Sequence requirements for cytochrome P-450IID1 catalytic activity. A single amino acid change (Ile380 Phe) specifically decreases Vmax of the enzyme for bufuralol but not debrisoquine hydroxylation

A cDNA coding for an allelic variant of rat IID1, designated IID1v, was isolated that produced a P-450 having a 10-fold lower catalytic activity toward the substrate bufuralol when expressed in COS-1 cells (Matsunaga, E., Zanger, U. M., Hardwick, J. P., Gelboin, H. V., Meyer, U. A., and Gonzalez, F. J. (1989) Biochemistry, 28, 7349-7355). IID1 and IID1v cDNA-deduced proteins differed in sequence by 4 amino acid residues. IID1 has Val, Phe, Arg, and Leu while IID1v has Ile, Leu, Gln, and Phe at amino acid positions 123, 124, 173, and 380, respectively. Chimeric cDNAs between IID1 and IID1v were constructed and expressed in hepatoma cells using vaccinia virus. A chimera having the Phe (IID1v) at amino acid 380, with the remaining 3 variant amino acid residues of IID1, was found to have a 17-fold decrease in Vmax and a 2 to 3-fold decrease in Km for (+)-bufuralol 1'-hydroxylation when compared to a converse chimera having Ile (IID1) in a background of IID1v sequence. Although this enzyme lacked significant bufuralol metabolism, it was able to carry out debrisoquine 4-hydroxylation. In contrast, the chimera having Ile (IID1) at position 380 was lacking in debrisoquine 4-hydroxylation. Type I difference spectra analysis revealed that both forms could bind debrisoquine with similar spectral dissociation constants. These data demonstrate that the single amino acid substitution Ile380----Phe differentially decreases the catalytic activity of IID1 toward bufuralol but not debrisoquine.     

Functional expression of mammalian cytochromes P450IIB in the yeast Saccharomyces cerevisiae

Three mammalian cytochromes P450 from the IIB subfamily, P450IIB11 from canine and P450IIB4 and P450IIB5 from rabbit, have been expressed in the yeast Saccharomyces cerevisiae by use of an autonomously replicating vector containing the galactose-inducible gal10 promoter. Cytochromes P450IIB4 and P450IIB5 are closely related proteins, with only 11 amino acid substitutions between them. P450IIB11 is a homologous protein, likely orthologous with IIB4 or IIB5, with 102 amino acid substitutions compared with the P450IIB4 protein and 106 compared with the P450IIB5 protein. The expressed proteins are functional in yeast microsomes, exhibiting activity toward androstenedione, 7-ethoxycoumarin, and, in some cases, progesterone. Expressed cytochromes P450IIB4 and P450IIB11 hydroxylate androstenedione with regio- and stereoselectivity characteristic of the purified, reconstituted proteins. A striking difference in the androstenedione metabolite profiles of IIB4 and IIB5 was observed, with IIB4 producing almost exclusively the 16 beta-hydroxy metabolite and IIB5 producing the 16 alpha-hydroxy and 15 alpha-hydroxy products. This is the first time that 15 alpha-hydroxylase activity has been associated with IIB4/IIB5. This activity has also been detected in liver microsomes from some, but not all, individual phenobarbital-induced rabbits tested and is largely inhibited by anti-rabbit P450IIB immunoglobulin G. These studies illustrate the utility of the yeast expression system for defining catalytic activities of individual mammalian cytochromes P450 and identifying new marker activities that can be utilized in liver microsomes.     

Regioselectivity and stereoselectivity of androgen hydroxylations catalyzed by cytochrome P-450 isozymes purified from phenobarbital-induced rat liver

The regioselectivity and stereoselectivity of androgen hydroxylations catalyzed by five isozymes of cytochrome P-450 purified from phenobarbital-induced rat liver were studied in a reconstituted monooxygenase system using testosterone (T) and androst-4-ene-3,17-dione (delta 4-A) as substrates. P-450 PB-3, an isozyme exhibiting low catalytic activity with many xenobiotic substrates, catalyzed efficient (turnover = 15.7 to 18.5 min-1 P-450-1 at 25 microM substrate) and highly stereoselective B-ring hydroxylations of both steroid substrates, with the corresponding 7 alpha- and 6 alpha-hydroxy alcohols formed in ratios of approximately 20 to 30:1, respectively. P-450 PB-2c metabolized testosterone to a mixture of 16 alpha OH-T, 2 alpha OH-T, and delta 4-A (product ratio = 1.0/0.78/0.33; turnover = 10.2 min-1 P-450-1). PB-2c is present in significantly larger amounts in mature male rats as compared to immature males, and probably catalyzes the male-specific testosterone 16 alpha-hydroxylase activity known to be induced at puberty and subject to endocrine control. P-450 PB-4, the major phenobarbital-induced isozyme in rat liver, catalyzed efficient D-ring hydroxylations, yielding 16 beta OH- delta 4-A as the predominant product with delta 4-A as substrate (turnover = 12.0 min-1 P-450-1) and a mixture of 16 beta OH-T, 16 alpha OH-T, and delta 4-A (the latter compound presumably formed via 17 alpha hydroxylation) with testosterone as substrate (turnover = 5.2 min-1 P-450-1). P-450 isozymes PB-1 and PB-5 hydroxylated both steroids with essentially the same regioselectivity as PB-4 but at only 5 to 10% the catalytic rate. Cytochrome b5 stimulated most of these steroid hydroxylations up to 2-fold with no change in regio- or stereoselectivity. The identification of specific steroid metabolites as diagnostic of particular P-450 isozymes should be useful for the assessment of isozymic contributions to microsomal activities and, in addition, facilitate comparisons of P-450 isozymes isolated in different laboratories.     

Cytochrome P-450 hPCN3, a novel cytochrome P-450 IIIA gene product that is differentially expressed in adult human liver. cDNA and deduced amino acid sequence and distinct specificities of cDNA-expressed hPCN1 and hPCN3 for the metabolism of steroid hormones and cyclosporine

Immunoblotting analysis of human liver microsome preparations revealed that human cytochrome P-450 PCN1 (hPCN1, Mr approximately 52,000) was expressed in each of 40 individual specimens examined. In about 10-20% of the livers, an immunologically related protein having a lower electrophoretic mobility (Mr approximately 52,500) was also detected. A single liver was found that expressed only the lower mobility protein, designated hPCN3, and RNA isolated from this liver was used to construct a lambda gt11 library. The library was screened with an hPCN1 cDNA probe resulting in the isolation of a unique full-length cDNA that was sequenced and shown to encode hPCN3. The deduced amino acid sequence of this cDNA contained 502 residues, a calculated molecular mass of 57,115 daltons, and displayed 84% similarity with hPCN1. The deduced amino-terminal sequence of hPCN3 was identical to that of HFLa, a major cytochrome P-450 expressed in human fetal liver that is immunologically cross-reactive with several family III cytochrome P-450s. hPCN1 and hPCN3 cDNAs were expressed in Hep G2 cells using a vaccinia virus expression system and shown to encode active enzymes, both characterized by reduced CO-binding spectra with lambda max at 450 nm. Enzymatic analysis revealed that both cytochrome P-450s were similarly active in catalyzing oxidation of the calcium channel blocking drug nifedipine. Both enzymes also catalyzed 6 beta-hydroxylation of the steroid hormones testosterone, progesterone, and androstenedione, although hPCN1 exhibited several-fold higher expressed activity than hPCN3. Several minor oxidation products of these steroids (e.g. 15 beta-hydroxytestosterone), comprising up to approximately 20% of the total metabolites, were formed by hPCN1 but not hPCN3, indicating that hPCN3 is a more highly regiospecific monooxygenase catalyst with steroid substrates. Clear differences were also detected in their catalytic activities toward the immunosuppressive drug cyclosporine, with two hydroxylated metabolites (M1 and M17) and one demethylated metabolite (M21) formed by hPCN1 but only one metabolite (M1) formed by hPCN3. These studies establish that hPCN3 is a newly described cytochrome P-450 that is differentially expressed in the adult human population and that has overlapping substrate specificity compared to hPCN1 for metabolism of steroid and drug substrates.     

Regio- and stereoselective regulation of monooxygenase activities by isoenzyme-selective phosphorylation of cytochrome P450

The phosphorylation of the two major phenobarbital-inducible cytochrome P450 isoenzymes IIB1 and IIB2 was increased in hepatocytes by the action of the membrane permeating cAMP derivatives N6-dibutyryl-cAMP and 8-thiomethyl-cAMP. Under these conditions the dealkylation of 7-pentoxyresorufin, a selective substrate of cytochrome P450IIB1 and P450IIB2 was markedly reduced. 16 beta-Hydroxylation of testosterone which is catalyzed specifically only by cytochrome P450IIB1 and IIB2 was strongly reduced; for 16 alpha-hydroxylation which is also catalyzed by cytochrome P450IIB1 and IIB2 but additionally by 3 further cytochrome P450 isoenzymes, this reduction was less pronounced; for the oxidation of the 17 beta-hydroxyl group which besides cytochromes P450IIB1 and IIB2 is additionally catalyzed not only by other cytochromes P450 but also by 17 beta-hydroxysteroid dehydrogenase there was a clear tendency of reduction which, however, no longer reached statistical significance. Hydroxylation at other positions of testosterone which are catalyzed by other cytochrome P450 isoenzymes were not significantly changed. Hence isoenzyme-selective phosphorylation of cytochrome P450 leads to a corresponding isoenzyme-selective modulation of monooxygenase activity which holds promise to be especially important as a fast regulation of the control of genotoxic metabolites.     

Replacing the carboxy-terminal 28 residues of rabbit liver P-450 (laurate (omega-1)-hydroxylase) with those of P-450 (testosterone 16 alpha-hydroxylase) produces a new stereospecific hydroxylase activity

cDNA for chimeric P-450 consisting of the amino-terminal 462 residues of P-450 (laurate (omega-1)-hydroxylase) and the remaining 28 residues of P-450 (testosterone 16 alpha-hydroxylase) was constructed and expressed in yeast cells. The resulting chimera could catalyze laurate (omega-1)-hydroxylation and benzphetamine N-demethylation at much higher rates than the parental P-450s, but exhibited the same specificity towards fatty acid substrates as the wild-type laurate hydroxylase. When testosterone was examined as a substrate, the 16 beta-hydroxylated product, which cannot be formed by either of the parental P-450s, was detected, suggesting that the laurate hydroxylase contains a structure that is capable of binding testosterone at a proper orientation so that it can be hydroxylated at the 16 beta position.     

Metabolism of two hydroxy-7-oxocholanic acids in isolated perfused rat liver

The metabolism of 7-oxolithocholic acid and 7-oxodeoxycholic acid in isolated perfused rat livers was compared. The metabolites extracted from the bile of perfused livers were analysed by gas chromatography. The amount of bile acids excreted in bile was greater after infusion with 7-oxolithocholic acid than with 7-oxodeoxycholic acid. When 7-oxolithocholic acid was infused almost all of the bile acids excreted in bile were taurine conjugates; with 7-oxodeoxycholic acid about 10 percent remained unconjugated. 7-Oxolithocholic acid was more susceptible to reduction than 7-oxodeoxycholic acid. 7-Oxolithocholic acid was preferably reduced to 7 beta-hydroxy rather than to 7 alpha-hydroxy metabolites. In contrast, 7-oxodeoxycholic acid was reduced predominantly to the 7 alpha-hydroxy rather than to the 7 beta-hydroxy metabolite.     

Isolation and characterization of 17β-hydroxy steroid dehydrogenase from human erythrocytes

1. The 17beta-hydroxy steroid dehydrogenase was solubilized during haemolysis of erythrocytes and was isolated from the membrane-free haemolysate. Membrane preparations isolated in different ways did not contain 17beta-hydroxy steroid dehydrogenase activity. The 17beta-hydroxy steroid dehydrogenase activity in the haemolysate was concentrated by repeated ammonium sulphate precipitation and gel filtration on Sephadex G-150. The 17beta-hydroxy steroid dehydrogenase activity of the purified preparation per unit weight of protein was 350-3000 times higher than the activity of the crude erythrocyte haemolysate. The 20alpha-hydroxy steroid dehydrogenase activity was lost during this purification procedure. 2. The 17beta-hydroxy steroid dehydrogenase was NADP-dependent and had a pH optimum for conversion of testosterone between 8.5 and 10. For the molecular weight of the enzyme a value of 64000 was calculated from Sephadex chromatography results. 3. p-Chloromercuribenzoate inhibited the enzymic activity. The oxidative activity of the enzyme for the 17beta-hydroxyl group was only partly inhibited when a large excess of 17-oxo steroids was added. The catalysing activity of the enzyme was influenced by the NADP(+)/NADPH ratio. The oxidation of the 17beta-hydroxyl group in the presence of NADP(+) proceeded faster than the reduction of the 17-oxo group with NADPH. When both reduced and oxidized cofactors were present the oxidation of the 17beta-hydroxyl group was inhibited to a considerable extent. 4. The enzyme had a broad substrate specificity and not only catalysed the conversion of androstanes with a 17beta-hydroxyl group, or 17-oxo group, but also the conversion oestradiolleft arrow over right arrowoestrone. In addition the steroid conjugates dehydroepiandrosterone sulphate and oestrone sulphate were also converted. There were no indications that more than one 17beta-hydroxy steroid dehydrogenase was present in the partially purified preparation.     

Genetically engineered V79 Chinese hamster cell expression of purified cytochrome P-450IIB1 monooxygenase activity

Chinese hamster V79 fibroblasts, frequently used as target cells in short-term tests for mutagenicity, do not possess measurable monooxygenase activity; in particular, enzymatic oxidation of testosterone (T) cannot be demonstrated. If these V79 cells, however, had been transfected with the cDNA-encoding rat liver cytochrome P-450IIB1 under control of the SV40 early promoter, they stably expressed monooxygenase activity. These so-called SD1 cells then oxidatively metabolized T at a rate of 27 pmol/mg protein/min, converting it to 16 alpha- and 16 beta-hydroxy-T as well as 4-androsten-3,17-dione as sole metabolites in a ratio of 1.1:1.0:1.6. The regio- and stereoselective conversion of T by SD1 cells, as well as the quantitative distribution of the metabolites, corresponds well with the results reported for pure cytochrome P-450IIB1 in a reconstituted system.     

The role of the hypophysis and the hypophyseal hormone prolactin in maintenance of the sexual specificity of the metabolism of testosterone and 5-alpha-dihydrotestosterone in rat liver slices]

Hypophysectomy of rats 55 days after birth causes profound changes in the sexually differentiated liver metabolism of testosterone and 5alpha-dihydrotestosterone, which were studied when the rats were 80 days old. 1. Metabolism of testosterone after hypophysectomy: The turnover of testosterone decreased significantly to the same level in both sexes. The effect was especially marked in the female, which normally has a high turnover of this compound. The sexual differences in the patterns of metabolites were also lost, owing to the following changes: In the male, the high level of metabolites of the 3beta-hydroxy-5alpha-androstane type falls to the low level found in the female controls. The low level of 4-androstene-3,17-dione in the female increases to the high level found in the male controls. The concentrations of testosterone increase and those of the metabolites of the 3-oxo- and 3alpha-hydroxy-5alpha-androstane decrease to values that are significantly much higher or lower, respectively, than the normal values found in the control animals. 2. Metabolism of 5alpha-dihydrotestosterone after hypophysectomy: In comparison with the controls, the turnover of this substrate is significantly decreased by the same factor in both sexes; thus the difference between the sexes persists. In the pattern of metabolites, the sexual differences are still apparent, but less marked. The levels of metabolites show two opposing changes: a significant increase in the concentration of 3beta-hydroxy metabolites, and a significant decrease in the concentration of 3alpha-hydroxy metabolites; although the activity of the microsomal 3alpha-hydroxysteroid dehydrogenase increases by a factor of 3 - 4 in both sexes after hypophysectomy[1]. This discrepancy indicates a compartmentalization of androgen metabolism in the liver cell, in which delta4-5alpha- and 3beta-hydrogenation occur on the membranes of the endoplasmic reticulum, whereas 3alpha-hydrogenation occurs in the cytosol. 3. Action of prolactin on the metabolism of testosterone in hypophysectomized animals: Prolactin (125 mug twice daily from the 70th to the 79th day of life) causes a significant acceleration of the delta4-5alpha-hydrogenation, which is recognized as a significant increase in the concentrations of 5alpha-androstane metabolites; the 3beta-hydroxy compounds in both sexes reach the normal level of male control animals. The significant increase in the concentration of 3alpha-hydroxy compounds is accompanied by a partial reestablishment of the sexual differences. The sex differences in androgen turnover and metabolite pattern are subject to a hypophyseal regulation, which is separate from the gonadotropic partial function. The hydroxylation activity of the liver, measured as the production of C19O3-steroids, is not significantly affected by hypophysectomy or by treatment with prolactin.     

Rat testosterone 7 alpha-hydroxylase. Isolation, sequence, and expression of cDNA and its developmental regulation and induction by 3-methylcholanthrene

A P-450, designated P-450a, with high testosterone 7 alpha-hydroxylase activity was purified from rat liver microsomes. Specific polyclonal antibody against this P-450 was used to screen a lambda gt11 expression cDNA library and a 1687-base pair cDNA was isolated and sequenced. The deduced protein had 492 amino acids, a calculated Mr of 56,016, and it shared 51 and 45% amino acid similarities to P-450e and P-450f, respectively. Regions of similarity were distributed in distinct areas of high and low similarity along the P-450a primary sequence. P-450a cDNA was introduced into yeast cells using the expression vector pAAH5, and the resultant yeast microsomes contained both a protein of identical electrophoretic mobility to that of rat P-450a and testosterone 7 alpha-hydroxylase activity. These results confirm enzyme reconstitution data and antibody inhibition data that P-450a possesses testosterone 7 alpha-hydroxylase activity. The antibody and cDNA probes were used to examine the mechanism of regulation of P-450a by inducers and during development. P-450a and its mRNA were present at low level in newborn rats and increased to maximal level at 1 week of age in both males and females. At age 12 weeks, however, the P-450a level decreased in males but remained elevated in females. Concomitant with the decrease in P-450a in adult males was an increase in level of another immunologically related P-450. In adult male rats, P-450a was induced almost 5-fold by administration of 3-methylcholanthrene and this induction was the result of an increase in its mRNA. These results establish testosterone 7 alpha-hydroxylase as a member of the P-450e gene family that is developmentally regulated, sex-dependent, and markedly inducible by 3-methylcholanthrene.     

Sterol synthesis. Preparation and characterization of fluorinated and deuterated analogs of oxygenated derivatives of cholesterol.

Oxygenated sterols, including both autoxidation products and sterol metabolites, have many important biological activities. Identification and quantitation of oxysterols by chromatographic and spectroscopic methods is greatly facilitated by the availability of authentic standards, and deuterated and fluorinated analogs are valuable as internal standards for quantitation. We describe the preparation, purification and characterization of 43 oxygenated sterols, including the 4 beta-hydroxy, 7 alpha-hydroxy, 7 beta-hydroxy, 7-keto, and 19-hydroxy derivatives of cholesterol and their analogs with 25,26,26,26,27,27,27-heptafluoro (F7) and 26,26,26,27,27,27-hexadeuterio (d6) substitution. The 7 alpha-hydroxy, 7 beta-hydroxy, and 7-keto derivatives of (25R)-cholest-5-ene-3 beta, 26-diol (1d) and their 16,16-dideuterio analogs were also prepared. These d2-26-hydroxysterols and [16,16-2H2]-(25R)-cholest-5-ene-3 beta, 26-diol (1e) were synthesized from [16,16-2H2]-(25R)-cholest-5-ene-3 beta, 26-diol diacetate (2e), which can be prepared from diosgenin. The highly specific deuterium incorporation at C-16 in 1e and 2e should be useful in mass spectral analysis of 26-hydroxycholesterol samples by isotope dilution methods. The delta 5-3 beta, 7 alpha, 26- and delta 5-3 beta, 7 beta, 26-triols were regioselectively oxidized/isomerized to the corresponding delta 4-3-ketosteroids with cholesterol oxidase. Also described are 5,6 alpha-epoxy-5 alpha-cholestan-3 beta-ol, its 5 beta,6 beta-isomer, cholestane-3 beta, 5 alpha,6 beta-triol, their F7 and d6 derivatives, and d3-25-hydroxycholesterol, which was prepared from 3 beta-acetoxy-27-norcholest-5-en-25-one (30). The 43 oxysterols and most synthetic intermediates were isolated in high purity and characterized by chromatographic and spectroscopic methods, including mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy. Detailed mass spectral assignments are presented, and 1H NMR stereochemical assignments are derived for the C-19 protons of 19-hydroxysterols and for the side-chain protons of 30.     

Testosterone metabolism by cytochrome P-450 isozymes RLM3 and RLM5 and by microsomes. Metabolite identification

Testosterone metabolism by cytochrome P-450 isozymes RLM3 and RLM5 in a reconstituted system and by rat liver microsomes was examined. Eleven metabolites were detected. Two of these, found in spots 2 and 4 of a thin layer plate, were only formed by the rat liver microsomes and may represent reductive metabolites of testosterone. A number of monohydroxy metabolites were conclusively identified by gas chromatography-mass spectrometry. These include the 2-, 6 beta-, 7 alpha-, and 16 alpha-hydroxy isomers. Liver microsomes formed the 2 alpha- and 2 beta-epimers in a 1:2 ratio and both co-chromatographed with a third reduced metabolite in thin layer plate spot 4. In contrast with RLM5 about 90% of the 2-hydroxy isomer was the 2 alpha-epimer. RLM3 did not perform the 2-hydroxylation in detectable amounts. The 6 beta-isomer was a major metabolite of RLM3 and microsomes, but a minor product of metabolism by RLM5. In contrast, the 7 alpha-isomer was a minor metabolite of RLM3, was not formed by RLM5, and was a major microsomal metabolite. Hydroxylation at position 16 alpha was a major activity of RLM5 and the heterogeneous microsomal cytochromes, but with RLM3 it was a minor reaction. One new metabolite was found which appeared to be hydroxylated in the D-ring, had a mass spectrum different from both 16 alpha- and 16 beta-hydroxytestosterone, and was tentatively identified as a 15-hydroxy isomer. In agreement with the literature, androstene-3,17-dione was found to be an oxidative metabolite of testosterone by both microsomes and purified cytochrome P-450. It was a major metabolite of RLM5 but was not produced by RLM3. Studies with 18O2 and H218O conclusively show that oxidation of testosterone at C-17 does not involve transient incorporation of an oxygen atom in this position. A mechanism is suggested whereby cytochrome P-450 acts as a peroxidase in the formation of androstenedione.     

Human placenta estrogen synthetase (aromatase) purified by affinity chromatography

Microsomal estrogen synthetase (cytochrome P-450ES), also known as aromatase, was purified from fresh human placenta microsomes by DEAE-Trisacryl and testosterone-agarose chromatography. Estrogen synthetase assays were done with androstenedione as substrate, NADPH as electron donor, and a partially purified P-450 reductase from human placenta as the electron carrier. The specific cytochrome P-450 content of the purified P-450 was 0.67 nmol mg-1 of protein, and the preparation contained no cytochrome P-420. The absorbance maximum was 448.5 nm. The specific estrogen synthetase activity of the purified P-450ES fraction was 35 nmol min-1 nmol-1 of cytochrome P-450 or 23.3 nmol min-1 mg-1 of protein. The latter value shows a 179-fold purification with a yield greater than 1% in the two-step procedure. Kinetic constants for the reaction were measured with androstenedione as the aromatizable substrate. The Km was 1.4 nM and the Vmax was 37 nmol min-1 nmol-1 of P-450. The purified enzyme aromatized androstenedione and testosterone at identical rates; androstenedione gave only estrone, and testosterone gave only estradiol-17 beta. Dehydroepiandrosterone was not detectably aromatized or otherwise metabolized. Neither 16 alpha-hydroxytestosterone nor 16 alpha-hydroxyandrostenedione was aromatized. No hydroxysteroid dehydrogenase or reductase was detected in direct assays. No free reaction intermediates were detected in aromatization assay incubation mixtures. The purity of the product and the simplicity of the preparation recommend it for use in further studies of the enzyme.     

Cloning and expression of a cDNA for human cytochrome P-450aldo as related to primary aldosteronism

A cDNA clone encoding human aldosterone synthase cytochrome P-450 (P-450aldo) has been isolated from a cDNA library derived from human adrenal tumor of a patient suffering from primary aldosteronism. The insert of the clone contains an open reading frame encoding a protein of 503 amino acid residues together with a 3 bp 5'-untranslated region and a 1424 bp 3'-untranslated region to which a poly(A) tract is attached. The nucleotide sequence of P-450aldo cDNA is 93% identical to that of P-450(11) beta cDNA. Catalytic functions of these two P-450s expressed in COS-7 cells are very similar in that both enzymes catalyze the formation of corticosterone and 18-hydroxy-11-deoxycorticosterone using 11-deoxycorticosterone as a substrate. However, they are distinctly different from each other in that P-450aldo preferentially catalyzes the conversion of 11-deoxycorticosterone to aldosterone via corticosterone and 18-hydroxycorticosterone while P-450(11)beta substantially fails to catalyze the reaction to form aldosterone. These results suggest that P-450aldo is a variant of P-450(11)beta, but this enzyme is a different gene product possibly playing a major role in the synthesis of aldosterone at least in a patient suffering from primary aldosteronism.     

Photoperiod-induced changes in the testicular metabolism of [4-14C]17 alpha-hydroxyprogesterone in the bank vole (Clethrionomys glareolus)

Minces of the testes of bank voles, born and reared in a long (18L:6D) photoperiod until weaning (18-22 days of age) and subjected thereafter to a short (6L:18D, Group S) or a long (18L:6D, Group L) photoperiod for 6-9 weeks, were incubated with [4-14C]17 alpha-hydroxyprogesterone in the presence of cofactors (NADP/NADPH, 1.3 mmol/1) for 1 h at 37 degrees C. The radioactive metabolites were characterized and identified by thin-layer chromatography with derivative formation and chromatography to constant specific activity and isotope ratio. In Group L virtually all of the substrate was utilized and it was readily converted to androgens (48% of the radioactivity recovered) such as androstenedione and testosterone. The only pregnane metabolite identified was 17 alpha-hydroxy,20 alpha-dihydroxyprogesterone (43.3%). In Group S there was a decreased production of 17 alpha-hydroxy,20 alpha-dihydroprogesterone and androgens (25.4% and 10.4% respectively) and a substantial portion of the substrate was not metabolized (38.8%). The main androgen metabolites identified, androst-4-ene-3 beta,17 beta-diol and 5 alpha-androstane-3,17-dione are hormonally quite inert steroids. No androstenedione or testosterone was found. The results indicate that exposure to short photoperiod induces a decrease in the testicular C17-C20 lyase and 20 alpha-hydroxysteroid dehydrogenase.     

Mutant rho factors with increased transcription termination activities. II. Identification and functional dissection of amino acid changes

We have determined the nucleotide sequences of three mutant rho genes encoding hyperfunctional rho proteins (rho S) together with their parent allele, rho-ts702. These mutant rho factors contain the following amino acid changes as deduced from their sequences: (1) the thermo-labile mutant, rho-ts702, has Thr304 substituting for Ala; (2) rho S-77 and rho S-81, which are selectively altered in the primary polynucleotide binding site, share an identical mutation, Leu3----Phe; (3) rho S-82, which is altered in both the primary and secondary polynucleotide binding sites, carries three amino acid substitutions together, Leu3----Phe, Asp156----Asn and Thr323----Ile. Dissection and functional characterization of each mutation in rho S-82 have revealed that Ile323 alone is responsible for alterations in both the secondary RNA interaction and the terminator selectivity observed with the original mutant, rho S-82. Taken together, these results not only confirm our proposal in the accompanying paper that the primary and secondary RNA binding sites differently contribute in determining the overall efficiency and site-specificity of termination, respectively, but also support the possibility that these binding sites exist as structurally distinct domains in rho protein. In contrast, Asn156 was shown to cause decreased termination efficiency, though it had no influence on RNA interactions. Thus, this amino acid residue appears to be associated with still another rate-determining step of termination, for instance, interactions between rho and RNA polymerase. On the basis of Chou-Fasman secondary structure predictions as well as amino acid sequence comparison with F1-ATPase, we discuss how the proposed domains are structurally and functionally related to the putative ATPase reactive center of rho protein.     

The chemistry of 9 alpha-hydroxysteroids. 3. Methods for selective formation and dehydrations of 17 beta-cyano-9 alpha,17 alpha-dihydroxyandrost-4-en-3-one

Two methods to produce the 17-cyanohydrin, using potassium cyanide in acetic acid/methanol or acetone cyanohydrin with aqueous sodium hydroxide, were followed with 9 alpha-hydroxyandrost-4-ene-3,17-dione, both providing 17 beta-cyano-9 alpha,17 alpha-dihydroxyandrost-4-en-3-one. The selectivity of one of these methods, that which uses acetone cyanohydrin, is not in agreement with a comparable reaction with the 9 alpha-unsubstituted androst-4-ene-13,17-dione to give the 17 alpha-cyano-17 beta-hydroxy product, as reported in the literature and confirmed by us. The 9 alpha-hydroxy and 17 alpha-hydroxy groups were used for the regioselective introduction of 9(11)- and 16(17)-double bonds by dehydrating 17 beta-cyano-9 alpha,17 alpha-dihydroxyandrost-4-en-3-one under different conditions.