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At least 19 recordsLinked to original sources
PubMed1989

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.

Binding, Competitive↗
PubMed1984

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.

Aerobiosis↗
PubMed1989

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.

Chemical Phenomena↗
PubMed1991

Lanosterol 14 alpha-demethylase (P45014DM): effects of P45014DM inhibitors on sterol biosynthesis downstream of lanosterol.

Lanosterol 14 alpha-demethylase (P45014DM) is the cytochrome P450 enzyme complex responsible for an early step in cholesterol biosynthesis, namely the 14 alpha-demethylation of lanosterol. We have synthesized a novel series of steroidal substrate analogues, designed to be specific and potent inhibitors of P45014DM. We describe here the effects of these compounds on sterol biosynthesis downstream from lanosterol, focusing ultimately on their efficacy as inhibitors of cholesterol biosynthesis. Results using a radio-high performance liquid chromatography (HPLC) assay show that in rat liver microsomal preparations, with [24,25-3H]dihydrolanosterol as substrate, the compounds do indeed inhibit the biosynthesis of sterols downstream from lanosterol. A range of inhibitory potencies was observed, and the key enzyme being inhibited was believed to be P45014DM. Inhibitor efficacy was readily correlated with non-metabolized [24,25-3H]dihydrolanosterol, formation of 4,4-dimethyl-cholest-8-en-3 beta-ol, and formation of lathosterol, a sterol believed to be an excellent indicator of whole body cholesterol biosynthesis in humans.

Animals↗
PubMed1991

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.

Catalysis↗
PubMed1989

Role of the 8-double bond of lanosterol in the enzyme-substrate interaction of cytochrome P-450(14DM) (lanosterol 14 alpha-demethylase).

The role of the 8-double bond of lanosterol in the enzyme-substrate interaction of yeast cytochrome P-450(14DM) (lanosterol 14 alpha-demethylase) was studied by analyzing metabolism of 8-lanostene-3 beta,32-diol, 7-lanostene-3 beta,32-diol, 6-lanostene-3 beta,32-diol and lanostane-3 beta,32-diol by the cytochrome. 8-Lanostene-3 beta,32-diol was actively metabolized by cytochrome P-450(14DM) and converted to the 32-nor-14-unsaturated metabolite. 7-Lanostene-3 beta,32-diol was also metabolized by the cytochrome, but the rate of metabolism was low. However, the cytochrome failed to catalyze the conversion of 6-lanostene-3 beta,32-diol and lanostane-3 beta,32-diol to their 32-nor metabolites. Spectral analysis of the sterol-cytochrome complexes and kinetics of cytochrome P-450(14DM) reduction in the presence of the sterols indicated that 6-lanostene-3 beta,32-diol and lanostane-3 beta,32-diol could not interact with the substrate site of the cytochrome. These results revealed that the 8-double bond of lanosterol plays an important role in the enzyme-substrate interaction of cytochrome P-450(14DM).

Cytochrome P-450 Enzyme System↗
PubMed1990

Determination of serum levels of unesterified lanosterol by isotope dilution-mass spectrometry.

The synthesis of 2H3-labelled lanosterol is described. This compound was used for assay of unesterified lanosterol in serum by isotope dilution-mass spectrometry. After addition of a fixed amount of internal standard (150 ng) to a fixed amount of serum (250 microliters) the steroids were extracted with chloroform and subjected to Lipidex 5000 chromatography. The fraction containing lanosterol was eluted with methanol. This fraction was converted into trimethylsilyl derivative and subjected to mass spectrometric analysis with selected monitoring of the ions at m/z 498 (molecular ion of unlabelled lanosterol) and m/z 501 (molecular ion of 2H3-labelled lanosterol). A standard curve was used for quantification of lanosterol in serum. Under the conditions employed the coefficient of variation was less than 4%. In a recovery experiment the maximal difference between expected and found value was about 5%. Sera from 10 healthy subjects were found to have a mean concentration of lanosterol of 225 ng/ml with a SD of 63 ng/ml. Using a less accurate method for analysis of lanosterol we have shown previously that there is a high correlation between the hepatic HMG CoA reductase and the absolute and relative concentration of lanosterol in serum (concentration of lanosterol relative to cholesterol). When using the same (old) sera as in the previous investigation this could not be confirmed, most probably because of the degradation of lanosterol as a consequence of prolonged storage and/or freezing and thawing. It appears that serum lanosterol must be analysed as soon as possible after the collection of the blood sample in order to obtain accurate results.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1987

Modulation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase by azole antimycotics requires lanosterol demethylation, but not 24,25-epoxylanosterol formation.

The lanosterol 14 alpha-methyl demethylase inhibitors miconazole and ketoconazole have been used to assess their effects upon cholesterol biosynthesis in cultured Chinese hamster ovary cells. In Chinese hamster ovary cells treated with either agent, an initial accumulation of lanosterol and dihydrolanosterol has been observed. At elevated concentrations, however, ketoconazole, but not miconazole, causes the preferential accumulation of 24,25-epoxylanosterol and squalene 2,3:22,23-dioxide. These metabolites accumulate at the expense of lanosterol, thereby demonstrating a second site of inhibition for ketoconazole in the sterol biosynthetic pathway. Both demethylase inhibitors produced a biphasic modulation of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, the rate-limiting enzyme in the cholesterol biosynthetic pathway. The biphasic modulation is characterized by low levels of the drugs suppressing HMG-CoA reductase activity which is restored to either control or above control values at higher drug concentrations. This modulatory effect of the lanosterol demethylase inhibitors upon HMG-CoA reductase was not observed in the lanosterol 14 alpha-methyl demethylase-deficient mutant AR45. Suppression of HMG-CoA reductase activity is shown to be due to a decrease in the amount of enzyme protein consistent with a steroidal regulatory mechanism. Collectively, the results establish that lanosterol 14 alpha-methyl demethylation, but not 24,25-epoxylanosterol formation, is required to suppress HMG-CoA reductase in the manner described by lanosterol demethylase inhibitors.

Animals↗
PubMed1985

The effect of lanosterol on platelet aggregation in human platelets.

It has been reported that lanosterol can sensitize isolated rat platelets to agonists such as ADP and thrombin (4). The purpose of this paper was to determine whether lanosterol had similar effects on human platelets and whether this was achieved by changes in membrane fluidity. Lanosterol did increase the sensitivity of human platelets, particularly to adrenaline and ADP at concentrations as low as 5 mg.L-1 when added from solutions in ethanol. At similar concentrations cholesterol, 4-cholesten-3-one or ethynyloestradiol had either no effect or were inhibitory. Measurement of membrane fluidity with diphenylhexatriene indicated that lanosterol did not affect membrane fluidity. Incubation of platelets with [4C]-mevalonic acid gave rise to a very small incorporation into lanosterol, squalene and farnesol. Sudden activation of the platelets did not accelerate lanosterol synthesis during or after platelet aggregation. It was concluded that lanosterol could only influence platelet behaviour if it came from the plasma. However the concentration of the steroid in both platelets and plasma is ten fold less than that required to sensitise the platelets.

Blood Platelets↗
PubMed1989

Regulation of hepatic cholesterol biosynthesis. Effects of a cytochrome P-450 inhibitor on the formation and metabolism of oxygenated sterol products of lanosterol.

The involvement of oxygenated cholesterol precursors in the regulation of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase activity was studied by examining the effect of ketoconazole on the metabolism of mevalonic acid, lanosterol and the lanosterol metabolites, lanost-8-ene-3 beta,32-diol,3 beta-hydroxylanost-8-en-32-al and 4,4-dimethylcholesta-8,14-dien-3 beta-ol, in liver subcellular fractions and hepatocyte cultures. Inhibition of cholesterol synthesis from mevalonate by ketoconazole at concentrations up to 30 microM was due exclusively to a suppression of cytochrome P-450LDM (LDM = lanosterol demethylase) activity, resulting in a decreased rate of lanosterol 14 alpha-demethylation. No enzyme after the 14 alpha-demethylase step was affected. When [14C]mevalonate was the cholesterol precursor, inhibition of cytochrome P450LDM was accompanied by the accumulation of several labelled oxygenated sterols, quantitatively the most important of which was the C-32 aldehyde derivative of lanosterol. There was no accumulation of the 24,25-oxide derivative of lanosterol, nor of the C-32 alcohol. Under these conditions the activity of HMG-CoA reductase declined. The C-32 aldehyde accumulated to a far greater extent when lanost-8-ene-3 beta,32-diol rather than mevalonate was used as the cholesterol precursor in the presence of ketoconazole. With both precursors, this accumulation was reversed at higher concentrations of ketoconazole in liver subcellular fractions. A similar reversal was not observed in hepatocyte cultures.

Animals↗
PubMed1976

Binding of squalene, lanosterol, desmosterol, and cholesterol to proteins in brain and liver 105,000 g supernatant fractions: evidence for specific binding sites.

The binding of squalene, lanosterol, desmosterol, and cholesterol to proteins in 105,000 g supernatant fraction (S105) from brain and liver of rats was investigated. The S105 fractions from both tissues contain specific binding sites for sterols, which are sensitive to trypsin. The dissociation constants for squalene and sterol protein complexes were in the range of 10(-6) M and were not appreciably different for proteins in brain and liver S105. Competition studies revealed that both brain and liver S105 contain one receptor protein which binds lanosterol and is specific for methyl sterols, and a second receptor which binds both desmosterol and cholesterol. Binding of 7-dehydrocholesterol reported by others must occur at a third independent site since this compound does not interfere with the binding of lanosterol, desmosterol, or cholesterol. Although binding of squalene to proteins in brain and liver S105 does occur, we were unable to show the specificity of squalene binding. The concentration of desmosterol and cholesterol binding sites, which ranged from 6 to 10 nmol/mg protein, was 3- to 5-fold higher than the concentration of squalene and lanosterol binding sites (1.6-2.3 nmol/mg protein). The brain S105 from suckling rats contained fewer binding sites for desmosterol and cholesterol than the brain S105 from weaned rats. However, the concentration of lanosterol binding sites in brain S105 did not show an age-dependent change. The receptor proteins in brain and liver appear to be identical.

Animals↗
PubMed1992

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.

Binding Sites↗
PubMed1984

Aphidicolin, a specific inhibitor of DNA polymerase alpha, inhibits conversion of lanosterol to C-27 sterols in mouse L cells.

Aphidicolin, a fungal metabolite which is a specific inhibitor of DNA polymerase alpha, inhibited the incorporation of [14C]acetate into desmosterol in mouse L cells by 50% at a concentration of 8.8 microM. It had no effect on acetate metabolism into fatty acids or CO2. The site of inhibition was determined to be distal to the formation of mevalonic acid since aphidicolin also inhibited the incorporation of [14C]mevalonolactone into desmosterol but had no effect on the activity of 3-hydroxy-3-methylglutaryl-CoA reductase (EC 1.1.1.34) or the incorporation of [14C]acetate into total nonsaponifiable lipids. High pressure liquid chromotographic analysis of the distribution of radioactivity among the nonsaponifiable lipids formed from [14C]acetate in the presence of aphidicolin indicated an accumulation of lanosterol accompanied by a proportional decrease in radiolabeled desmosterol and two of its precursors, delta 5,7,24-cholestatrienol, and 4 alpha-methyl-delta 8,24-cholestadienol. In cells exposed to aphidicolin, lanosterol accumulation was rapid (15 min) and reversible after a 3-h exposure when cells were rinsed and fresh medium added. It was concluded that aphidicolin inhibits the conversion of lanosterol to C-27 sterols. Although the exact mechanism of this inhibition has not yet been determined, addition of aphidicolin to 20,000 X g supernatant fractions of mouse liver homogenates inhibited the incorporation of [14C]mevalonolactone into cholesterol in a concentration-dependent manner, suggesting that aphidicolin may act directly on one or more of the enzymatic steps involved in lanosterol demethylation. The ubiquitous occurrence of an aphidicolin binding site on eukaryotic DNA alpha polymerases and the inhibitory action of aphidicolin at a proposed secondary regulatory site in sterol biosynthesis (lanosterol metabolism) suggest that a naturally occurring compound may exist which can regulate both DNA replication and cholesterogenesis.

Acetates↗