A comparison of alkaline phosphatase, beta-galactosidase, penicillinase and peroxidase used as labels for progesterone determination in milk by heterologous microtitre plate enzymeimmunoassay

Assays for alkaline phospatase, beta-galactosidase, penicillinase and peroxidase were optimised for quantitation in microtitre plate wells. Their value as labels in microtitre plate enzymeimmunoassay (EIA) for progesterone was assessed following coupling with 11 alpha-hydroxyprogesterone 11-glucuronide using an active ester procedure. Bridge-heterologous antiserum (11 alpha-hydroxyprogesterone 11-hemisuccinate-bovine serum albumin as immunogen) was used to minimize bridge recognition. The limits of detection of the enzymes were in the order penicillinase greater than peroxidase greater than alkaline phosphatase greater than beta-galactosidase. Under appropriate conditions it was possible to achieve greater than 50% displacement of label with 50 pg of progesterone for all four labels.     

Use of progesterone 11-glucuronide-alkaline phosphatase conjugate in a sensitive microtitre-plate enzymeimmunoassay of progesterone in milk and its application to pregnancy testing in dairy cattle

A simple direct-addition microtitre plate enzymeimmunoassay (EIA) for progesterone in whole milk is described. The assay used antiserum raised against 11 alpha-hydroxyprogesterone 11-hemisuccinate (progesterone 11-hemisuccinate) and a heterologous label prepared by conjugation of 11 alpha-hydroxyprogesterone 11-glucuronide (progesterone 11-glucuronide) with alkaline phosphatase using an active ester procedure. The sensitivity, analytical recovery, linearity of response and precision of the assay compared favourably with radioimmunoassay (RIA). Results from EIA of milk samples were compared with determinations made after isolation of progesterone by HPLC (r = 0.910). Milk samples (200) were assayed by RIA at both the Milk Marketing Board and the Cattle Breeding Centre and the results were correlated with EIA performed at the Cattle Breeding Centre (r = 0.890 and r = 0.833 respectively). Calving data were obtained from a further 110 cows for which the milk progesterone EIA had provided a pregnancy test 24 days after AI; 46 cows were correctly identified as non-pregnant and 58 as pregnant and there were 4 false positive and 2 inconclusive results.     

Identification of monohydroxy progesterones produced by CYP106A2 using comparative HPLC and electrospray ionisation collision-induced dissociation mass spectrometry

Two previously uncharacterised products, produced by recombinant CYP106A2 of Bacillus megaterium ATCC 13368 using progesterone as substrate, were identified. For this purpose a combination of comparative HPLC and electrospray ionisation collision induced dissociation mass spectrometry (ESI CID MS) was established and applied for rapid identification of the steroids, which were identified as 11alpha-hydroxyprogesterone and 9alpha-hydroxyprogesterone. The pharmaceutical relevance of these steroids is discussed. Furthermore, the hydroxylation activity was quantified for all monohydroxylation products (15beta-hydroxyprogesterone, 6beta-hydroxyprogesterone, 11alpha-hydroxyprogesterone, and 9alpha-hydroxyprogesterone). The V(max) values for 15beta-hydroxyprogesterone, 6beta-hydroxyprogesterone, 11alpha-hydroxyprogesterone, and 9alpha-hydroxyprogesterone were determined as 337.3+/-43.7, 22.3+/-0.9, 17.5+/-0.9, and 6.5+/-0.3nmol product/min/nmol CYP106A2, respectively.     

Regioselective progesterone hydroxylation catalyzed by eleven rat hepatic cytochrome P-450 isozymes

Quantitative high-pressure liquid chromatographic assays were developed that separate progesterone and 17 authentic monohydroxylated derivatives. The assays were utilized to investigate the hydroxylation of progesterone by 11 purified rat hepatic cytochrome P-450 isozymes and 8 different rat hepatic microsomal preparations. In a reconstituted system, progesterone was most efficiently metabolized by cytochrome P-450h followed by P-450g and P-450b. Seven different monohydroxylated progesterone metabolites were identified. 16 alpha-Hydroxyprogesterone, formed by 8 of the 11 isozymes, was the only detectable metabolite formed by cytochromes P-450b and P-450e. 2 alpha-Hydroxyprogesterone was formed almost exclusively by cytochrome P-450h, and 6 alpha-hydroxyprogesterone and 7 alpha-hydroxyprogesterone were only formed by P-450a. 6 beta-hydroxylation of progesterone was catalyzed by four isozymes with cytochrome P-450g being the most efficient, and 15 alpha-hydroxyprogesterone was formed as a minor metabolite by cytochromes P-450g, P-450h, and P-450i. None of the isozymes catalyzed 17 alpha-hydroxylation of progesterone, and only cytochrome P-450k had detectable 21-hydroxylase activity. 16 alpha-Hydroxylation catalyzed by cytochrome P-450b was inhibited in the presence of dilauroylphosphatidylcholine (1.6-80 microM), while this phospholipid either stimulated (up to 3-fold) or had no effect on the metabolism of progesterone by the other purified isozymes. Results of microsomal metabolism in conjunction with antibody inhibition experiments indicated that cytochromes P-450a and P-450h were the sole 7 alpha- and 2 alpha-hydroxylases, respectively, and that P-450k or an immunochemically related isozyme contributed greater than 80% of the 21-hydroxylase activity observed in microsomes from phenobarbital-induced rats.     

Cyclic analogs of substance P. I. Cyclo(11----epsilon 5)-[Lys5] substance P-(5-11)

Two solution syntheses of cyclo(11----5 epsilon)-[Lys5]substance P-(5-11) (CLP) were carried out. The first synthesis involved the stepwise elongation of the peptide chain starting from glycine tert-butyl ester. At the stage of hexapeptide deprotection, the cleavage of Boc and But groups was accompanied by tert-butylation of the Met residue. Cyclization was carried out via a pentafluorophenyl ester intermediate. The benzyloxycarbonyl-cyclopeptide (Z-CLP) formed was deprotected by catalytic transfer hydrogenation. A (3 + 4) block coupling strategy was used in course of the repeated preparation of the linear precursor of CLP. Optimization of the cyclization and subsequent deprotecting stages lead to increased yields and facilitated the synthetic procedure. Z-CLP was found to possess myotropic activity on isolated guinea pig ileum (alpha = 0.55 +/- 0.18; pD2 = 7.97 +/- 0.20), whereas CLP was inactive in these experiments. Z-CLP causes a slight two-phase effect on arterial pressure in rats, CLP being inactive. Similar to substance P, CLP displays an antidepressant-like effect in mice as indicated by the swimming test.     

Studies on the metabolism of steroid hormones in a virilizing adrenal cortex adenoma.

Slices of an adreno-cortical adenoma which had been obtained at operation from an 11-year-old girl with clinical signs of virilism were incubated with each of the following steroids: [1,2-3H]progesterone, [4-14C]pregnenolone, [1,2-3H]testosterone, [4-14C]androstenedione and [7-3H]dehydroepiandrosterone, respectively. Isolation and identification of the free radioactive metabolites were achieved by gel column chromatography on Sephadex LH-20, thin-layer chromatography, radio gas chromatography and isotope dilution. After incubation of progesterone, the following metabolites were identified: 11beta-hydroxyprogesterone, 16alpha-hydroxyprogesterone, 17alpha-hydroxyprogesterone, 21-deoxycortisol, corticosterone and cortisol. Pregnenolone was metabolized to 17alpha-hydroxypregnenolone, progesterone, dehydroepiandrosterone, androstenedione and 11beta-hydroxyandrostenedione. When testosterone was used as substrate, 11beta-hydroxytestosterone, androstenedione and 11beta-hydroxyandrostenedione were found as metabolites, whereas androstenedione was metabolized to testosterone and 11beta-hydroxyandrostenedione. After incubation of dehydroepiandrosterone, only androstenedione and 11beta-hydroxyandrostenedione were isolated and identified. From these results, it appears that cortisol was formed in the adenoma tissue via 21-deoxycortisol and corticosterone. Delta4-3oxo steroids of the C19-series arose exclusively from pregnenolone via 17alpha-hydroxypregnenolone and dehydroepiandrosterone, and not from progesterone and 17alpha-hydroxyprogesterone. Calculated on the amounts of metabolites formed, the highest enzyme activities were those of the 11beta-hydroxylase and the 17alpha-hydroxylase. It is interesting to note that only traces of testosterone were detected after incubation of androstenedione, whereas testosterone yielded large amounts of androstenedione.     

The rate-determining step in P450 C21-catalyzing reactions in a membrane-reconstituted system

Adrenal cytochrome P450 C21 in a membrane-reconstituted system catalyzed 21-hydroxylation of 17alpha-hydroxyprogesterone at a rate higher than that for progesterone in the steady state at 37 degrees C. The rate of product formation in the steady state increased with the concentration of the complex between P450 C21 and the reductase in the membranes. The complex formation was independent of the volume of the reaction, showing that the effective concentrations of the membrane proteins should be defined with the volume of the lipid phase. The rates of conversion of progesterone and 17alpha-hydroxyprogesterone to the product in a single cycle of the P450 C21 reaction were measured with a reaction rapid quenching device. The first-order rate constant for the conversion of progesterone by P450 C21 was 4.3 +/- 0.7 s(-)1, and that for 17alpha-hydroxyprogesterone was 1.8 +/- 0.5 s(-)1 at 37 degrees C. It was found from the analysis of kinetic data that the rate-determining step in 21-hydroxylation of progesterone in the steady state was the dissociation of product from P450 C21, whereas the conversion to deoxycortisol was the rate-determining step in the reaction of 17alpha-hydroxyprogesterone. The difference in the rate-determining steps in the reactions for the two substrates was clearly demonstrated in the pre-steady-state kinetics.     

Solution structure of marinostatin, a natural ester-linked protein protease inhibitor

Marinostatin is a unique protein protease inhibitor containing two ester linkages. We have purified a 12-residue marinostatin [MST(1-12), (1)FATMRYPSDSDE(12)] and determined the residues involved in the formation of the ester linkages and the solution structure by (1)H NMR spectroscopy and restrained molecular dynamics calculation. The two ester linkages of MST(1-12) are formed between hydroxyl and carboxyl groups, Thr(3)-Asp(9) and Ser(8)-Asp(11), indicating that MST(1-12) has two cyclic regions which are fused at the residues of Ser(8) and Asp(9). A strong NOE cross-peak between Tyr(6) H(alpha) and Pro(7) H(alpha) was observed, indicating that the Pro(7) residue takes a cis-conformation. Well-converged structures and hydrogen-deuterium experiments of MST(1-12) showed that the backbone NH proton of the P1'residue, Arg(5), is hydrogen-bonded to the carbonyl oxygen of the ester linkage between Thr(3) and Asp(9). To reveal the significance of the ester linkages, a marinostatin analogue, MST-2SS ((1)FACMRYPCCSCE(12)) with two disulfide bridges of Cys(3)-Cys(9) and Cys(8)-Cys(11), was also synthesized. The inhibitory activity of MST-2SS was as strong as that of MST(1-12), and the Pro(7) residue of MST-2SS also takes a cis-conformation. However, the exchange rate of the Arg(5) NH proton of MST-2SS was about 100 times faster than that of MST(1-12), and the structure calculation of MST-2SS was not converged on account of the small number of NOEs, indicating that MST-2SS takes a more flexible structure. The hydrogen acceptability of the ester linkage formed by the P2 position residue, Thr(3), is crucial for suppressing the fluctuation of the reactive site and sustaining the inhibitory activity, which enables marinostatin to be one of the smallest protease inhibitors in nature.     

Specific accumulation of 17 alpha-hydroxyprogesterone in microsomal membranes during the process of cytochrome P-450(C-17)-catalysed androgen biosynthesis. A dynamic study of intermediate formation and turnover.

A complete dynamic analysis of cytochrome P-450(C-17)-catalysed androgen biosynthesis from a single dose of progesterone and 17 alpha-hydroxyprogesterone in a double-label double-substrate experiment was performed in order to elucidate the controversial intermediacy of 17 alpha-hydroxyprogesterone. Label distribution within the steroid fractions as well as in the membrane and buffer compartments yields direct evidence that the endogenously formed 17 alpha-hydroxyprogesterone (which is in an 'intermediate state') accumulates to a higher degree in microsomal membranes than does the exogenously added 17 alpha-hydroxyprogesterone (which is in a 'substrate state') under certain conditions. It is also demonstrated that endogenously formed 17 alpha-hydroxyprogesterone may partly leave the membrane compartment (in terms of a 'leakage' or 'overflow' phenomenon) and is then able to equilibrate with the pool of exogenously added 17 alpha-hydroxyprogesterone. Since only the label distribution in the membrane-associated (but not always in the aqueous) 17 alpha-hydroxyprogesterone pool corresponds to the label distribution in the androgen fraction, it is concluded that only the membrane-associated 17 alpha-hydroxyprogesterone pool is directly accessible to cytochrome P-450(C-17)-catalysed conversion into androgens.     

Differences in steroidogenesis by the subcellular fractions of adrenocortical special zone and cortex proper of the female possum (Trichosurus vulpecula)

Steroidogenesis by subcellular fractions of adrenal cortex proper (C.P.) and special zone (S.Z.) of female possum (Trichosurus vulpecula) was studied. Mitochondrial, microsomal and cytosol cell fractions were incubated with appropriate substrates in the presence of an NADPH-generating system. The major products formed from [3H]progesterone and [3H]17 alpha-hydroxyprogesterone by the microsomal fraction of the C.P. were 11-deoxycortisol and 11-deoxycorticosterone and 3 alpha (beta)-hydroxy-5 alpha-androstan-17-one by the S.Z. The mitochondrial fraction converted [3H]11-deoxycortisol to cortisol in yields twenty times higher by the C.P. than by the S.Z. and to 17 alpha, 20 beta,21-trihydroxy-4-pregn-3-one thirty times higher by the S.Z. The conversion of [3H]androstenedione to 11 beta-hydroxyandrostenedione by the C.P. was approximately double that of the S.Z., while 18-hydroxyandrostenedione (tentatively identified) formed the highest yield in both zones. Incubation of the same substrates with cytosol formed two 5 beta-pregnane and two 5 beta-androstane derivatives in total yields less than 5% by C.P. and greater than 60% by S.Z. Aromatase activity, estimated by the release of [3H2O] from [1 beta 3H]testosterone, in the adrenals of 8 possums, was in each experiment negligibly low. Determination of total enzyme activities in the two zones revealed that 11 beta, 18 and 21-hydroxylases were higher in the C.P., while 17 alpha-hydroxylase was higher in the S.Z. Similar results were obtained when the rates of formation of hydroxylated products were estimated in the presence of saturating amounts of substrates. Active 5 alpha- and 5 beta-reductases, C17-20-lyase and 3 alpha (beta) and 20 beta-hydroxysteroid dehydrogenases were found almost exclusively in the S.Z. We conclude that the S.Z. at lower levels of activity than the C.P. could contribute to the basal secretion of corticosteroids. In addition, the S.Z. has a high capacity to form C19 steroids and 5 alpha- and 5 beta-reduced steroids. The possible role of the S.Z. in possum is discussed.     

Interaction between substrate and oxygen ligand responsible for effective O-O bond cleavage in bovine cytochrome P450 steroid 21-hydroxylase proved by Raman spectroscopy

We investigated structural and functional properties of bovine cytochrome P450 steroid 21-hydroxylase (P450c21), which catalyzes hydroxylation at C-21 of progesterone and 17alpha-hydroxyprogesterone. The uncoupled H(2)O(2) formation was higher in the hydroxylation of progesterone (26% of NADPH consumed) than that of 17alpha-hydroxyprogesterone (15% of NADPH consumed), indicating that 17alpha-hydroxyprogesterone can better facilitate the O-O bond scission. In relation to this, it is noted that the O-O stretching mode (nu(O-O)) of the oxygen complex of P450c21 was sensitive to the substrate; the progesterone- or 17alpha-hydroxyprogesterone-bound enzyme gave single (at 1137 cm(-1)) or split nu(O-O) bands (at 1124 and 1138 cm(-1)), respectively, demonstrating the presence of two forms for the latter. In contrast to nu(O-O), no corresponding difference was observed for the Fe-O(2) stretching mode between two different substrate-bound forms. The Fe-S(Cys) stretching mode in the ferric state was also identical (349 cm(-1)) for each substrate-bound form, suggesting that modulation through the axial thiolate by the substrate is unlikely. Therefore, it is deduced that the hydroxyl group at C-17 of 17alpha-hydroxyprogesterone forms a hydrogen bond with the terminal oxygen atom of the FeOO complex in one form, yielding a lower nu(O-O) frequency with higher reactivity for O-O cleavage, whereas the other form in which the substrate does not provide a hydrogen bond to the oxygen ligand is essentially the same between the two kinds of substrates. In the hydrogen-bonded species, the substrate changes the geometry of the FeOO moiety, thereby performing the hydroxylation reaction more effectively in 17alpha-hydroxyprogesterone than in progesterone.     

Expression of cytochrome P450c17 and other steroid-converting enzymes in the rat kidney throughout the life-span

We have investigated the metabolism of [14C]-labelled progesterone (P4) and dehydroepiandrosterone (DHEA) by kidney tissues of newborn and 7-, 15-, 30-, 60- and 365-day-old rats of both sexes. The following enzymes were revealed at all ages by radiochemical identification of the corresponding products: 5alpha-reductase, cytochromes P450c17 and P450c21, 3beta-hydroxysteroid dehydrogenase (HSD)/delta5-delta4 isomerase, and 17beta- and 20alpha-HSDs, catalyzing reductive reactions. The major P4 metabolites were 5alpha-reduced C21 steroids, whose formation was almost completely suppressed by the 5alpha-reductase 4-azasteroid inhibitor, PNU 156765. Androstenedione and testosterone were also formed via 17alpha-hydroxyprogesterone, together with 11-deoxycorticosterone and 20alpha-dihydroprogesterone. DHEA was mainly converted to androst-5-ene-3beta,17beta-diol, with smaller amounts of the above androgens. Cytochrome P450c17 mRNA and protein were demonstrated by Northern blotting and Western blotting analyses, respectively. P450c17 mRNA, assessed by Northern blotting, protein and catalytic activity all peaked in the kidney samples at 15 days of life and declined thereafter. Cytochrome P450arom was below the level of detection of semi-quantitative RT-PCR. Since the rat kidney has been previously shown to contain cytochrome P450scc as well as androgen and estrogen receptors, it is suggested that it is capable of autonomous hormonal steroidogenesis and that renal steroids, or nephrosteroids, may act locally, in a paracrine or autocrine fashion.     

Expression of bovine cytochrome P450c21 and its fused enzymes with yeast NADPH-cytochrome P450 reductase in Saccharomyces cerevisiae

Recombinant plasmids for expression of bovine cytochrome P450c21 (pA gamma 2), both P450c21 and yeast NADPH-cytochrome P450 reductase (pAR gamma 1), P450c21/yeast reductase fused enzymes (pAF gamma R1, pAF gamma R2, and pAF gamma R20), and yeast reductase/P450c21 fused enzymes (pAFR gamma 1 and pAFR gamma 2) were constructed by using expression vector pAAH5. The plasmids were each introduced into the yeast Saccharomyces cerevisiae AH22 cells. The recombinant yeast strains AH22/pA gamma 2 (Y21) and AH22/pAR gamma 1 (Y21R) produced 2-3 X 10(3) molecules of P450c21 per cell. The cultures of both strains converted progesterone and 17 alpha-hydroxyprogesterone into 11-deoxycorticosterone and 11-deoxycortisol, respectively. The 21-hydroxylase activity per cell of the strain Y21R was about three times higher than that of the strain Y21, probably due to overproduction of yeast reductase. The recombinant yeast strains AH22/pAF gamma R1 (Y21RF1), AH22/pAF gamma R2 (Y21RF2), and AH22/pAF gamma R20 (Y21RF20) produced about 1.1-2.0 X 10(4) molecules per cell of the corresponding P450c21/yeast reductase fused enzymes. The specific 21-hydroxylase activity toward 17 alpha-hydroxyprogesterone per cell of the strains Y21RF1, Y21RF2, and Y21RF20 was about 21, 28, and 49 times higher than that of the strain Y21, respectively. Thus, the fused enzymes were superior to P450c21 in the specific activity and in the expression level in the yeast. The Km values for 17 alpha-hydroxyprogesterone of P450c21 in the strains Y21 and Y21R, and of the fused enzymes in the strains Y21RF1 and Y21RF2 were 0.29, 0.30, 0.67, and 0.65 microM, respectively. The Vmax values of P450c21 in the strains Y21 and Y21R, and of the fused enzymes in the strains Y21RF1 and Y21RF2 were 28, 124, 151, and 222 moles/min.mole P450c21 or fused enzyme, respectively. These results indicated that the fused enzymes showed lower affinity for the substrate, probably due to structural modification and higher reaction rates through efficient intramolecular electron transfer as compared with those of P450c21. While the strain AH22/pAFR gamma 2 (YR21F2) produced about 3 X 10(4) molecules per cell of the reductase/P450c21 fused enzyme, the specific 21-hydroxylase activity of the fused enzyme toward 17 alpha-hydroxyprogesterone was extremely low, suggesting that the structure of the fused enzyme might not be suited for electron transfer in yeast microsomes.     

A double-antibody radioimmunoassay for serum progesterone using progesterone-3-(O-carboxymethyl) oximino-[125I]-iodo-histamine as radioligand.

A reliable, convenient and economical radioimmunoassay (RIA) for serum progesterone has been established and tested. This procedure employs diethyl ether extraction followed by RIA utilizing rabbit anti-11 alpha-hydroxyprogesterone 11-hemisuccinyl-bovine serum albumin (progesterone-11 alpha-BSA) serum, progresterone-3-(O-carboxymethyl) oximino-[125I]-iodohistamine (progesterone-3-[125I]) as radioligand and goat anti-rabbit gamma globulin as second antibody. In conjunction with antiprogesterone-11 alpha-BSA serum, the overall assay specificity of the progesterone-3-[125I] RIA is similar to that of the [3H]-progesterone method using dextran-coated charcoal. The results of serum progesterone measurements during the menstrual cycle obtained by the progesterone-3-[125I] RIA appear comparable to those of [3H]-progesterone assays which employ similar anti-progesterone-11 alpha-BSA sera. The progesterone-3-[125I] double-antibody RIA, however, is more convenient and less expensive than the [3H]-progesterone RIA method.     

Tributyltin disturbs bovine adrenal steroidogenesis by two modes of action

Tributyltin, an environmental pollutant, affected adrenal steroid hormone biosynthesis by two modes of action. Treatment of bovine adrenal cultured cells with 10-100 nM tributyltin for 48 h suppressed cortisol and androstenedione secretion, but induced the accumulation of 17alpha-hydroxyprogesterone and deoxycortisol, indicating that the P450(C21) and P450(11beta) activities were specifically suppressed. Direct inhibition of the enzymatic activities due to tributyltin was not observed in isolated organelles of untreated cells at concentrations less than 10 microM. Western blotting experiments using specific antibodies against steroidogenic enzymes showed that treatment with 1-100 nM tributyltin caused a decrease in cellular P450(C21) and P450(11beta) protein levels, and real-time PCR experiments showed that the decrease in protein content was attributable to decreases in mRNA of the enzymes. Tributyltin at concentrations higher than 100 nM suppressed all steroid biosynthesis in the adrenal cells. This suppression was closely correlated to the decrease in steroidogenic acute regulatory protein. Since nanomolar concentrations of tributyltin disturbed steroidogenesis in mammalian cells, there is the possibility that steroid hormone synthesis in polluted wild animals is affected by this compound.     

11-cis-Acyl-CoA:retinol O-acyltransferase activity in the primary culture of chicken Muller cells

A novel retinoid cycle has recently been identified in the cone-dominated chicken retina, and this cone cycle accumulates 11-cis-retinyl esters upon light adaptation. The purpose of this study is to investigate how 11-cis-retinyl esters are formed in the retina. Primary cultures of chicken Muller cells and cell membrane were incubated with all-trans- or 11-cis-retinol to study retinyl ester synthesis. In Muller cells, esterification of 11-cis-retinol was four times greater than esterification of all-trans-retinol. In the presence of palmitoyl-CoA and CRALBP, Muller cell membranes synthesized 11-cis-retinyl ester from 11-cis-retinol at a rate which was 20-fold higher than that of all-trans-retinyl ester. In the absence of CRALBP, 11-cis-retinyl ester synthesis was greatly reduced (by 7-fold). In the absence of palmitoyl-CoA, retinyl ester synthesis was not observed. Muller cell membranes incubated with radiolabeled palmitoyl-CoA resulted in the transfer of the labeled acyl group to retinol. This acyl transfer was greatly reduced in the presence of progesterone, a known ARAT inhibitor. 11-cis-ARAT activity remained unchanged when assayed in the presence of all-trans-retinol, suggesting a distinct catalytic activity from that of all-trans-ARAT. Apparent kinetic rates for 11-cis-ARAT were 0.135 nmol min(-)(1) mg(-)(1) (V(max)) and 11.25 microM (K(M)) and for all-trans-ARAT were 0.0065 nmol min(-)(1) mg(-)(1) (V(max)) and 28.88 microM (K(M)). Our data indicate that Muller cells in the chicken retina possess 11-cis-ARAT activity, thus providing an explanation for the accumulation of 11-cis-retinyl esters in the cone cycle.     

Product inhibition of steroid-17 alpha-monooxygenase by endogenous 17 alpha-hydroxyprogesterone in microsomes and isolated Leydig cells from rat testis

Use of integrated rate equations for analysis of progesterone metabolism by isolated Leydig cells and microsomes from rat testis in presence of several progesterone concentrations within several periods reveals competitive product inhibition by endogenously formed 17 alpha-hydroxyprogesterone (Kpm = 0.1 microM) of steroid-17 alpha-monooxygenase activity (Ksm = 0.8 microM). The discrepancy between this very low interaction constant of endogenous 17 alpha-hydroxyprogesterone with the steroid-17 alpha-monooxygenase, and the respective values (from the literature) for exogenous 17 alpha-hydroxyprogesterone which are about 50-fold higher, may be explained by accumulation of endogenous 17 alpha-hydroxyprogesterone at the catalytic site of the steroid-17 alpha-monooxygenase. This mechanism may be important for intratesticular regulation of androgen biosynthesis from precursor steroids.     

Chemical structure of corticosteroids and its relationship with their acute induction of lordosis in the female rat

The intravenous injection of several corticosteroids in the spayed estrogen-primed rat resulted, 5 min later, in a remarkable induction of lordosis response for deoxycorticosterone and 11-deoxycortisol. A lower but important effect was induced with corticosterone and 17 alpha-hydroxyprogesterone. A chemical structure-biological effect analysis showed that C-21 hydroxylation (e.g., deoxycorticosterone, 11-deoxycortisol, and corticosterone) induces the behavioral effect. However, C-11 or C-17 hydroxylation alone promotes the lordosis response to a lesser degree (corticosterone and 17 alpha-hydroxyprogesterone, respectively). However, the effect of these latter compounds is significant. From the results, it is concluded that deoxycorticosterone and 11-deoxycortisol along with other steroids such as 20 alpha-hydroxyprogesterone are suitable candidates for synergizing the well-known estrogen-progesterone induction of lordotic behavior.     

Evidence for concerted kinetic oxidation of progesterone by purified rat hepatic cytochrome P-450g

Purified cytochrome P-450g, a male-specific rat hepatic isozyme, was observed to metabolize progesterone to two primary metabolites (6 beta-hydroxyprogesterone and 16 alpha-hydroxyprogesterone), two secondary metabolites (6 beta,16 alpha-dihydroxyprogesterone and 6-ketoprogesterone), and one tertiary metabolite (6-keto-16 alpha-hydroxyprogesterone). The Km,app for the formation of these products from progesterone was determined to be approximately 0.5 microM, while the Km,app for metabolism of 6 beta- and 16 alpha-hydroxyprogesterone was found to be 5-10 microM. The ratio of primary to secondary metabolites did not change significantly at progesterone concentrations from 6 to 150 microM, and a lag in formation of secondary metabolites was not observed in 1-min incubations. Concerted oxidation of progesterone to secondary products without the intermediate products leaving the active site was suggested by these results and confirmed by isotopic dilution experiments in which little or no dilution of metabolically formed 6 beta,16 alpha-dihydroxyprogesterone and 6-keto-16 alpha-hydroxyprogesterone was observed in incubations containing a mixture of radiolabeled progesterone and unlabeled 6 beta-hydroxyprogesterone or 16 alpha-hydroxyprogesterone. Incubation of 6 beta-hydroxyprogesterone with a reconstituted system in an atmosphere of 18O2 resulted in greater than 90% incorporation of 18O in the 16 alpha-position of 6 beta,16 alpha-dihydroxyprogesterone but no incorporation of 18O into 6-ketoprogesterone, even though the reaction was dependent upon enzyme and O2, and not inhibited by mannitol, catalase, or superoxide dismutase. Factors which characterize the metabolism of progesterone by cytochrome P-450g in terms of active-site constraints and the catalytic competence of the enzyme in microsomes were also explored.     

Synthesis and biological activity of 7,8-dihydro derivatives of batrachotoxinin interacting with fast sodium channels

7,8-Dihydrobatrachotoxinin (A) (I) was synthesized from 11 alpha-hydroxyprogesterone (III) by a 37-stage procedure. Trimethylpyrrolcarboxylate, benzoate as well as 2-azido-benzoate derivatives of (I) were obtained by mixed anhydride technique, the latter two derivatives being prepared also with tritium atoms in aromatic rings (sp. radioactivity about 28 Cu/mmol). Upon interaction with rat brain synaptosomes the apparent Kd of 7,8-dihydrobatrachotoxinin A 20 alpha-[4-3H]benzoate (Iv) was about 2,5 x 10(-6) M. The (Iv) specific binding was inhibited by aconitine with K0,5 = 1,3 x 10(4) M. Anemonia sulcata toxin II (ATX II) enhanced (Iv) affinity for the receptor up to 7 x 10(-7) M, the maximum binding capacity being 2,5 pmol/mg of protein. Benzocaine and tetracaine competitively displaced specifically bound toxin with K0,5 = 3,1 x 10(-4) M and 5,7 x 10(-7) M, respectively, in the presence of 10(-5) M ATX II. 2-Azido[5-3H]benzoate derivative (Id) was shown to be an effective probe for covalent labeling of the alkaloid toxin receptor of the sodium channel.