Purification of squalene epoxidase from rat liver microsomes

Squalene epoxidase was purified from rat liver microsomes by DEAE-cellulose, alumina C_ν gel, hydroxylapatite, CM-Sephadex C-50 and Cibacron Blue Sepharose 4B in the presence of Triton X-100. The specific activity was increased 50 fold with a yield of about 10%. On SDS-polyacrylamide gel electrophoresis, the preparation gave one major band and one minor band with apparent molecular weights of 47,000 and 27,000 daltons, respectively. The protein of 47,000 was the most probable candidate for squalene epoxidase. Squalene epoxidase activity could be reconstituted in the squalene epoxidase preparation with the addition of NADPH-cytochrome P-450 reductase, FAD, and Triton X-100.     

Solubilization and partial characterization of rat liver squalene epoxidase.

The microsomal enzyme system from rat liver which catalyzes squalene epoxidation requires a supernatant protein and phospholipids (Tai, H., and Bloch, K. (1972) J. Biol. Chem. 247, 3767). It has now been found that these two cytoplasmic components can be replaced by Triton X-100. The same detergent solubilizes the microsomal squalene epoxidase and the resulting supernatant can be separated into two components, A and B, by DEAE-cellulose chromatography. Neither Fraction A nor B alone has significant squalene epoxidase activity but combining the two affords a reconstituted system 5-fold higher in specific epoxidase activity than that of the original microsomes. FAD and Triton X-100 in addition to molecular oxygen and NADPH are required in the reconstituted system. Subjecting Fraction A to a second DEAE-cellulose chromatography does not change its specific activity but lowers NADH-ferricyanide reductase activity and the protoheme content to 1/25 and 1/4, respectively. When Fraction B was chromatographed on Sephadex G-200, the specific epoxidase activity tested in the presence of Fraction A was increased 3-fold. This procedure also raised the specific activity of NADPH-cytochrome c reductase activity in Fraction B 3-fold. The reconstituted epoxidase system is not inhibited by either carbon monoxide, potassium cyanide, or o-phenanthrolien but Tiron at 1 mM was inhibitory (50%). Erythrocuprein has no effect on epoxidation. No evidence has been found for the participation of hemoproteins (P450 or cytochrome b5) in squalene epoxidation. Component B appears to be identical with the flavoprotein NADPH-cytochrome c reductase. Component A may be a flavoprotein with an easily dissociable prosthetic group.     

Purification and partial characterization of squalene epoxidase from rat liver microsomes

Squalene epoxidase (EC 1.14.99.7, squalene 2,3-monooxygenase (epoxidizing) was purified to an apparent homogeneity from rat liver microsomes. The purification was carried out by solubilization of microsomes by Triton X-100, fractionation with ion exchangers, hydroxyapatite, Cibacron Blue Sepharose 4B, and chromatofocusing column chromatography. A total purification of 143-fold over the first DEAE-cellulose fraction was achieved. The purified enzyme gave a single major band on SDS-polyacrylamide gel electrophoresis and the Mr was estimated to be 51 000 as a single polypeptide chain. The enzyme showed no distinct absorption spectrum in the visible regions. The squalene epoxidase activity was reconstituted with the purified enzyme, NADPH-cytochrome P-450 reductase (EC 1.6.2.4), FAD, NADPH and molecular oxygen in the presence of Triton X-100. The apparent Michaelis constants for squalene and FAD were 13 microM and 5 microM, respectively. The Vmax was about 186 nmol per mg protein per 30 min for 2,3-oxidosqualene. The enzyme activity was not inhibited by potent inhibitors of cytochrome P-450. It is suggested that squalene epoxidase is distinct from cytochrome P-450 isozymes.     

Regulation of rat liver microsomal squalene epoxidase: inactivation of the supernatant protein factor by nucleotides

Modulation of squalene epoxidase activity by nucleotides was studied in rat liver microsomal preparations. Supernatant protein factor (SPF) stimulates hepatic microsome-associated squalene epoxidase. The stimulatory effect of this activator was abolished by some nucleotides, and the effect of ATP on SPF was examined in detail. The inhibition by ATP was time- and concentration-dependent and was increased remarkably by the addition of Mg2+. Binding studies employing Sephadex column chromatography showed that ATP and SPF formed a complex (molar ratio, 1:1). These results suggest that nucleotides may regulate cholesterol metabolism through inactivation of the supernatant protein activator in the presence of bivalent metal ions.     

Regulation of transglutaminase activity in Chinese hamster ovary cells

We have investigated the regulation of transglutaminase activity (epsilon-(gamma-glutamyl)lysine crosslinking enzyme) in Chinese hamster ovary cells in culture. We report that transglutaminase activity increases several-fold in CHO cells at maximum density in suspension culture. This increase cannot be explained by the presence of soluble regulators of the enzyme activity or the appearance of a new enzyme activity with a different affinity for substrate, but appears to be due to an increase in total enzyme activity. Treatment of CHO cells at low cell density with 8-bromo cyclic AMP results in a small increase (20--70%) in transglutaminase activity. By studying CHO mutants which have altered or absent cyclic-AMP-dependent protein kinases, we have demonstrated that the effect of cyclic AMP on transglutaminase activity at low cell density is mediated by cyclic-AMP-dependent protein kinase. However, the protein kinase mutants show normal increases in transglutaminase activity at high cell density, indicating that cyclic AMP-dependent protein kinase does not mediate density-dependent changes in transglutaminase activity.     

Regulation of transglutaminase activity in Chinese hamster ovary cells

We have investigated the regulation of transglutamine activity (-(γ-glutamyl)lysine crosslinking enzme) in Chinese hamster ovary cells in culture. We report that transglutaminase activity increases several-fold in CHO cells at maximum density in suspension culture. This increase cannot be explained by the presence of the soluble regulators of the enzyme activity or the appearance of a new enzyme activity with a different affinity for substrate, but appears to be due to an increase in total enzyme activity. Treatment of CHO cells at low cell density with 8-bromo cyclic AMP results in a small increase (20–70%) in transglutaminase activity. By studying CHO mutants which have altered or absent cyclic-AMP-dependent protein kinases, we have demonstrated that the effect of cyclic AMP on transglutaminase activity at low cell density is mediated by cyclic-AMP-dependent protein kinase. However, the protein kinase mutants show normal increases in transglutaminase activity at high cell density, indicating that cyclic AMP-dependent protein kinase does not mediate density-dependent changes in transglutaminase activity.     

Regulation of squalene epoxidase in HepG2 cells

Regulation of squalene epoxidase in the cholesterol biosynthetic pathway was studied in a human hepatoma cell line, HepG2 cells. Since the squalene epoxidase activity in cell homogenates was found to be stimulated by the addition of Triton X-100, enzyme activity was determined in the presence of this detergent. Incubation of HepG2 cells for 18 h with L-654,969, a potent competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, increased squalene epoxidase activity dose-dependently. On the other hand, low density lipoprotein (LDL) and 25-hydroxy-cholesterol decreased the enzyme activity. These results demonstrate that squalene epoxidase is regulated by the concentrations of endogenous and exogenous sterols. The affinity of the enzyme for squalene was not changed by treatment with L-654,969. Cytosolic (S105) fractions, prepared from HepG2 cells treated with or without L-654,969, had no effect on microsomal squalene epoxidase activity of HepG2 cells, in contrast to the stimulating effect of S105 fractions from rat liver homogenate. Mevalonate, LDL, and oxysterol treatment abolished the effect of L-654,969. Simultaneous addition of cycloheximide and actinomycin D also prevented enzyme induction in HepG2 cells. From these results, the change in squalene epoxidase activity is thought to be caused by the change in the amount of enzyme protein. It is further suggested that squalene epoxidase activity is suppressed only by sterols, not by nonsterol derivative(s) of mevalonate, in contrast to the regulation of HMG-CoA reductase.     

Involvement of NADPH-cytochrome c reductase in the rat liver squalene epoxidase system.

Microsomal squalene epoxidase has previously been solubilized with Triton X-100 and resolved into fractions, FA and FB, by DEAE-cellulose chromatography (Ono T. and Bloch K (1975) J biol. Chem. 250, 1571-1579). It has now been found that FB is identical with NADPH-cytochrome c reductase (denoted FPT, EC 1.6.2.3). Although both NADPH and NADH served as electron donors, the former was preferred for squalene epoxidase activity in the reconstituted system of FA and FB. FB is characterized by its ability to reduce cytochrome c by NADPH. In place of FB, partially purified FPT was tested for its ability to support squalene epoxidation in the presence of FA. A stepwise purification of the deoxycholate-solubilized FPT yielded an increase in specific FPT activity with a parallel increase in squalene epoxidase activity. Bromelain-solubilized FPT was less effective. Rabbit antisera preparations to the purified FPT solubilized with trypsin were shown to inhibit concomitantly FPT activity and squalene epoxidase activity. These observations support the concept that squalene epoxidation is primarily mediated via a flavoprotein, NADPH-cytochrome c reductase, and a terminal oxidase, squalene epoxidase, which is distinct from cytochrome P-450.     

Photoaffinity labeling and site-directed mutagenesis of rat squalene epoxidase

Squalene epoxidase (SE) (EC 1.14.99.7) is a flavin-requiring, non-cytochrome P-450 oxidase that catalyzes the conversion of squalene to (3S)-2,3-oxidosqualene. Photolabeling and site-directed mutagenesis were performed on recombinant rat SE (rrSE) to elucidate the location and roles of active-site residues important for catalysis. Two new benzophenone-containing analogs of NB-598, a nanomolar inhibitor of vertebrate SE, were synthesized in tritium-labeled form. These photoaffinity analogs (PDA-I and PDA-II) became covalently attached to SE when irradiated at 360 nm. Lys-C digestion and HPLC purification of [3H]PDA-I-labeled rrSE resulted in isolation of a single major peptide. MALDI-TOF mass spectrometry of this peptide indicated a covalent adduct between PDA-I and a tripeptide, Asp-Ile-Lys, beginning at Asp-426 of rat SE. Based on the labeling results, three mutant constructs were made. First, the D426A and K428A constructs showed a 5- to 8-fold reduction in SE activity compared with wild-type enzyme, while little change was observed in the I427A mutant. Second, a set of five mutant constructs was prepared for the conserved region based on the structure of the flavoprotein p-hydroxybenzoate hydroxylase (PHBH). Compared with wild-type, D284A and D407A showed less than 25% SE activity. This reduction also appeared to correlate with reduced affinity of the mutant proteins for FAD. Finally, each of the seven Cys residues of rrSE were individually mutated to Ala. Three Cys substitutions had no effect on SE activity, and substitutions at Cys-500 and Cys-533 showed a 50% lower SE activity. Mutations at Cys-490 and Cys-557 produced proteins with negligible SE activity, implicating these residues as being either structurally or catalytically essential. Chemical modification of wildtype and Cys mutants with a thiol-modifying reagent support the existence of a disulfide bond between Cys-490 and Cys-557.     

Epoxidation of 6,7- and 10,11-oxidosqualenes by the squalene epoxidase present in rat liver microsomes

Incubation of 6,7-oxidosqualene (2) or 10,11-oxidosqualene (3) with rat liver microsomes led to the formation of mixtures of the corresponding dioxidosqualenes (4 and 5, or 6 and 7, respectively), resulting from the epoxidation of 2 and 3 at their terminal double bonds. The epoxidation requires the presence of both NADPH and FAD. In addition, the HPLC analysis of the Mosher esters resulting from the controlled hydrolysis of dioxide 5 to give the corresponding epoxydiols 9 followed by derivatization with (R)-MTPA, showed that the epoxidation had been stereoselective. These facts support the hypothesis that these dioxidosqualenes had been generated by the squalene epoxidase present in the incubation medium.     

Site-directed mutagenesis of conserved aromatic residues in rat squalene epoxidase

Squalene epoxidase catalyzes the conversion of squalene to (3S)2,3-oxidosqualene, which is a rate-limiting step of the cholesterol biogenesis. To evaluate the importance of conserved aromatic residues, 15 alanine-substituted mutants were constructed and tested for the enzyme activity. Except F203A, all the mutants significantly lost the enzyme activity, confirming the importance of the residues, either for correct folding of the protein, or for the catalytic machinery of the enzyme. Further, interestingly, F223A mutant no longer accepted (3S)2,3-oxidosqualene as a substrate, while Y473A mutant converted (3S)2,3-oxidosqualene to (3S,22S)2,3:22,23-dioxidosqualene twice more efficiently than wild-type enzyme. It is remarkable that the single amino acid replacement yielded mutants with altered substrate and product specificities. These aromatic residues are likely to be located at the substrate-binding domain of the active-site, and control the stereochemical course of the enzyme reaction.     

Determination of Chinese hamster ovary cell-derived recombinant thyrotropin by reversed-phase liquid chromatography

A reversed-phase high-performance liquid chromatography (RP-HPLC) methodology for the qualitative and quantitative analysis of human thyrotropin (hTSH) in CHO cell conditioned medium and in purified preparations has been set up and validated for accuracy, precision and sensitivity. A recovery test indicated a bias of less than 2% and intra-day and inter-day quantitative determinations presented relative standard deviations (RSD) always <7%, while sensitivity was 0.2 microg (RSD=5.6%). The novel methodology was applied to the study of the best cultivation conditions and was able to detect a significant difference in retention time (t(R)) between pituitary and recombinant hTSH, probably reflecting the influence of the heterogeneity of the carbohydrate moiety on the hydrophobic properties of the molecule.     

Regulation of polyamine transport in Chinese hamster ovary cells

Control Chinese hamster ovary (CHO) cells and mutant CHO cells lacking ornithine decarboxylase activity (CHODC-) were used to study the regulation of polyamine uptake. It was found that the transport system responsible for this uptake was regulated by intracellular polyamine levels and that this regulation was responsible for the maintenance of physiological intracellular levels under extreme conditions such as polyamine deprivation or exposure to exogenous polyamines. Polyamine transport activity was enhanced by decreases in polyamine content produced either by inhibition of ornithine decarboxylase with alpha-difluoromethylornithine in CHO cells or via polyamine starvation of CHODC- cells. The provision of exogenous polyamines resulted in rapid and large increases in intracellular polyamine content followed by decreased polyamine transport activity. Soon after this decrease in uptake activity, intracellular polyamine levels then fell to near control values. Cells grown in the presence of exogenous polyamines maintained intracellular polyamine levels at values similar to those of control cells. Protein synthesis was necessary for the increase in transport in response to polyamine depletion, but appeared to play no role in decreasing polyamine transport. Bis(ethyl) polyamine analogues mimicked polyamines in the regulation of polyamine transport but this process was relatively insensitive to regulation by methylglyoxal bis(guanylhydrazone), a spermidine analogue known to enter cells via this transport system and to accumulate to very high levels.     

Vitamin K epoxidase activity of rough and smooth microsomes from rat liver

The distribution of vitamin K epoxidase activity in rough and smooth microsomes has been studied and compared to the prothrombin precursor and vitamin K-dependent carboxylase activity. All three activities were high in rough microsomes as compared to the low levels found in smooth microsomes. The results are in agreement with the suggestion that there might be a linkage between the vitamin K-dependent carboxylation and epoxidation reaction in vivo.     

Inhibition and activation of porcine squalene epoxidase.

Pig liver squalene epoxidase (SE) has been partially purified from solubilized microsomes by DEAE-Sephacel and Blue Sepharose 4B chromatography. This stable and reproducible preparation was used to investigate the mechanism of several substrate-like inhibitors of SE and to study the effects of pH, metals, detergents, and cofactors on enzyme activity. Most divalent (1 mM) and trivalent (0.1 mM) metal cations had little effect on SE at pH 7.4; only ferrous and cupric ions showed ca. 50% reduction in SE activity. Interestingly, at pH 8.8, EDTA (10 mM) shows 1.8-fold enhancement of enzyme activity. Among the detergents, Triton X-100 was clearly superior for solubilization and purification of porcine SE; Tween 80, Lubrol-PX, 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonic acid, octyl beta-glucoside, and three different Zwittergents were much less effective for SE solubilization. Partially purified pig liver SE showed maximal activity at pH 8.8-9.0. Trisnorsqualene alcohol and trisnorsqualene cyclopropylamine were noncompetitive inhibitors at pH 8.8, with Ki values of 4 microM and 180 nM, respectively; these two inhibitors were not substrates for SE. In contrast, 26-hydroxysqualene was both a competitive inhibitor with a Ki value of 4 microM at pH 8.8 and a substrate for SE. An unexpected enhancement (up to 350%) of SE activity was observed at pH 7.4 following preincubation with selected nonpolar derivatives of farnesol and farnesoic acid. At pH 8.8, this effect was less dramatic but still evident.     

Synthesis and biological activity of a novel squalene epoxidase inhibitor, FR194738

The synthesis and biological properties of a novel squalene epoxidase inhibitor, FR194738, are described. This compound displayed potent in vitro inhibitory activities against squalene epoxidase and cholesterol synthesis, and lowered plasma cholesterol and triglyceride levels in dogs.     

Regulation of squalene synthetase activity in rat liver: elevation by cholestyramine, but no diurnal variation

Squalene synthetase activity in liver microsomes from rats sacrificed at three different times of the diurnal cycle showed no significant differences. Addition of 4% cholestyramine to the food resulted in a marked increase in activity (280% of control), independent of the time of killing. 3-Hydroxy-3-methylglutaryl coenzyme A reductase and cholesterol 7 alpha-hydroxylase activity, determined as positive controls, were also found to be elevated by cholestyramine and additionally showed a diurnal variation. On the other hand, five control enzyme activities, not directly related to cholesterol metabolism, i.e. glutamate dehydrogenase, NADPH cytochrome-c reductase, beta-hexosaminidase, catalase and acyl coenzyme A oxidase, showed neither an influence of cholestyramine feeding nor a time of sacrifice dependent variation.