Yeast cytochrome P-450 catalyzing lanosterol 14 alpha-demethylation. II. Lanosterol metabolism by purified P-450(14)DM and by intact microsomes

A reconstituted monooxygenase system containing a form of cytochrome P-450, termed P-450(14)DM, and NADPH-cytochrome P-450 reductase, both purified from yeast microsomes, catalyzed the conversion of lanosterol (4,4,14 alpha-trimethyl-5 alpha-cholesta-8,24-dien-3 beta-01) to a sterol metabolite in the presence of NADPH and molecular oxygen. This conversion did not occur anaerobically or when either P-450(14)DM, the reductase, or NADPH was omitted from the system. In both free and trimethylsilylated forms, this metabolite showed a relative retention time (relative to lanosterol) of 1.10 in gas chromatography on OV-17 columns. Comparison of its mass spectrum and retention time with those of lanosterol and 4,4-dimethylzymosterol (4,4-dimethyl-5 alpha-cholesta-8,24-dien-3 beta-ol) indicated that the metabolite was 4,4-dimethyl-5 alpha-cholesta-8,14,24-trien-3 beta-ol. Upon aerobic incubation of microsomes from semianaerobically grown yeast cells in the presence of NADPH and cyanide, endogenous lanosterol was converted to 4,4-dimethylzymosterol. This metabolism was inhibited by CO, metyrapone, SKF-525A, and antibodies to P-450(14)DM. It is concluded that in yeast microsomes lanosterol is 14 alpha-demethylated by a P-450(14)DM-containing monooxygenase system to give rise to 4,4-dimethyl-5 alpha-cholesta-8,14,24-trien-3 beta-ol, which is then reduced to 4,4-dimethylzymosterol by an NADPH-linked reductase.     

Purification and characterization of cytochrome P-45014DM (lanosterol 14 alpha-demethylase) from pig liver microsomes.

Cytochrome P-45014DM, which catalyzes lanosterol 14 alpha-demethylation, from pig liver microsomes was purified to a state of virtually homogeneous by gel electrophoresis. Its apparent monomeric molecular weight was estimated to be 53,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the amino-terminal amino acid sequence was Gly-Leu-Leu-Thr-Gly(Leu)-Asp-Leu-Leu-Gly-Ile. When reconstituted with NADPH-cytochrome P-450-reductase, the enzyme showed a high activity for lanosterol and 24,25-dihydrolanosterol 14 alpha-demethylation. Furthermore, the oxygenated intermediates of 24,25-dihydrolanosterol 14 alpha-demethylation, 32-hydroxy-24,25-dihydrolanosterol and 32-oxo-24,25-dihydrolanosterol, were converted to the 32-nor compound, 4,4-dimethylcholesta-8,14-dien-3 beta-ol, by the reconstituted enzyme system.     

Induction and substrate specificity of the lanosterol 14 alpha-demethylase from Saccharomyces cerevisiae Y222.

The potential inducibility of the lanosterol 14 alpha-demethylase (P-45014DM) from Saccharomyces cerevisiae Y222 by xenobiotics was investigated. This enzyme and NADPH-cytochrome P-450 reductase were unaffected by a number of compounds known to induce mammalian and some yeast cytochrome P-450 monooxygenases. Furthermore, dibutyryl cyclic AMP did not affect P-45014DM or P-450 reductase levels, while growth at 37 degrees C resulted in a slight decrease. P-45014DM was found to be specific for lanosterol and did not metabolize a number of P-450 substrates including benzo[a]pyrene.     

Deformylation of 32-oxo-24,25-dihydrolanosterol by the purified cytochrome P-45014DM (lanosterol 14 alpha-demethylase) from yeast evidence confirming the intermediate step of lanosterol 14 alpha-demethylation

32-Oxo-24,25-dihydrolanosterol (32-oxo-DHL) was deformylated to 4,4-dimethylcholesta-8,14-dien-3 beta-ol, the product of 14 alpha-demethylation of 24,25-dihydro-lanosterol (DHL), by the reconstituted lanosterol 14 alpha-demethylase system consisting of cytochrome P-45014DM and NADPH-cytochrome P-450 reductase of yeast. Affinity of 32-oxo-DHL to the cytochrome was considerably higher than those of lanosterol and DHL, and the rate of deformylation of 32-oxo-DHL was faster than the rate of demethylation of lanosterol and DHL. Spectral analysis of the 32-oxo-DHL complex of cytochrome P-45014DM suggested the interaction between the 32-aldehyde group and the heme iron. These observations, together with our preceding findings on the metabolism of 32-hydroxy-24,25-dihydrolanosterol (Aoyama, Y., Yoshida, Y., Sonoda, Y., and Sato, Y. (1987) J. Biol. Chem. 262, 1239-1243), indicate that the 14 alpha-demethylation of lanosterol catalyzed by cytochrome P-45014DM proceeds with three step monooxygenations via the 32-hydroxy and 32-oxo intermediates, and the cytochrome mediates this sequential reaction without releasing the intermediates.     

Role of the side chain of lanosterol in substrate recognition and catalytic activity of lanosterol 14 alpha-demethylase (cytochrome P-450 (14DM)) of yeast

The 14 alpha-demethylation of 24,25-dihydrolanosterol (DHL) derivatives having trimmed side chains, 27-nor-DHL, 26,27-dinor-DHL, 25,26,27-trinor-DHL, 24,25,26,27-tetranor-DHL, 23,24,25,26,27-pentanor-DHL and 22,23,24,25,26,27-hexanor-DHL, was studied with the reconstituted lanosterol 14 alpha-demethylase system consisting of cytochrome P-450(14DM) and NADPH-cytochrome P-450 reductase both purified from yeast microsomes. The demethylase catalyzed the 14 alpha-demethylation of the derivatives having the side chains longer than tetranor but the activities for the trinor- and tetranor-derivatives were lower. Kinetic analysis indicated that affinity of the trinor-derivative for the demethylase was considerably higher than that of DHL. The affinities of the 27-nor- and dinor-derivatives were increased by this order and were the intermediates of DHL and the trinor derivative. On the other hand, Vmax values of the demethylase for the DHL derivatives were decreased depending on their side-chain lengths, and the substrate-dependent reduction rate of cytochrome P-450(14DM) was also decreased in the same manner. Based on these observations, it was concluded that interaction of the side chain of lanosterol especially C-25, 26 and 27 with the substrate site of lanosterol 14 alpha-demethylase was necessary for enhancing the catalytic activity of the enzyme. However, this interaction was considered not to be essential for substrate binding.     

Metabolism of 32-hydroxy-24,25-dihydrolanosterol by purified cytochrome P-45014DM from yeast. Evidence for contribution of the cytochrome to whole process of lanosterol 14 alpha-demethylation

Metabolism of 32-hydroxy-24,25-dihydrolanosterol (lanost-8-ene-3 beta,32-diol), a posturated intermediate of the 14 alpha-demethylation (removal of C-32) of 24,25-dihydrolanosterol (lanost-8-en-3 beta-ol), by a reconstituted system consisting of yeast cytochrome P-450 which catalyzes lanosterol 14 alpha-demethylation (cytochrome P-45014DM) (Yoshida, Y., and Aoyama, Y. (1984) J. Biol. Chem. 259, 1655-1660 and Aoyama, Y., Yoshida, Y., and Sato, R. (1984) J. Biol. Chem. 259, 1661-1666) and NADPH-cytochrome P-450 reductase was studied. The reconstituted system converted both 32-hydroxy-24,25-dihydrolanosterol and 24,25-dihydrolanosterol to 4,4-dimethyl-5 alpha-cholesta-8,14-dien-3 beta-ol, the 14 alpha-demethylated product of the latter. The metabolism of these compounds was inhibited by a low concentration of ketoconazole which is a potent cytochrome P-45014DM inhibitor. Affinity of cytochrome P-45014DM for 32-hydroxy-24,25-dihydrolanosterol was about 20 times higher than for 24,25-dihydrolanosterol and the cytochrome metabolized the former about 4 times faster than the latter under the experimental conditions. Spectral analysis suggested that the 32-hydroxyl group of 32-hydroxy-24,25-dihydrolanosterol interacted with the heme iron of the oxidized cytochrome and this interaction might support the high affinity of this compound for the cytochrome. These lines of evidence indicate that 32-hydroxy-24,25-dihydrolanosterol is the intermediate of the 14 alpha-demethylation of 24,25-dihydrolanosterol by cytochrome P-45014DM. It is also clear that the cytochrome catalyzes further metabolism of the 32-hydroxylated intermediate to the 14 alpha-demethylated product with higher efficiency than the 32-hydroxylation of the substrate. Cytochrome P-45014DM is thus classified as lanosterol C14-C32 lyase.     

Stereo-selective interaction of enantiomers of diniconazole, a fungicide, with purified P-450/14DM from yeast

R(-) isomer of diniconazole [S-3308L, (E)-1-(2,4-dichlorophenyl)-4,4-dimethyl-2-(1,2,4-triazol-l-yl)-1-+ ++penten-3-ol], a newly developed fungicide strongly inhibited lanosterol 14 alpha-demethylation catalyzed by a yeast cytochrome P-450 (P-450/14DM). On the other hand, S(+) isomer of diniconazole was a weaker inhibitor for P-450/14DM. The R(-) isomer combined with both ferric and ferrous P-450/14DM and interfered binding of CO to the cytochrome. The S(+) isomer also interacted with both forms of P-450/14DM but the absorption spectra of the S(+)-diniconazole complexes were different from those of the R(-)-diniconazole complexes. Furthermore, S(+) isomer did not significantly interfere the binding of CO to P-450/14DM. These observations suggest that P-450/14DM discriminates enantiomers of diniconazole and the R(-) isomer is more favorably fit for the active site of the cytochrome.     

Isolation and characterization of an altered cytochrome P-450 from a yeast mutant defective in lanosterol 14 alpha-demethylation

A cytochrome P-450 (P-450SG1) was purified from a lanosterol 14 alpha-demethylase (P-450(14DM)) defective mutant of Saccharomyces cerevisiae, strain SG1, by a method similar to that used in the purification of the wild type enzyme (Yoshida, Y., and Aoyama, Y. (1984) J. Biol. Chem. 259, 1655-1660). P-450SG1 had the same apparent Mr as and was immunochemically identical to P-450(14DM). Peptide maps of P-450SG1 made by limited proteolysis with Staphylococcus aureus V8 proteinase, chymotrypsin, or papain followed by gel electrophoresis were identical to corresponding peptide maps of P-450(14DM). However, P-450SG1 showed no lanosterol 14 alpha-demethylase activity and its mode of interaction with diniconazole [(E)-1-(2,4-dichlorophenyl)-4,4-dimethyl-2-(1,2,4-triazol-1-y1)-1- penten-3- o1], a specific inhibitor of P-450(14DM), was fundamentally different from that of P-450(14DM). The absorption spectrum of ferric P-450SG1 was unusual for a native low-spin cytochrome P-450 and was superimposable on that of 1-methylimidazole complex of P-450(14DM), indicating that P-450SG1 has a histidine 6th ligand trans to the thiolate 5th ligand, while the 6th ligand of other ferric low-spin cytochrome P-450s is a water molecule or a hydroxyl group of an oxyamino acid. It is concluded that P-450SG1 is an altered P-450(14DM). Difference in the primary structure between P-450SG1 and P-450(14DM) may be slight and was not detected by peptide mapping. However, the alteration caused significant change in the substrate site and heme environments of the cytochrome. P-450SG1 is the first example of a cytochrome P-450 having a histidine axial ligand trans to thiolate and of a genetically altered cytochrome P-450 isolated in a homogeneous state.     

Yeast cytochrome P-450 catalyzing lanosterol 14 alpha-demethylation. I. Purification and spectral properties

A form of cytochrome P-450 catalyzing lanosterol 14 alpha-demethylation (tentatively called "P-450(14)DM") was purified from microsomes of semi-anaerobically grown cells of Saccharomyces cerevisiae to gel electrophoretic homogeneity. An apparent monomeric Mr = 58,000 was estimated for the purified cytochrome by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both optical and EPR spectra of oxidized P-450(14)DM are characteristic of low spin ferric heme proteins, and its reduced CO complex showed a Soret absorption peak at 447 nm. As in the case of hepatic microsomal cytochromes P-450, the ethyl isocyanide complex of reduced P-450(14)DM was in a pH-dependent equilibrium between two states having Soret peaks at 429 and 453 nm, the equilibrium being considerably shifted toward the 453-nm state. Oxidized P-450(14)DM was peculiar in that in its CD spectrum there was a negative shoulder at 425 nm and the 350- and 414-nm troughs possessed larger and relatively smaller [theta] values, respectively, than those reported for other low spin ferric cytochromes P-450. Lanosterol was the only compound which caused a Type I spectral change in oxidized P-450(14)DM. The lanosterol-induced low to high spin state change was, however, only slight even at saturating concentrations of the sterol, indicating that the lanosterol-P-450(14)DM adduct was in a spin state equilibrium.     

Structural analysis of the interaction between the side-chain of substrates and the active site of lanosterol 14 alpha-demethylase (P-450(14)DM) of yeast.

The role of the side-chain of lanosterol in the enzyme-substrate interaction of yeast P-450(14)DM (lanosterol 14 alpha-demethylase) was analyzed with lanosterol derivatives having functional groups on the side-chain. Purified P-450(14)DM from Saccharomyces cerevisiae catalyzed 14 alpha-demethylation of 26-hydroxylanosterol and 25-hydroxy-24,25-dihydrolanosterol with a lower activity than lanosterol and 24,25-dihydrolanosterol. This enzyme demethylated the (Z)-24-ethylidene-24,25-dihydrolanosterol with a low rate, but did not metabolize the E-isomer. The apparent Km of 26-hydroxylanosterol was 10.8 microM, which was higher than that of lanosterol, but lower than that of 24,25-dihydrolanosterol. On the other hand, competition experiments suggested that the affinity of 25-hydroxy-24,25-dihydrolanosterol and (Z)-24-ethylidene-24,25-dihydrolanosterol for P-450(14)DM was significantly lower than that of 24,25-dihydrolanosterol. Integration of the present results with the preceding ones (Aoyama, Y., Yoshida, Y., Sonoda, Y. and Sato, Y. (1991) Biochim. Biophys. Acta, 1081, 262-266 and Aoyama, Y. and Yoshida, Y. (1991) Biochem. Biophys. Res. Commun., 178, 1064-1071) suggests that yeast P-450(14)DM recognizes two parts of the side-chain, the structure around C-24 and the terminal fork consisting of C-25, C-26 and C-27.     

Collagenolytic activity of rabbit V2-carcinoma growing at multiple sites.

Interaction between lanosterol and cytochrome P-450 purified from microsomes of anaerobically-grown Saccharomyces cerevisiae was studied. Lanosterol (4,4,14α-trimethyl-5α-cholesta-8,24-dien-3β-ol) stimulated the oxidation of NADPH by molecular oxygen in the presence of cytochrome P-450 and NADPH-cytochrome P-450 reductase both purified from S. cerevisiae microsomes. Lanosterol stimulated the reduction of cytochrome P-450 by NADPH with the cytochrome P-450 reductase, and induced Type I spectral change of cytochrome P-450. These observations suggest that lanosterol interacts to the substrate region of cytochrome P-450 of S. cerevisiae. Based on these facts, possible role of cytochrome P-450 in lanosterol metabolism in yeast cell is discussed.     

7-Oxo-24,25-dihydrolanosterol: a novel lanosterol 14 alpha-demethylase (P-45014DM) inhibitor which blocks electron transfer to the oxyferro intermediate

7-Oxo-24,25-dihydrolanosterol (3 beta-hydroxy-8-lanosten-7-one, 7-oxo-HDL) was a potent competitive inhibitor for lanosterol 14 alpha-demethylase (cytochrome P-45014DM) of Saccharomyces cerevisiae. Affinity of 7-oxo-DHL for the enzyme was more than 50-times higher than those of the inherent substrates, lanosterol and 24,25-dihydrolanosterol. 7-Oxo-DHL accelerated NADPH-dependent reduction of cytochrome P-45014DM in the reconstituted system consisting of the cytochrome and NADPH-cytochrome P-450 reductase. These observations indicated that 7-oxo-DHL interacted with the substrate site of cytochrome P-45014DM. However, 7-oxo-DHL was not metabolized by the reconstituted system. Incubation of 7-oxo-DHL with the reconstituted system caused accumulation of oxyferro intermediate of cytochrome P-45014DM. It can thus be concluded that 7-oxo-DHL interfered with electron transfer to the oxyferro intermediate of the cytochrome, though it stimulated reduction of the heme iron. So far as we know, 7-oxo-DHL is the first example of a cytochrome P-450 inhibitor which selectively interferes with the electron transfer to oxyferro intermediate. 7 alpha-Hydroxy-24,25-dihydrolanosterol was also a competitive inhibitor of cytochrome P-45014DM. However, this compound was metabolized by the reconstituted system and could not block the electron transfer to oxyferro intermediate. 11-Oxo-24,25-dihydrolanosterol, an isomer of 7-oxo-DHL, did not have such inhibitory effects. These lines of evidence suggest a possibility that the keto group at C-7 of lanost-8-ene skeleton may interact with a certain site of cytochrome P-45014DM which has an important role in the electron transfer to oxyferro intermediate.     

Evidence for the presence of cytochrome P-450 functional in lanosterol 14 alpha-demethylation in microsomes of aerobically grown respiring yeast

Recent studies have shown that a cytochrome P-450 present in microsomes of semi-anaerobically grown cells of Saccharomyces cerevisiae is functional in the 14 alpha-demethylation of lanosterol (4,4,14 alpha-trimethyl-5 alpha-cholesta-8,24-dien-3 beta-ol), but the occurrence of the same cytochrome P-450 in microsomes of aerobically grown yeast cells has not yet been reported. In this study, the microsomal fraction from aerobically grown cells was found to catalyze the lanosterol demethylation in the presence of NADPH and O2 and that this activity was sensitive to CO. In Ouchterlony double diffusion test, antibodies to the yeast cytochrome P-450 formed a single precipitin line with the microsomal fraction as well as with the purified yeast cytochrome P-450 and the two precipitin lines fused with each other. Furthermore, the antibodies inhibited the lanosterol demethylation activity of the microsomal fraction from aerobically grown cells. The quadratic-derivative absorption spectrum of the microsomal fraction measured in the presence of both Na2S2O4 and CO showed an absorption band at 450 nm which is attributable to the reduced CO compound of cytochrome P-450. These facts led to the conclusion that cytochrome P-450 actually exists in aerobically grown yeast and participates in the lanosterol 14 alpha-demethylation which is essential for the ergosterol (5 alpha-ergosta-5,7,22-trien-3 beta-ol) biogenesis by yeast.     

Involvement of cytochrome b5 and a cyanide-sensitive monooxygenase in the 4-demethylation of 4,4-dimethylzymosterol by yeast microsomes

According to Ohba et al. (Ohba, M., Sato, R., Yoshida, Y., Nishino, T. and Katsuki, H. (1978) Biochem. Biophys. Res. Commun. 85, 21-27), yeast microsomes catalyze the removal of three methyl groups attached to the C-4 and C-14 positions of [1,7,15,22,26,30-14C]lanosterol (4,4,14 alpha-trimethyl-5 alpha-cholesta-8,24-dien-3 beta-ol) in the presence of NADPH, NAD+ and molecular oxygen, concomitant with the liberation of 14CO2 derived from C-30 (one of the two methyl groups at the C-4 position). In this process the methyl group at the C-14 position is first removed in a cyanide-insensitive reaction and then the two methyl groups at the C-4 position are removed by a cyanide-sensitive enzyme system. In this study it was found that the 14CO2 formation from the 14C-labeled lanosterol was inhibited by antibodies to yeast cytochrome b5 and by palmitoyl-CoA, a substrate of the cytochrome b5-containing fatty acyl-CoA desaturase system of yeast microsomes. However, neither the antibodies nor palmitoyl-CoA inhibited the conversion of lanosterol to 4,4-dimethyl zymosterol (4,4-dimethyl-5 alpha-cholesta-8,24-dien-3 beta-ol). It is concluded that cytochrome b5 and a cyanide-sensitive enzyme are involved in the 4-demethylation of 4,4-dimethylzymosterol, but not the 14 alpha-demethylation of lanosterol, by yeast microsomes. It is suggested that a cyanide-sensitive enzyme acts as the terminal 4-demethylase and cytochrome b5 transfers reducing equivalents from NADPH to the terminal enzyme, as in the case of fatty acyl-CoA desaturation. The cyanide sensitivity of the 4-demethylation was, however, much greater than that of the desaturation.     

Meiosis-activating sterol synthesis in rat preovulatory follicle: is it involved in resumption of meiosis?

Meiosis-activating sterol (MAS) was shown to overcome the inhibitory effect of hypoxanthine on spontaneous maturation of mouse oocytes and was suggested to mediate the stimulation of meiosis by gonadotropins. Follicular fluid (FF)-MAS is synthesized by cytochrome P450 lanosterol 14alpha-demethylase (LDM). Follicular LDM was preferentially localized in oocytes by immunohistochemistry. Using [3H]acetate or R-[5-3H]mevalonate as precursors as well as high-performance liquid chromatographic and thin-layer chromatographic separation, we have measured the concentrations of de novo-synthesized lanosterol, FF-MAS, and cholesterol in rat graafian follicles, cumulus-oocyte complexes (COCs), and denuded oocytes (DOs) treated with LH, AY-9944 (an inhibitor of Delta14-reductase, which was anticipated to increase FF-MAS levels by inhibiting its metabolism), or both after 8 h of culture. In follicles, both LH and AY-9944 increased the accumulation of FF-MAS as compared to controls. In COCs, AY-9944 caused a marked increase in FF-MAS, but we were unable to detect accumulation of FF-MAS in DOs. Neither the endogenous increases in FF-MAS accumulation nor the addition of FF-MAS to the culture medium could overcome the inhibition on resumption of meiosis by phosphodiesterase inhibitors. Compared to LH-induced resumption of meiosis in follicles, that induced by AY-9944 was much delayed. These results call into question any role of FF-MAS as an obligatory mediator of LH activity on germinal vesicle breakdown. The discrepancy between the positive staining for LDM in oocytes and our inability to detect de novo synthesized FF-MAS in DOs may relate to the sensitivity of the methodology employed and either the number of oocytes used or a deficiency in LDM synthetic activity in such oocytes. Further studies are required to confirm any of these alternatives.     

The 14 alpha-demethylation of obtusifoliol by a cytochrome P-450 monooxygenase from higher plants' microsomes

Microsomes isolated from corn embryos (Zea mays) can demethylate the 14 alpha-methyl group of obtusifoliol 2. An enzymatic assay has been developed for obtusifoliol 14 alpha-methyl-demethylase in higher plants. The enzymatic reaction was shown to occur sequentially, converting obtusifoliol 2 to 4 alpha-methyl-5 alpha-ergosta-8,24(28)-dien-3 beta-ol 4 via the trienol 4 alpha-methyl-5 alpha-ergosta-8,14,24(28)-trien-3 beta-ol 3 which was thoroughly identified. This enzymatic reaction is dependent of NADPH and molecular oxygen. It is inhibited by CO, menadione and specific inhibitors of cytochrome P-450, the CO inhibition being partially reversed by light. It is concluded that in Zea mays microsomes, obtusifoliol is demethylated at C-14 by a cytochrome P-450 containing monooxygenase system.     

The 3-hydroxy group of lanosterol is essential for orienting the substrate site of cytochrome P-450(14DM) (lanosterol 14 alpha- demethylase)

Interaction of lanosterol, 3-epilanosterol, 3-oxolanosta-8,24-diene, 3-methylenelanost-8-ene and lanosterol acetate with cytochrome P-450(14DM) were studied. The cytochrome mediated the 14alpha-demethylation of 3-epilanosterol with nearly the same activity as lanosterol but could not mediate the 14alpha-demethylation of the 3-methylene derivative and the 3-acetate. The cytochrome catalyzed the 14alpha-demethylation of the 3-oxo derivative with low rate. Based on these and some additional observations the hydrogen bond formation between the 3-hydroxy group of lanosterol and the specific amino acid residue in the substrate site is assumed to be essential for orienting the substrate in the substrate site of the cytochrome.     

Buthiobate: a potent inhibitor for yeast cytochrome P-450 catalyzing 14 alpha-demethylation of lanosterol

Buthiobate (S-n-butyl S'-p-tert-butylbenzyl N-3-pyridyldithiocarbon-imidate), a fungicide, inhibited 14 alpha-demethylation of lanosterol catalyzed by a reconstituted enzyme system consisting of cytochrome P-450 (P-450(14)-DM) and NADPH-cytochrome P-450 reductase both purified from Saccharomyces cerevisiae. Concentration of buthiobate necessary for the 50% inhibition was 0.3 microM and this value was markedly lower than those of metyrapone and SKF-525A. Buthiobate bound stoichiometrically to P-450(14)-DM and induced Type II spectral change of the cytochrome. Buthiobate inhibited lanosterol-dependent enzymatic reduction of the cytochrome. These facts indicate that buthiobate binds to P-450(14)-DM with high affinity and acts as a potent inhibitor on the cytochrome.     

Effect of inhibition of sterol delta 14-reductase on accumulation of meiosis-activating sterol and meiotic resumption in cumulus-enclosed mouse oocytes in vitro

Two sterols of the cholesterol biosynthetic pathway induce resumption of meiosis in mouse oocytes in vitro. The sterols, termed meiosis-activating sterols (MAS), have been isolated from human follicular fluid (FF-MAS, 4,4-dimethyl-5 alpha-cholest-8,14,24-triene-3 beta-ol) and from bull testicular tissue (T-MAS, 4,4-dimethyl-5 alpha-cholest-8,24-diene-3 beta-ol). FF-MAS is the first intermediate in the cholesterol biosynthesis from lanosterol and is converted to T-MAS by sterol delta 14-reductase. An inhibitor of delta 7-reductase and delta 14 reductase, AY9944-A-7, causes cells with a constitutive cholesterol biosynthesis to accumulate FF-MAS and possibly other intermediates between lanosterol and cholesterol. The aim of the present study was to evaluate whether AY9944-A-7 added to cultures of cumulus-oocyte complexes (COC) from mice resulted in accumulation of MAS and meiotic maturation. AY9944-A-7 stimulated dose dependently (5-25 mumol l-1) COC to resume meiosis when cultured for 22 h in alpha minimal essential medium (alpha-MEM) containing 4 mmol hypoxanthine l-1, a natural inhibitor of meiotic maturation. In contrast, naked oocytes were not induced to resume meiosis by AY9944-A-7. When cumulus cells were separated from their oocytes and co-cultured, AY9944-A-7 did not affect resumption of meiosis, indicating that intact oocyte-cumulus cell connections are important for AY9944-A-7 to exert its effect on meiosis. Cultures of COC with 10 mumol AY9944-A-7 l-1 in the presence of [3H]mevalonic acid, a natural precursor for steroid synthesis, resulted in accumulation of labelled FF-MAS, which had an 11-fold greater amount of radioactivity incorporated per COC compared with the control culture without AY9944-A-7. In contrast, incorporation of radioactivity into the cholesterol fraction was reduced 30-fold in extracts from the same oocytes. The present findings demonstrate for the first time that COC can synthesize cholesterol from mevalonate and accumulate FF-MAS in the presence of AY9944-A-7. Furthermore, AY9944-A-7 stimulated meiotic maturation dose dependently, indicating that FF-MAS, and possibly other sterol intermediates of the cholesterol synthesis pathway, play a central role in stimulating mouse oocytes to resume meiosis. The results also indicate that oocytes may not synthesize steroids from mevalonate.     

Microsomal enzymes of cholesterol biosynthesis. Purification of lanosterol 14 alpha-methyl demethylase cytochrome P-450 from hepatic microsomes

Employing reconstitution assays and measurement of cytochrome P-450 content, lanosterol 14 alpha-demethylase and cholesterol 7 alpha-hydroxylase have been studied in solubilized preparations of rat hepatic microsomes. Both activities have been resolved from other cytochrome P-450 isozymes and each other by chromatography on DEAE-Sephacel and adsorption on hydroxylapatite. The demethylase has been further purified to homogeneity by cation exchange chromatography on Mono-S resin. The purified cytochrome displays a specific content of 15.8 nmol of heme/mg of protein and a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent Mr of 51,000. A Soret maximum for the reduced/CO binding complex at 448 nm is observed. Reconstitution of the purified cytochrome with NADPH-cytochrome-c reductase, dilaurylphosphatidylcholine, NADPH, and O2 supports the demethylation process which is inhibited by CO. Reconstitution also affords accumulation of oxygenated, metabolic intermediates with single catalytic turnover of the cytochrome, thus supporting the hypothesis that a single isozyme of cytochrome P-450 is responsible for all three oxidations and the lyase activity involved in the lanosterol C-32 demethylation sequence. Low oxidase activity toward several xenobiotic substrates and selectivity toward endogenous sterol substrates is observed for the purified cytochrome. These results indicate a high degree of substrate specificity for the cytochrome, which would be expected for a constitutive P-450 involved in anabolic biochemical processes.