) and placental microsomes (K_m = 16.7 μM). The 3β-HSD activity (V_max = 14.5 nmol/min/mg) is 1.6-fold higher in the Sf-9 cell homogenate compared to placental microsomes (V_max = 9.1 nmol/min/mg). The K_m values are almost identical for the isomerase substrate, 5-androstene-3,17-dione, in the Sf-9 cell homogenate (K_m = 14.7 μM) and placental microsomes (K_m = 14.4 μM). The specific isomerase activity is 1.5-fold higher in the Sf-9 cells (V_max = 25.7 nmol/min/mg) relative to placenta (V_max = 17.2 nmol/min/mg). These studies show that our recombinant baculovirus system over-expresses fully active enzyme that is kinetically identical to native 3β-HSD/isomerase in human placenta.     

Regulation of mRNA expression of 3 beta-hydroxy-5-ene steroid dehydrogenase in porcine granulosa cells in culture: a role for the protein kinase-C pathway

We studied the effect of the tumor-promoting phorbol ester phorbol 12-myristate 13-acetate (PMA), which activates protein kinase-C, on porcine granulosa cells in culture. PMA as well as cholera toxin, forskolin, and hCG increased cAMP accumulation. PMA further augmented the elevation in cAMP accumulation induced by cholera toxin, forskolin, and hCG. In the same cell culture model, hCG induced a time-dependent increase in the 3 beta-hydroxy-5-ene steroid dehydrogenase (3 beta HSD) mRNA levels with a maximal 3-fold stimulation obtained at 8-16 h of incubation with 1 IU hCG/ml. PMA inhibited the increase in 3 beta HSD mRNA levels induced by hCG in a dose-dependent manner. The phorbol ester also inhibited the increase in 3 beta HSD mRNA levels stimulated by LH as well as cholera toxin and forskolin and the cAMP analogs (Bu)2cAMP and 8-bromo-cAMP. Activation of protein kinase-C by mezerein similarly inhibited hCG stimulation of 3 beta HSD mRNA levels. The present data indicate that activation of the protein kinase-C pathway induces generation of cAMP, but causes a near-complete inhibition of the stimulatory effects of hCG, LH, forskolin, cholera toxin, and cAMP analogs on 3 beta HSD mRNA levels in porcine granulosa cells in culture.     

Enzymes of vitamin B6 degradation. Purification and properties of isopyridoxal dehydrogenase and 5-formyl-3-hydroxy-2-methylpyridine-4-carboxylic-acid dehydrogenase

Two NAD+-dependent, highly specific pyridine-5-aldehyde dehydrogenases, 5-formyl-3-hydroxy-2-methylpyridine-4-carboxylic-acid (Compound 1) dehydrogenase and isopyridoxal dehydrogenase, were purified to homogeneity from Pseudomonas MA-1 and Arthrobacter Cr-7, respectively. Both enzymes are induced in response to growth of the organisms on pyridoxine and catalyze steps in the degradation of this compound by these organisms. Compound 1 dehydrogenase (Mr = 65,000) contains two subunits of equal size with methionine as the NH2-terminal amino acid and acts optimally at pH 7.8-8.5. It catalyzes with equal facility (turnover number = 400-670 s-1 molecule-1) both the oxidation of Compound 1 (Km = 65 microM) by NAD+ (Km = 25 microM) to 3-hydroxy-2-methylpyridine-4,5-dicarboxylic acid and the reduction of Compound 1 by NADH (Km = 20 microM) to 4-pyridoxic acid and appears to act as a true dismutase. The possible advantage to the organism of its ability to act as a dismutase is discussed briefly. No oxidation of 4-pyridoxic acid by this enzyme was observed. Isopyridoxal dehydrogenase (Mr = 242,000) contains four subunits of equal size, again with methionine at the NH2 terminus. At its optimal pH of 8.0-8.6, it catalyzes the oxidation of isopyridoxal (Km = 40 microM, turnover number = 10 s-1 molecule-1) by NAD+ (Km = 40 microM) to a mixture of 5-pyridoxic acid and 5-pyridoxolactone, which are produced in constant ratio throughout the course of the reaction. Formation of the two products, although unusual, is readily understandable in terms of the structure of isopyridoxal in solution or the structure of a possible acyl-enzyme intermediate in the oxidative reaction.