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At least 19 recordsLinked to original sources
PubMed1980

Non-specific biosynthesis of gammacerane derivatives by a cell-free system from the protozoon Tetrahymena pyriformis. Conformations of squalene, (3S)-squalene epoxide and (3R)-squalene epoxide during the cyclization.

1. A cell-free system from the protozoon Tetrahymena pyriformis was incubated with either [12-3H]squalene or (RS)-2,3-epoxy-2,3-dihydro-[12,13-3H]squalene. Squalene was cyclized into tetrahymanol whereas racemic squalene epoxide was transformed into gammacerane-3 alpha,21 alpha-diol and gammacerane-3 beta,21 alpha-diol. After cyclization of (RS)-2,3-epoxy-2,3-dihydro-[3-3H]squalene, both epimeric gammaceranediols were labelled with a tritium atom located at C-3, showing that no isomerization via a 3-oxo compound occurred. 2. The proton NMR spectra of the cyclization products of synthetic (2E, 22E)-(1,1,1,24,24,24-2H6)squalene and (RS)-(22E)-2,3-epoxy-2,3-dihydro-(1,1,1,24,24,24-2H6)squalene show that squalene and the (3S)enantiomer of its epoxide are cyclized in an all pre-chair conformation, whereas the (3R) enantiomer of squalene epoxide is cyclized in a pre-boat conformation as concerns the cycle A. 3. The squalene cyclase of T. pyriformis presents the same lack of substrate specificity as the cyclase of Acetobacter pasteurianum: in addition to squalene, its normal substrate, it also cyclizes both enantiomers of its epoxide. This conformational versatility is characteristic of squalene cyclases but no longer exists in the squalene epoxide cyclases from eukaryotes.

Animals↗
PubMed1987

Squalene synthetase. Solubilization and partial purification of squalene synthetase, copurification of presqualene pyrophosphate and squalene synthetase activities.

Squalene synthetase (farnesyldiphosphate:farnesyldiphosphate farnesyltransferase, EC 2.5.1.21) is an intrinsic microsomal protein that catalyzes the synthesis of squalene from farnesyl pyrophosphate via the intermediate presqualene pyrophosphate. We have solubilized this enzyme from yeast with a mixture of the detergents N-octyl beta-D-glucopyranoside and Lubrol PX. Approximately 50-fold purification of the solubilized activities has been achieved by chromatography on DEAE-cellulose and hydroxylapatite and by isoelectric focusing. The most highly purified preparation has one major band of protein with a molecular weight of 53,000 as estimated by electrophoresis under denaturing conditions. The enzyme may also have been modified by proteolysis during isolation since a 47,000 molecular weight species was also found. The two activities, presqualene pyrophosphate synthetase and squalene synthetase, copurified during isolation.

Chromatography, DEAE-Cellulose↗
PubMed1997

A simplified squalene epoxidase assay based on an HPLC separation and time-dependent UV/visible determination of squalene.

A novel and highly simplified enzyme assay for squalene epoxidase (EC 1.14.99.7) has been developed. The assay relies on the UV/visible determination of squalene at 195 nm, as it elutes from an octadecylsilane HPLC column. An acetonitrile/water (95.5/0.5, v/v) mixture was found to provide an ideal mobile phase, into which aqueous enzyme reaction mixture aliquots could be injected. Squalene, the natural substrate for squalene epoxidase, may be quantitatively determined within the concentration range 0-30 microM, with a calibration curve exhibiting an r2 (where r2 is the square of the Pearson correlation coefficient r) of 0.995. The HPLC retention time for squalene was significantly longer (> 15 min) than that for any other component required to prepare an enzyme assay reaction mixture, so facilitating its identification and quantification. In this way HPLC was used to follow enzymic squalene consumption within aliquots taken over a 30-min period. Previously reported squalene epoxidase assays rely on the radiolabeling and subsequent monitoring of squalene as it is metabolized by the enzyme. A highly simplified enzyme assay for squalene epoxidase is therefore reported.

Chromatography, High Pressure Liquid↗
PubMed1980

In vitro conversion of squalene from squalene-phospholipid liposomes into sterols by rat liver microsomes and cytosol.

When rat liver cytosol, possessing sterol carrier protein (SCP) activity was incubated with [3H]-squalene-phospholipid liposomes, cofactors, and rat liver microsomes, squalene from the liposomes was converted into sterols. When cytosol was omitted from the incubation mixture, only insignificant amounts of sterols were produced. Liposomes of squalene with either phosphatidylserine or phosphatidylcholine were equally effective as substrates. The liposomes were stable at 4 degrees C for 3 weeks. The ratio of squalene to phospholipid in the liposomes could be varied over a range of 0.004 to 0.23. Multilamellar liposomes with squalene were not effective as a substrate for the conversion of squalene to sterols. The mechanism for transfer of squalene from the liposomes to the enzymes appears to be initial binding of liposomes to microsomes, with subsequent transfer of the substrate to the enzyme site by the SCP in the cytosol. Microsome-liposome complexes prepared in the absence or presence of cytosol are effective in converting squalene to sterols only if cytosol is added again, indicating that cytosol is not required for the binding of liposomes to microsomes.

Animals↗
PubMed2001

Supernatant protein factor, which stimulates the conversion of squalene to lanosterol, is a cytosolic squalene transfer protein and enhances cholesterol biosynthesis.

Squalene epoxidase, a membrane-associated enzyme that converts squalene to squalene 2,3-oxide, plays an important role in the maintenance of cholesterol homeostasis. In 1957, Bloch and colleagues identified a factor from rat liver cytosol termed "supernatant protein factor (SPF)," which promotes the squalene epoxidation catalyzed by rat liver microsomes with oxygen, NADPH, FAD, and phospholipid [Tchen, T. T. & Bloch, K. (1957) J. Biol. Chem. 226, 921-930]. Although purification of SPF by 11,000-fold was reported, no information is so far available on the primary structure or biological function of SPF. Here we report the cDNA cloning and expression of SPF from rat and human. The encoded protein of 403 amino acids belongs to a family of cytosolic lipid-binding/transfer proteins such as alpha-tocopherol transfer protein, cellular retinal binding protein, yeast phosphatidylinositol transfer protein (Sec14p), and squid retinal binding protein. Recombinant SPF produced in Escherichia coli enhances microsomal squalene epoxidase activity and promotes intermembrane transfer of squalene in vitro. SPF mRNA is expressed abundantly in the liver and small intestine, both of which are important sites of cholesterol biosynthesis. SPF is expressed significantly in isolated hepatocytes, but the expression level was markedly decreased after 48 h of in vitro culture. Moreover, SPF was not detectable in most of the cell lines tested, including HepG2 and McARH7777 hepatomas. Transfection of SPF cDNA in McARH7777 significantly stimulated de novo cholesterol biosynthesis. These data suggest that SPF is a cytosolic squalene transfer protein capable of regulating cholesterol biosynthesis.

Amino Acid Sequence↗
PubMed1989

Regulation of squalene synthetase and squalene epoxidase activities in Saccharomyces cerevisiae.

Squalene synthetase (EC 2.5.1.21) and squalene epoxidase (EC 1.14 99.7) activities have been measured in cell-free extracts of wild type yeast grown in aerobic and semi-anaerobic conditions as well as in sterol-auxotrophic mutant strains grown aerobically. The results show that both enzymes are induced resulting in an almost two- to five-fold increase in enzymatic activities in mutant strains containing limited sterol amounts and are repressed in the wild type strain cultured in anaerobiosis in excess of sterol. The results show also that squalene epoxidase is repressed by lanosterol, and that the mevalonic acid pool may regulate squalene synthetase levels. The large change in the activities of the two enzymes, depending on the sterol needs of the cells, as well as their low specific activities in comparison with those of the enzymes involved in the early stages of sterol synthesis strongly suggests that squalene synthetase and squalene epoxidase are of importance in regulating the amount of sterol synthesized by yeast.

Anaerobiosis↗
PubMed1994

An NMR and molecular mechanics study of squalene and squalene derivatives.

Various squalene derivatives, including squalene, squalene 2,3-epoxide (monoepoxide, SQME), squalene 2,3;22,23-diepoxide (SQDE), 2-aza-2,3-dihydrosqualene (SQN) and 2-aza-2,3-dihydrosqualene N-oxide (SQNO), were studied in chloroform solutions using ID high-resolution 1H spectra and 13C longitudinal relaxation studies, 2D proton NOESY and COSY and 2D proton-carbon HETCOR spectroscopy. A full interpretation of the 1H and 13C-NMR spectra is presented. Staggered conformations along the C11-C12 bond are favoured and a relatively rigid structure of the central part of the chain is indicated in relaxation and coupling data, while further away from the central part the molecular mobility grows. A detected NOE dipolar interaction between terminal and central parts of the molecule indicates the presence of dynamically folded structures in solution. The proposed model also explains the selective reactivity of the mobile chain endings with respect to the central part which is protected by these moving ends. Different solvents at different concentrations induce some variations in this molecular model with a shortening or a lengthening of the mean path covered by the tail endings. Molecular mechanics and molecular dynamics calculations on the free squalene molecule indicate that the mobility of the chain is almost equivalent in all its isoprenic moieties, and the greater mobility of the chain ends may be ascribed to co-operative movements from the center to the tails. The solvent probably plays an important role in hindering the motion of the central part of the molecule.

Carbon Isotopes↗
PubMed1992

Epoxidation of 2,3-oxidosqualene to 2,3;22,23-squalene dioxide by squalene epoxidase.

Partially purified squalene epoxidase (SE) from pig liver converts 2,3-oxidosqualene (SO) to 2,3;22,23-squalene dioxide (SDO) with approximately one-half the efficiency of the epoxidation of squalene at pH 7.4. The SO to SDO conversion is independent of pH and shows an absolute requirement for exogenous FAD. Two noncompetitive SE inhibitors show selectivity in blocking squalene (Sq) to SO versus SO to SDO epoxidations. Finally, SDO inhibits the activity of crude pig liver oxidosqualene cyclase (OSC) with an IC50 of 16 microM. Thus, SE inhibitors not only reduce the production of SO from Sq, but also strongly suppress "leakage" of SO to the potentially harmful SDO in vitro.

Animals↗
PubMed1997

Inhibition of squalene synthase but not squalene cyclase prevents mevalonate-mediated suppression of 3-hydroxy-3-methylglutaryl coenzyme A reductase synthesis at a posttranscriptional level.

Previously, we found that mevalonate-derived products together with an oxysterol regulated reductase synthesis at a posttranscriptional level. To determine which products were responsible for this regulation, either the squalene synthase inhibitor zaragozic acid A or the squalene cyclase inhibitor 4,4,10-beta-trimethyl-trans-decal-3beta-ol (TMD) was added to lovastatin-treated Syrian hamster cells in conjunction with mevalonate. Mevalonate alone decreased reductase synthesis 50% compared with lovastatin-treated cells. In contrast, when both zaragozic acid A and mevalonate were added to lovastatin-treated cells, there was no change in reductase synthesis. With either treatment, reductase mRNA levels did not change compared with lovastatin-treated cells. When both 25-hydroxycholesterol and mevalonate were added to lovastatin-treated cells, reductase synthesis and mRNA levels were decreased 95 and 50%, respectively. The 10-fold difference between changes in reductase synthesis and mRNA levels under these conditions reflects a specific effect of mevalonate-derived isoprenoids on reductase synthesis at the translational level. In contrast, coincubation of cells with mevalonate plus 25-hydroxycholesterol in the presence of zaragozic acid decreased reductase synthesis and mRNA levels 60 and 50%, respectively, compared with lovastatin-treated cells. Moreover, degradation of reductase was increased approximately 7-fold in cells treated with mevalonate alone but only 3-fold in cells treated with mevalonate and zaragozic acid A. These results indicate that isoprenoid products between mevalonate and squalene affect reductase at a posttranslational level by increasing degradation but do not regulate reductase synthesis at a posttranscriptional level. In contrast, when both TMD and mevalonate were added to lovastatin-treated cells, reductase synthesis was decreased approximately 50% with no corresponding decrease in reductase mRNA levels, similar to mevalonate only. Reductase degradation was increased approximately 7-fold under these conditions. Cellular incubation in TMD, mevalonate, and 25-hydroxycholesterol decreased reductase synthesis and mRNA levels 95 and 50%, respectively. From these results we concluded that mevalonate-derived nonsterols synthesized between squalene and lanosterol decrease reductase synthesis at a translational level-either alone or in combination with 25-hydroxycholesterol-and also increase reductase degradation.

Animals↗
PubMed1995

Retention of the label during the conversion of [3-3H] squalene into (3S)-2,3-oxidosqualene catalyzed by mammalian squalene oxidase.

Squalene epoxidase is the only known flavoprotein that catalyzes the epoxidation of an olefin. In order to test the possibility of a catalytic non-heme metal-based mechanism, the conversion of chemically synthesized [3-3H]squalene into [3H]2,3-oxidosqualene, by partially purified pig liver squalene epoxidase, was studied. No exchange of the labeled hydrogen could be observed, ruling out a mechanism involving, e.g., an iron carbene type species at C-3.

Animals↗
PubMed1989

Squalene analogues containing isopropylidene mimics as potential inhibitors of pig liver squalene epoxidase and oxidosqualene cyclase.

Several squalene analogues containing 1,1-dihaloalkene, acetylene, allene, diene, and cyclopropane functionalities were synthesized and evaluated as potential inhibitors of pig liver squalene epoxidase and oxidosqualene cyclase. Both monofunctionalized and bisfunctionalized analogues were prepared. Poor inhibition of squalene epoxidase and oxidosqualene cyclase was found for most compounds (IC50 much greater than 400 microM), with the exception of the monofunctionalized alkynol (IC50 = 300 microM). This alkynol showed mixed-function inhibition with KI = 0.95 mM. Oxidation of the alcohol to the alkynone resulted in loss of epoxidase activity, indicating that the hydroxyl group is necessary for inhibition and that the alkynol is not a proinhibitor. Molecular mechanics calculations indicated that a good inhibitor should possess hydrophobic substituents on an unpolarized, unsaturated system; additionally, the presence of a pro-C-3 hydroxyl group can confer inhibitory potency.

Alkenes↗
PubMed1986

A possible role for squalene in the pathogenesis of acne. I. In vitro study of squalene oxidation.

An in vitro study of the oxidation of squalene, and a description of factors acting on this transformation are presented. Thin layer chromatography was used to quantify the products generated by different oxidation processes. The results clearly show that squalene is a highly effective oxygen-scavenging agent. Its oxidation may first induce comedogenesis and, as a secondary event, cause a large reduction in oxygen tension in the human pilo-sebaceous duct. Porphyrins were confirmed to be highly efficient catalytic factors in the squalene oxidation process. The relationships between comedogenesis, bacterial colonization, and the role of sebum in the pathogenesis of acne are discussed in the light of these findings.

Acne Vulgaris↗
PubMed1981

Squalene epoxidase, oxido-squalene cyclase and cholesterol biosynthesis in normal and tumoral mucosa of the human gastro-intestinal tract. Evidence of post-HHG CoA regulation.

Cholesterol biosynthesis was evaluated in different parts of the human gastro-intestinal tract by incorporation of labelled acetic or mevalonic acid into non-saponifiable metabolites and also by measuring squalene epoxidase activity, oxido=squalene cyclase activity and dihydrolanosterol demethylation rate. With respect to rat liver, whole cholesterol biosynthesis was generally low and we found an anatomic localization of activity towards the end of the gastro-intestinal tract. The results of analysis of mevalonic acid metabolites implied the existence of post-HMG CoA regulation. A comparison with the corresponding dat obtained from homologous cancerous tissues (inthe case of high cholesterogenic activity) did not show a greater activity in the tumor, in spite of higher levels of epoxidase concentration.

Acetates↗
PubMed1969

Assay of the possible organization of particle-bound enzymes with squalene synthetase and squalene oxidocyclase systems.

Lanosterol was biosynthesized in pig liver homogenate from [4,8,12-(14)C(3)]farnesyl pyrophosphate and [4S-4-(3)H]NADPH through the intermediary formation of squalene labelled asymmetrically with (3)H. The biosynthetic lanosterol, freed from labelled 24,25-dihydrolanosterol, which was also synthesized, was converted into 24,25-dihydrolanosteryl acetate and subjected to chemical degradations to locate the position(s) of the (3)H label in the molecule. The ratio of (3)H at C-11 to that at C-12 was found to be 1.28. Although a certain inequality of labelling was thus indicated, experimental uncertainties did not permit the conclusion that the asymmetrically labelled squalene might have been cyclized preferentially from one end.

Animals↗
PubMed1986

A possible role for squalene in the pathogenesis of acne. II. In vivo study of squalene oxides in skin surface and intra-comedonal lipids of acne patients.

Skin surface lipids and lipids from open and closed comedones in acne patients were analysed by thin layer chromatography. The results showed that these lipids were enriched in polar lipids, as compared with the skin surface lipids obtained from controls without acne. In both open and closed comedones, these polar lipids appeared to be derived mainly from the oxidation of squalene, which is in agreement with our previous in vitro results. We suggest that squalene oxidation is the link between comedogenesis and bacterial colonization, and based on this, we propose a hypothesis of the pathogenesis of acne.

Acne Vulgaris↗
PubMed1997

Cloning and characterization of the Arabidopsis thaliana SQS1 gene encoding squalene synthase--involvement of the C-terminal region of the enzyme in the channeling of squalene through the sterol pathway.

Squalene synthase (SQS) catalyzes the first committed step of the sterol biosynthetic pathway. A full-length Arabidopsis thaliana SQS cDNA has been isolated by combining library screening and PCR-based approaches. Arabidopsis SQS is encoded by a small gene family of two genes (SQS1 and SQS2) which are organized in a tandem array. SQS1 and SQS2 have an identical organization with regard to intron positions and exon sizes and encode SQS isoforms showing a high level of sequence conservation (79% identity and 88% similarity). The isolated cDNA has been assigned to the SQS1 gene product, SQS1. RNA blot analysis has shown that the 1.6-kb SQS1 mRNA is detected in all plant tissues analyzed (inflorescenses, leaves, stems and roots) although the transcript is especially abundant in roots. Arabidopsis SQS1 isoform is unable to complement the SQS-defective Saccharomyces cerevisiae strain 5302, although SQS activity was detected in the microsomal fraction of the transformed yeast strain. However, a chimeric SQS resulting from the replacement of the 66 C-terminal residues of the Arabidopsis enzyme by the 111 C-terminal residues of the Schizosaccharomyces pombe enzyme was able to confer ergosterol prototrophy to strain 5302. Labeling studies using [3H]farnesyl-P2 and microsomal fractions obtained from yeast strains expressing either Arabidopsis SQS1 or chimeric Arabidopsis/S. pombe SQS derivatives indicated that the C-terminal region of the enzyme is involved in the channeling of squalene through the yeast sterol pathway.

Amino Acid Sequence↗
PubMed2000

Phenolic compounds and squalene in olive oils: the concentration and antioxidant potential of total phenols, simple phenols, secoiridoids, lignansand squalene.

The aim of this study was to evaluate the phenolic antioxidant and squalene content in a range of olive and seed oils. A mean of 290 +/- 38 (SEM) mg squalene/100 g was detected. However, while there was a weak significant difference between extra virgin (424 +/- 21 mg/kg) and refined virgin (340 +/- 31 mg/100 g; P<0.05) olive oils, highly significant differences were evident between extra virgin olive oils (P<0.0001) refined virgin olive oils (P<0.0001) and seed oils (24 +/- 5 mg/100 g). While seed oils were devoid, on average, the olive oils contained 196 +/- 19 mg/kg total phenolics as judged by HPLC analysis, but the value for extra virgin (232 +/- 15 mg/kg) was significantly higher than that of refined virgin olive oil (62 +/- 12 mg/kg; P<0.0001). Appreciable quantities of simple phenols (hydroxytyrosol and tyrosol) were detected in olive oils, with significant differences between extravirgin (41.87 +/- 6.17) and refined virgin olive oils (4.72 +/- 215; P<0.01). The major linked phenols were secoiridoids and lignans. Although extra virgin contained higher concentrations of secoiridoids (27.72 +/- 6.84) than refined olive oils (9.30 +/- 3.81) this difference was not significant. On the other hand, the concentration of lignans was significantly higher (P<0.001) in extra virgin (41.53 +/- 3.93) compared to refined virgin olive oils (7.29 +/- 2.56). All classes of phenolics were shown to be potent antioxidants. In future epidemiologic studies, both the nature and source of olive oil consumed should be differentiated in ascertaining cancer risk.

Antioxidants↗