Conversion of 5 alpha-pregnane-3,20-dione, 3 alpha-hydroxy-5 alpha-pregnan-20-one and 3 beta-hydroxy-5 alpha-pregnan-20-one to delta 16-C19 steroids by the reconstituted delta 16-C19-steroid synthetase system

The substrate specificity of the reconstituted delta 16-C19-steroid synthetase system, which catalyzes the formation of 5,16-androstadien-3 beta-ol or 4,16-androstadien-3-one from pregnenolone or progesterone, respectively, was studied. The reconstituted system consisted of a partially purified cytochrome P-450, NADPH-cytochrome P-450 reductase, cytochrome b5 and NADH-cytochrome b5 reductase all from pig testicular microsomes. It was found that 5 alpha-reduced C21 steroids such as 5 alpha-pregnane-3,20-dione, 3 alpha-hydroxy-5 alpha-pregnan-20-one and 3 beta-hydroxy-5 alpha-pregnan-20-one can be substrates for the enzyme system, resulting in the formation of 5 alpha-androst-16-en-3-one, 5 alpha-androst-16-en-3 alpha-ol and 5 alpha-androst-16-en-3 beta-ol, respectively. The results suggest that 5 alpha-reduced delta 16-C19 steroids might be synthesized from pregnenolone and progesterone via 5 alpha-reduced C21 steroids as intermediates. The pathways would bypass 5,16-androstadien-3 beta-ol and 4,16-androstadien-3-one which have been assumed as obligatory intermediates in the formation of 5 alpha-reduced delta 16-C19 steroids from pregnenolone and progesterone.     

Human placental delta5-3beta hydroxysteroid dehydrogenase activity (delta5-3beta HSDH): intracellular distribution, kinetic properties, retroinhibition and influence of membrane delipidation.

Delta5-3beta HSDH activity has been assayed either by spectrophotometric method or by use of radioactive substrates. The enzymatic activity is equally distributed between mitochondrial and microsomal fractions verified by electronic microscopy. The specific activity is comparable in both fractions, as well as the optimal pH and the Km for NAD and for the substrates. The delta5-3beta Hut optimal pH, specific activity and sensitivity to the inhibitory action of various steroids are different when C19 and C21 steroids are used as substrates. Estrogens and cyclic AMP have also an inhibitory action on the oxidation of C21 steroids. Treatment of microsomal or mitochondrial membranes with phospholipase A releases fatty acids (mainly arachidonic) and decreases the enzymatic activity. "Adsorbtion" of the fatty acids on bovine serum albumin partially reactivates the delta5-3beta HSDH.     

The effect of 4beta and 19 ester functionalities on some electrophilic addition reactions of delta5-steroids

The reactions of 3beta-acyloxyandrost-5-enes with bromine/silver acetate (Petrow reaction) and mercury(II) trifluoroacetate (modified Treibs oxidation) have been used previously to effect allylic oxidation on these substrates en route to biologically active compounds. In both these reactions, which involve electrophilic addition to the delta5-bond, the 3-acyloxy substituent plays a significant role. In this report, the effect of introducing other substituents proximate to the delta5-bond has been studied by using derivatives of 3beta-acetoxyandrost-5-en-17-one (1), namely, 3beta,4beta-diacetoxyandrost-5-en-17-one (13), 3beta,19-diacetoxyandrost-5-en-17-one (14), 3beta-acetoxyandrost-5-ene-7,17-dione (15), and 3beta-acetoxy-4,4-dimethylandrost-5-en-17-one (17). Our results indicate that in both sets of reactions the effect of the introduced functional groups was pronounced. In the Petrow reaction, electrophilic addition rather than allylic oxidation on the diacetates was observed. With the Treibs reaction, allylic oxidation on the diacetates occurred. The 7-keto and 4,4-dimethyl steroids proved to be poor substrates in both reactions.     

Microsomal 5-ane-3 beta-hydroxysteroid oxidoreductase from pubertal rat Leydig cells: partial purification and characterization

A 3 beta-hydroxysteroid oxidoreductase which acts on 5 alpha (beta)-reduced C19 and C21 steroids (5-ane-3 beta-hydroxysteroid oxidoreductase; 5-ane-3 beta-HSO) has been solubilized from pubertal rat Leydig cell microsomes and purified 300-fold by ion exchange and gel filtration chromatography. The partially purified enzyme is stable only in the presence of 0.4 M NaCl and appears to exist as a molecule having a molecular weight of 35,000 or as aggregates with a molecular weight in excess of 150,000. NAD+ and NADH+ are used exclusively as cofactors. The velocity of the steroid oxidation reaction was unaffected by either Ca2+ or Mg2+. The steroid oxidation reaction has a pH optimum between 8.0 and 8.5, a temperature optimum at 35 degrees C and an activation energy of 12,850 cal/mol. The pH optimum of the steroid reduction reaction is 6.6. A variety of 5 alpha-reduced C19 and C21 steroids can be utilized as substrates. Treatment of microsomes with phospholipase A2 resulted in a 26 to 90% loss of enzyme activity, paralleling decreased microsomal phospholipid content, and suggesting a role for phospholipids in 5-ane-3 beta-HSO activity. Assays with combined substrates indicate that one enzyme is responsible for activities observed with 5 alpha- and 5 beta-reduced C19- and 5 alpha-reduced C21-3 beta-hydroxysteroids. Purification data indicate that the 5-ane-3 beta-HSO and the 5-ene-3 beta-hydroxysteroid oxidoreductase:isomerase are distinct enzymes.     

The purification and properties of NADPH-dependent carbonyl reductases from rat ovary

Two carbonyl reductases have been highly purified from rat ovary to apparent homogeneity. Though they have similarities in terms of molecular weight (33,000), substrate specificities, inhibitor sensitivities, amino acid composition, and immunological properties, they differed in pI values (6.0 and 5.9). Both enzymes reduced aromatic aldehydes, ketones, and quinones at higher rates, compared to prostaglandins and 3-ketosteroids, whereas they showed higher affinity for prostaglandins and 3-ketosteroids. The enzymes also catalyzed oxidation of the 9-hydroxy group of prostaglandin F2 alpha. Moreover, they showed the remarkable characteristic of catalyzing the reduction of not only the 9-keto group of prostaglandin E2 but also the 15-keto group of 13,14-dihydro-15-keto-prostaglandin F2 alpha. Both enzymes were inhibited by SH-reagents, quercitrin, indomethacin, furosemide, and disulfiram. The results of immunoinhibition, using antibody against the purified enzymes, indicated that the enzymes were solely responsible for the overall catalytic activities of prostaglandin E series reduction, as well as 13,14-dihydro-15-keto-prostaglandin F2 alpha reduction and prostaglandin F2 alpha oxidation in rat ovarian cytosol. Western-blot analysis revealed that immunoreactive proteins were present in adrenal gland and various reproductive tissues except uterus of rats.     

Microsomal enzymes of cholesterol biosynthesis from lanosterol. Characterization, solubilization, and partial purification of NADPH-dependent delta 8,14-steroid 14-reductase

The membrane-bound enzyme of microsomes that catalyzes NADPH-dependent reduction of the 14-double bond of conjugated delta 8,14- and delta 7,14-sterols has been studied both as collected in microsomes from broken cell preparations of rat liver and after solubilization. Optimal incubation conditions for assay of the membrane-bound enzyme have been determined, and properties of the microsomal enzyme have been established with respect to cofactor requirements, kinetics, pH, addition of inhibitors, addition of glycerol phosphatides, and sterol substrate specificity. The 14-reductase is readily solubilized with a mixture of octylglucoside and taurodeoxycholic acid. The solubilized enzyme has been enriched by precipitation with polyethylene glycol and chromatography on DEAE-Sephacel and hydroxylapatite columns. The resulting partially purified enzyme has been obtained free of other microsomal enzymes of cholesterol biosynthesis: 4-methyl sterol oxidase, delta 5,7-sterol 7-reductase, delta 8,24-sterol 24-reductase, 3-ketosteroid reductase, and steroid 8----7-ene isomerase, plus microsomal cytochrome P-450, cytochrome P-450 reductase, cytochrome b5 reductase, and cytochrome b5. The partially purified enzyme is stimulated by addition of phospholipids. All of the properties exhibited by partially purified 14-reductase are consistent with the suggestion that the solubilized and enriched enzyme catalyzes the microsomal reduction of the 14-double bond of the sterol-conjugated dienes. However, presence of the enzyme does not prove that the sterol-conjugated dienes are obligatory precursors of cholesterol.     

Pluripotency of 17beta-hydroxysteroid dehydrogenase from the filamentous fungus Cochliobolus lunatus

Cochliobolus lunatus 17beta-hydroxysteroid dehydrogenase (17beta-HSD) is pluripotent for several steroidal and nonsteroidal substrates. In the presence of NADPH the enzyme was found to reduce 3-keto groups of 4,5-dihydro steroids, 20-keto groups, and most efficiently, 17-keto groups of steroidal substrates. In addition, several quinones were accepted and found to be even better substrates as steroids due to their higher affinity for the enzyme-coenzyme complex and faster conversion of the enzyme-coenzyme-substrate complex into the corresponding products. As suggested by the competition studies quinones and 17-ketosteroids are converted by the same active center of the enzyme. For all tested substrates, the equilibrium ordered mechanism was established with NADPH binding first to the enzyme. According to our knowledge, the investigated 17beta-HSD is the first known fungal pluripotent enzyme of this type.     

Identification of two dihydrodiol dehydrogenases associated with 3(17)alpha-hydroxysteroid dehydrogenase activity in mouse kidney

Dihydrodiol dehydrogenase activity was detected in the cytosol of various mouse tissues, among which kidney exhibited high specific activity comparable to the value for liver. The enzyme activity in the kidney cytosol was resolved into one major and three minor peaks by Q-Sepharose chromatography: one minor form cross-reacted immunologically with hepatic 3 alpha-hydroxysteroid dehydrogenase and another with aldehyde reductase. The other minor form was partially purified and the major form was purified to homogeneity. These two forms, although different in their charges, were monomeric proteins with the same molecular weight of 39,000 and had similar catalytic properties. They oxidized cis-benzene dihydrodiol and alicyclic alcohols as well as trans-dihydrodiols of benzene and naphthalene in the presence of NADP+ or NAD+, and reduced several xenobiotic aldehydes and ketones with NAD(P)H as a cofactor. The enzymes also catalyzed the oxidation of 3 alpha-hydroxysteroids and epitestosterone, and the reduction of 3- and 17-ketosteroids, showing much lower Km values (10(-7)-10(-6) M) for the steroids than for the xenobiotic alcohols. The results of mixed substrate experiments, heat stability, and activity staining on polyacrylamide gel electrophoresis suggested that, in the two enzymes, both dihydrodiol dehydrogenase and 3(17)alpha-hydroxysteroid dehydrogenase activities reside on a single enzyme protein. Thus, dihydrodiol dehydrogenase existed in four forms in mouse kidney cytosol, and the two forms distinct from the hepatic enzymes may be identical to 3(17)alpha-hydroxysteroid dehydrogenases.     

Properties of delta5-3beta-hydroxysteroid oxidoreductase isolated from Streptomyces griseocarneus.

Delta5-3beta-hydroxysteroid oxidoreductase was extracted in magnesium-containing Tris buffer from sonicated Streptomyces griseocarneus cells. The enzyme was partially purified (150 X) by ion exchange chromatography and gel filtration following (NH4)2SO4 fractionation. Upon gel filtration on Sephadex G-75 to G-200, the greatest part of the activity gave a peak in the fractionation range. The enzyme obtained from the gel yielded small enzyme molecules on repeated chromatography. A molecular weight of 32 to 36 000 was calculated for the activity appearing in the fractionation range of Sephadex G-75 to G-200. The enzyme is highly specific for the irreversible oxidation of the 3beta-hydroxyl group in steroids with a trans-anellated A : B ring system with either C5 or C6 double bond. Delta5-3-ketosteroids are converted into delta5-3-ketosteroids at a high rate, but the isomerase activity cannot be separated from the oxidoreductase activity either by chromatography or by selective heat inactivation. NAD, NADP, FMN or FAD did not influence the activity, but the enzyme is inactive in the absence of molecular oxygen.     

Assignment of the human 3 beta-hydroxysteroid dehydrogenase gene (HSDB3) to the p13 band of chromosome 1

Three-beta-hydroxysteroid dehydrogenase (HSDB3) is the enzyme which catalyses the oxidative conversion of delta 5-3 beta-hydroxy steroids to the delta 4-3-keto configuration and is therefore involved in the biosynthesis of all classes of hormonal steroids, namely progesterone, glucocorticoids, mineralocorticoids, androgens, and estrogens. Deficiency of the enzyme is associated with congenital adrenal hyperplasia and is usually lethal in early life. Despite its crucial role, chromosome assignment of the gene for this enzyme has not been reported. Using in situ hybridization, we report that hybridization with labeled human HSDB3-specific cDNA yielded 27% of silver grains associated with chromosome 1 with a maximal concentration in the p13 band.     

Purification and characterization of S-phenacylglutathione reductase from rat liver

An enzyme catalyzing the reduction of S-(2,4-dichlorophenacyl)glutathione to 2',4'-dichloroacetophenone was purified to apparent homogeneity by ion exchange, gel filtration, and hydroxylapatite chromatography from rat hepatic cytosol. The molecular weight was 30,000-37,000. The enzyme is distinct from the glutathione S-transferases, mercaptopyruvate sulfurtransferase, and glyoxalase I. Substrate specificity studies showed that S-phenacylglutathiones are the preferred first substrates and that several thiols (glutathione, mercaptoethanol, L-cysteine, or cysteamine) serve as reducing substrates. The enzyme serves to convert toxic alpha-haloketones, which react rapidly and nonenzymatically with glutathione, to nontoxic alkyl ketones.     

Expression of a full-length cDNA encoding bovine adrenal cytochrome P450C21

Two full-length cDNA clones encoding bovine adrenocortical P450C21 have been constructed in a eukaryotic expression vector using partial-length cDNAs whose structures have been previously reported. Following expression of these cDNAs in COS 1 cells, the substrate specificity of P450C21 was determined. Of the 18 steroids tested, progesterone, 17 alpha-hydroxyprogesterone, and 11 beta,17 alpha-dihydroxyprogesterone were found to be the only steroids to serve as substrates for this adrenal enzyme, a much higher degree of substrate specificity than has been reported for a hepatic 21-hydroxylase. The Vmax for 17 alpha-hydroxyprogesterone was 2.5 times greater than that for progesterone, whereas delta 5-steroids were unable to serve as substrate for this enzyme. A difference between the two cDNAs is located at amino acid 401 where one resultant enzyme contains tyrosine while the other contains histidine. This amino acid difference appears to have no effect on the kinetic properties of adrenal P450C21.     

Purification and characterization of NADP+-dependent 3 alpha-hydroxysteroid dehydrogenase from mouse liver cytosol

A monomeric 3 alpha-hydroxysteroid dehydrogenase with a molecular weight of 34,000 was purified to apparent homogeneity from mouse liver cytosol. The enzyme catalyzed the reversible oxidation of the 3 alpha-hydroxy group of C19-, C21-, and C24-steroids, reduced a variety of carbonyl compounds, and was inhibited by SH-reagents, synthetic estrogens, anti-inflammatory drugs, prostaglandins, and delta 4-3-ketosteroids. Although these properties are similar to those of the enzyme from rat liver cytosol, the mouse enzyme exhibited low dehydrogenase activity toward benzene dihydrodiol and some alicyclic alcohols, it showed a strict cofactor specificity for NADP(H), and high substrate inhibition was observed in the reverse reaction. In addition, dexamethasone, deoxycorticosterone, and medroxyprogesterone acetate inhibited the mouse enzyme competitively at low concentrations and noncompetitively at high concentrations, whereas hexestrol, indomethacin, and prostaglandin A1 were competitive inhibitors. Steady-state kinetic measurements in both directions indicated that the reaction proceeds through an ordered bi bi mechanism with the cofactors binding to the free enzyme. The 3-ketosteroid substrates inhibited the enzyme uncompetitively at elevated concentrations, suggesting that the substrates bind to the enzyme.NADPH complex and to the enzyme NADP+ complex.     

Characterization of a fourth type of 2-keto acid-oxidizing enzyme from a hyperthermophilic archaeon: 2-ketoglutarate ferredoxin oxidoreductase from Thermococcus litoralis.

Thermococcus litoralis is a strictly anaerobic archaeon (archaebacterium) that grows at temperatures up to 98 degrees C by fermenting peptides. It is known to contain three distinct ferredoxin-dependent, 2-keto acid oxidoreductases, which use pyruvate, aromatic 2-keto acids such as indolepyruvate, or branched-chain 2-keto acids such as 2-ketoisovalerate, as their primary substrates. We show here that T. litoralis also contains a fourth member of this family of enzymes, 2-ketoglutarate ferredoxin oxidoreductase (KGOR). In the presence of coenzyme A, KGOR catalyzes the oxidative decarboxylation of 2-ketoglutarate to succinyl coenzyme A and CO2 and reduces T. litoralis ferredoxin. The enzyme was oxygen sensitive (half-life of approximately 5 min) and was purified under anaerobic conditions. It had an M(r) of approximately 210,000 and appeared to be an octomeric enzyme (alpha2beta2gamma2delta2) with four different subunits with M(r)s of 43,000 (alpha), 29,000 (beta), 23,000 (gamma), and 10,000 (delta). The enzyme contained 0.9 mol of thiamine PPi and at least four [4Fe-4S] clusters per mol of holoenzyme as determined by metal analyses and electron paramagnetic resonance spectroscopy. Significant amounts of other metals (Cu, Zn, Mo, W, and Ni) were not present (<0.1 mol/mol of holoenzyme). Pure KGOR did not utilize other 2-keto acids, such as pyruvate, indolepyruvate, or 2-ketoisovalerate, as substrates, and the apparent Km values for 2-ketoglutarate, coenzyme A, T. litoralis ferredoxin, and thiamine PPi were approximately 250, 40, 8, and 9 microM, respectively. The enzyme was virtually inactive at 25 degrees C and exhibited optimal activity above 90 degrees C (at pH 8.0) and at pH 8.0 (at 80 degrees C). KGOR was quite thermostable, with a half-life at 80 degrees C (under anaerobic conditions) of about 2 days. An enzyme analogous to KGOR has been previously purified from a mesophilic archaeon, but the molecular properties of T. litoralis KGOR more closely resemble those of the other oxidoreductases from hyperthermophiles. In contrast to these enzymes, however, KGOR appears to have a biosynthetic function rather than a role in energy conservation.     

Characterization of the purified pituitary cytosolic NADPH:5 alpha-dihydroprogesterone 3 alpha-hydroxysteroid oxidoreductase

The purified cytosolic 3 alpha-hydroxysteroid oxidoreductase (3 alpha-HSOR) from female rat pituitary which catalyzes the reversible conversion of 5 alpha-dihydroprogesterone (5 alpha-DHP) to 3 alpha, 5 alpha-tetrahydroprogesterone (3 alpha, 5 alpha-THP) has been characterized in terms of its steroid substrate specificity, dihydrodiol dehydrogenase activity and inhibition by drugs such as medroxyprogesterone and indomethacin. The purified enzyme has a strong preference for the C21 progestin steroid substrates, 5 alpha-DHP and 3 alpha, 5 alpha-THP, over the corresponding C19 androgenic steroid substrates, 5 alpha-dihydrotesterone (5 alpha-DHT) and 3 alpha, 5 alpha-tetrahydrotestosterone (3 alpha, 5 alpha-THT). The apparent Km for 5 alpha-DHP (80 nM) is about 250 times lower than the Km for the androgenic steroid, 5 alpha-DHT (21 microM). In the oxidative direction, the apparent Km for 3 alpha, 5 alpha-TP (1.4 microM) is about 3-fold lower than the Km for the androgenic steroid, 3 alpha, 5 alpha-THT (4.2 microM). A number of other naturally occurring 3-keto- and 3 alpha(beta)-hydroxy-steroids were assessed for their ability to act as inhibitors (alternate substrates) of the 3 alpha-reduction of 5 alpha-DHP catalyzed by the purified 3 alpha-HSOR. None of the 3 beta- or 5 beta-isomers had any effect. Of the other 3-keto and 3 alpha- steroids tested, only deoxycorticosterone and the ovarian progestins showed any significant inhibition. These may be acting as inhibitors since there was little, if any, direct 3 alpha-reduction of progesterone to 3 alpha-hydroxy-4-pregnen-20-one. Unlike the liver cytosolic 3 alpha-HSOR, the pituitary enzyme has no associated dihydrodiol (quinone) dehydrogenase activity. This enzyme is similar to other cytosolic 3 alpha-HSORs from liver and brain in that it is potentially inhibited by indomethacin and by medroxyprogesterone.     

Inhibition of mammalian fatty acid synthetase activity by NADP involves decreased mobility of the 4'-phosphopantetheine prosthetic group

Mammalian fatty acid synthetase carrying a 3-keto, 3-hydroxy, or 2-enoyl acyl-enzyme intermediate on the 4'-phosphopantetheine thiol is reversibly inhibited by binding of NADP to the enoyl reductase domain. Acyl moieties which can normally leave the enzyme by thioester hydrolysis or by transfer to a CoA acceptor cannot readily be removed from the NADP-inhibited enzyme; in addition, 3-keto or 2-enoyl moieties attached to the enzyme 4'-phosphopantetheine cannot readily be reduced when NADP is replaced by NADPH, even though model substrates can be reduced immediately. Reactivation of the NADP-inhibited 3-ketoacyl-enzyme, by exposure to NADPH, is paralleled by reduction and dehydration of the 3-ketoacyl moiety to a saturated acyl moiety without accumulation of either the 3-hydroxy or 2-enoyl acyl-enzyme intermediates, indicating that once the 4'-phosphopantetheine engages the ketoacyl moiety in the ketoreductase domain, subsequent reactions occur very rapidly. The results are consistent with a hypothesis which proposes that NADP binding to the enoyl reductase domain of fatty acid synthetase carrying an acyl intermediate other than a saturated moiety induces a conformational change in the enzyme that results in decreased mobility of the 4'-phosphopantetheine prosthetic group. Normal mobility of the prosthetic group, essential for transfer of acyl-enzyme intermediates through the active sites of the various functional domains, is restored relatively slowly when NADP is replaced by NADPH. It remains to be determined whether this modulation by pyridine nucleotides observed in vitro plays a role in the regulation of fatty acid synthetase activity in vivo.     

Chromate reduction by rabbit liver aldehyde oxidase

Chromate was reduced during the oxidation of 1-methylnicotinamide chloride by partially purified rabbit liver aldehyde oxidase. In addition to 1-methylnicotinamide, several other electron donor substrates for aldehyde oxidase were able to support the enzymatic chromate reduction. The reduction required the presence of both enzyme and the electron donor substrate. The rate of the chromate reduction was retarded by inhibitors of aldehyde oxidase but was not affected by substrates or inhibitors of xanthine oxidase. These results are consistent with the involvement of aldehyde oxidase in the reduction of chromate by rabbit liver cytosolic enzyme preparations.     

Purification and characterization of delta helicase from fetal calf thymus.

A DNA helicase (delta helicase) which partially copurifies with DNA polymerase delta has been highly purified from fetal calf thymus. delta helicase differs in physical and enzymatic properties from other eukaryotic DNA helicases described thus far. The enzyme has an apparent mass of 57 kDa by gel filtration and is associated with polypeptides of 56 and 52 kDa by SDS-polyacrylamide gel electrophoresis. Photo-cross-linking of the purified enzyme with [alpha-32P]ATP resulted in labeling of a polypeptide of approximately 58 kDa, suggesting that the active site is present on the larger polypeptide. Unwinding of a partial duplex requires a nucleoside triphosphate which can be either ATP or dATP but not a nonhydrolyzable analogue of ATP. Other ribo- and deoxyribonucleoside triphosphates have little or no activity as cofactors. delta helicase also has DNA-dependent ATPase activity which has a relatively low Km for ATP (40 microM). delta helicase binds to single-stranded DNA but has little or no affinity for double-stranded DNA or single-stranded RNA. Similar to replicative DNA helicases from prokaryotes and the herpes simplex virus type 1 helicase-primase, delta helicase translocates in the 5'-3' direction along the strand to which it is bound and preferentially unwinds DNA substrates with a forklike structure.     

Regulation of hormonal and secretory granule membrane disulfides by adenohypophysial gluthion: disulfide oxidoreductase

An NADPH-dependent glutathione: disulfide oxidoreductase (thiol-transferase) has been identified in and partially purified (12.3-fold) from adenohypophysial cytosol. The enzyme is specific for NADPH and reduced glutatione, but the disulfide substrates include a wide size range (glutathione, cystine, RNase, oxytocin, vasopressin, monomeric and oligomeric growth hormone and prolactin). It also utilizes secretory granule membrane proteins. Substrate specificity studies (including utilization of cystine and failure to utilize insulin) and physico-chemical properties (M.W. 180,000) distinguish this enzyme from other glutathione: disulfide oxidoreductases. This thioltransferase may play a regulatory role in the hormone secretory process by control of the thiol: disulfide oxidation state of disulfide-bonded oligomers or of granule membrane proteins.     

Microbial oxidation of amines. Partial purification of a mixed-function secondary-amine oxidase system from Pseudomonas aminovorans that contains an enzymically active cytochrome-P-420-type haemoprotein

1. Crude extracts of Pseudomonas aminovorans grown on methylamine, di-methylamine, trimethylamine or trimethylamine N-oxide contain an enzyme or enzyme system catalysing the NADH- or NADPH- and oxygen-dependent oxidation of dimethylamine to methylamine and formaldehyde. 2. The enzyme has been partially purified about five-fold. It is unstable, but can be stabilized by addition of 5% (v/v) ethanol. 3. The partially purified enzyme will utilize either NADH (K(m) 6.5mum) or NADPH (K(m) 13.2mum): The following secondary amines have been shown to be substrates: dimethylamine, ethylmethylamine, diethylamine, methyl-n-propylamine, ethyl-n-propylamine, n-butylmethylamine and N-methylethanolamine. The K(m) values and comparative reaction rates for each substrate have been determined. Where the alkyl groups are different, the aldehyde products are derived from both groups. 4. The enzyme system has a pH optimum of 6.8 and is inhibited by mercurials, thiol compounds, cyanide and carbon monoxide. 5. The partially purified preparation had a spectral maximum at 412nm with shoulders at 427 and 550nm. Reduction with dithionite or NAD(P)H bleached the 412nm peak, and the shoulder at 427nm became a peak. Additional peaks appeared at 550 and 580-588nm. Reduction of a preparation bubbled with carbon monoxide enhanced and sharpened the Soret peak and caused it to shift to 422nm. 6. Analysis of the preparation showed the presence of flavin, acid-extractable iron and non-acid-extractable iron in the proportion 1.1:1.9:1. On reduction with dithionite or NADPH the preparation showed an electron-paramagnetic-resonance signal at around g=1.946.