Kinetic properties and solubilization of microsomal cholesterol ester hydrolase from rat liver

Some kinetic properties of the microsomal cholesterol ester hydrolase (CEH) have been examined in rat liver. The reaction was linear with time up to 60 min and with enzyme concentration up to 0.3 mg/mL, and a pH optimum of 6.7 for enzyme activity was observed. Cholesterol esterase exhibited the following apparent kinetic constants: Km, 68.88 microM and Vmax, 33 Units/mg protein. Cholesteryl palmitate was hydrolyzed to a much greater extent than cholesteryl oleate by the enzyme. Product inhibition with cholesterol and palmitic acid was not apparent; however, oleic acid added to the system reduced markedly microsomal CEH activity. The present paper also reports the solubilization of cholesteryl palmitate hydrolase from the microsomal fraction by pretreating it with Triton X-100, sodium deoxycholate, and sodium dodecylsulfate. All ionic and non-ionic detergents tested are capable of making the enzyme soluble, and maximal effects were found at higher concentrations of detergents although the esterase activity was strongly inhibited. Triton X-100 was found to be more effective than sodium deoxycholate and sodium dodecylsulfate in enzyme and protein solubilization. When the direct effects of detergents on CEH activity were studied, progressive concentration-dependent inhibitions were observed.     

Stereoselective formation of a K-region dihydrodiol from phenanthrene by Streptomyces flavovirens

The metabolism of phenanthrene, a polycyclic aromatic hydrocarbon (PAH), by Streptomyces flavovirens was investigated. When grown for 72 h in tryptone yeast extract broth saturated with phenanthrene, the actinomycete oxidized 21.3% of the hydrocarbon at the K-region to form trans-9,10-dihydroxy-9,10-dihydrophenanthrene (phenanthrene trans-9,10-dihydrodiol). A trace of 9-phenanthrol was also detected. Metabolites isolated by thin-layer and high performance liquid chromatography were identified by comparing chromatographic, mass spectral, and nuclear magnetic resonance properties with those of authentic compounds. Experiments using [9-¹⁴C]phenanthrene showed that the trans-9,10-dihydrodiol had 62.8% of the radioactivity found in the metabolites. Circular dichroism spectra of the phenanthrene trans-9,10-dihydrodiol indicated that the absolute configuration of the predominant enantiomer was (–)-9S,10S, the same as that of the principal enantiomer produced by mammalian enzymes. Incubation of S. flavovirens with phenanthrene is an atmosphere of ¹⁸O₂, followed by gas chromatographic/mass spectral analysis of the metabolites, indicated that one atom from molecular oxygen was incorporated into each molecule of the phenanthrene trans-9,10-dihydrodiol. Cytochrome P-450 was detected in 105,000×g supernatants prepared from cell extracts of S. flavovirens. The results show that the oxidation of phenanthrene by S. flavovirens was both regio- and stereospecific.Abbreviations CD circular dichroism - DMF N,N-dimethyl-formamide - GC/MS gas chromatography/mass spectrometry - HPLC high performance liquid chromatography - NMR nuclear magnetic resonance - ODS octadecylsilane - PAH polycyclic aromatic hydrocarbon - TLC thin-layer chromatography - TMS tetramethylsilane - UV ultraviolet     

Selectivity and contribution of lecithin: cholesterol acyltransferase to plasma cholesterol ester formation

Selectivity factors (Vm/Km) for human and rat lecithin: cholesterol acyltransferases (LCAT) for the transfer of various acyl groups from the 2-position of phosphatidylcholine were determined. By multiplying these values by the proportions of acyl groups at the 2-position of phosphatidylcholine, one can predict the proportions of molecular species of cholesterol ester which will be synthesized by LCAT. In human subjects fasted overnight, the molecular composition of plasma cholesterol ester was found to reflect the LCAT selectivity relatively accurately. This result supports the concepts that hepatic acyl-CoA:cholesterol acyltransferase (ACAT) does not contribute significantly to the synthesis of plasma cholesterol ester and that removal of cholesterol ester from plasma is not selective with respect to molecular species under these conditions. In contrast to the results with humans, the molecular composition of plasma cholesterol ester formed in spontaneously hypertensive rats fed a high-cholesterol diet and then fasted overnight differs from that which is predicted from LCAT selectivity and the proportion of various fatty acids at the 2-position of phosphatidylcholine: these results suggest that cholesterol ester is formed mainly via the ACAT reaction.     

Stereoselective formation and hydration of 12-methylbenz[a]anthracene 5,6-epoxide enantiomers by rat liver microsomal enzymes.

The K-region trans-5,6-dihydrodiols formed in the metabolism of 12-methylbenz[a]anthracene (12-MBA) by liver microsomal preparations from untreated, phenobarbital-treated and 3-methylcholanthrene-treated male Sprague-Dawley rats were found by chiral stationary-phase h.p.l.c. (c.s.p.-h.p.l.c.) analyses to contain (5S,6S)/(5R,6R) enantiomer ratios of 93:7, 88:12 and 97:3 respectively. The absolute stereochemistry of a 12-MBA trans-5,6-dihydrodiol enantiomer was elucidated by the exciton-chirality c.d. method. The 5,6-epoxides formed in the metabolism of 12-MBA by liver microsomal preparations from untreated, phenobarbital-treated and 3-methylcholanthrene-treated male Sprague-Dawley rats in the presence of the epoxide hydrolase inhibitor 3,3,3-trichloropropylene 1,2-oxide were isolated from a mixture of metabolites by normal-phase h.p.l.c., and their (5S,6R)/(5R,6S) enantiomer ratios were found by c.s.p.-h.p.l.c. analyses to be 73:27, 78:22 and 99:1 respectively. The absolute configurations of 12-MBA 5,6-epoxide enantiomers, resolved by c.s.p.-h.p.l.c., were determined via high-resolution (500 MHz) proton-n.m.r. and c.d. spectral analyses of the two isomeric methoxylation products derived from each of the 12-MBA 5,6-epoxide enantiomers. Enantiomeric pairs of the two methoxylation products were resolved by c.s.p.-h.p.l.c. The results indicate that enantiomeric 5S,6R-epoxide and 5S,6S-dihydrodiol were the major enantiomers preferentially formed in the metabolism at the K-region 5,6-double bond of 12-MBA by all three rat liver microsomal preparations. Optically pure 12-MBA 5S,6R-epoxide was hydrated predominantly at the C(6) position (R centre) to form 12-MBA trans-5,6-dihydrodiol with a (5S,6S)/(5R,6R) enantiomer ratio of 97:3. However, optically pure 12-MBA 5R,6S-epoxide was hydrated nearly equally at both C(5) and C(6) positions to form 12-MBA trans-5,6-dihydrodiol with a (5S,6S)/(5R,6R) enantiomer ratio of 57:43.     

Identification of neutral cholesterol ester hydrolase, a key enzyme removing cholesterol from macrophages

Unstable lipid-rich plaques in atherosclerosis are characterized by the accumulation of macrophage foam cells loaded with cholesterol ester (CE). Although hormone-sensitive lipase and cholesteryl ester hydrolase (CEH) have been proposed to mediate the hydrolysis of CE in macrophages, circumstantial evidence suggests the presence of other enzymes with neutral cholesterol ester hydrolase (nCEH) activity. Here we show that the murine orthologue of KIAA1363, designated as neutral cholesterol ester hydrolase (NCEH), is a microsomal nCEH with high expression in murine and human macrophages. The effect of various concentrations of NaCl on its nCEH activity resembles that on endogenous nCEH activity of macrophages. RNA silencing of NCEH decreases nCEH activity at least by 50%; conversely, its overexpression inhibits the CE formation in macrophages. Immunohistochemistry reveals that NCEH is expressed in macrophage foam cells in atherosclerotic lesions. These data indicate that NCEH is responsible for a major part of nCEH activity in macrophages and may be a potential therapeutic target for the prevention of atherosclerosis.     

Characterization of a naturally occurring new version of the cholesterol ester transfer protein (CETP) from small intestine

The cholesterol ester transfer protein (CETP) is found in plasma mediating the transfer of cholesterol esters and triacylglycerides between lipoproteins. The last 26 amino acids of its carboxy-end correspond to an amphipathic a-helix whose hydrophobic side has been directly involved in the transfer of lipids. Alterations in this region lead to the reduction or loss of lipid transfer activity. To date, the only variant of the CETP messenger that has been reported lacks exon 9, which translates into an inactive isoform regarding neutral lipid transfer. In this study, we describe a new version of the messenger RNA of rabbit CETP identified exclusively in the small intestine of wild type (WT) rabbits. This isoform includes several of the intron bases prior to exon 16. The presence of a stop codon within this sequence prevents translation of exon 16, substituting the original carboxy-end sequence and therefore generating a random structure that does not contain the region responsible for neutral lipid transfer. Antibodies generated against a peptide within the carboxy-end sequence of the new isoform show the presence of this new protein in human plasma.     

Simultaneous transfer of cholesteryl ester and phospholipid by protein(s) isolated from human lipoprotein-free plasma

Protein(s) catalyzing the transfer of [³H]cholesteryl ester and [¹⁴C]-phosphatidylcholine from high density lipoproteins to low density lipoproteins have been purified 4829-fold from human plasma by chromatography of the d > 1.21 g/ml infranatant fraction of human plasma on phenyl-Sepharose, CM-cellulose, concanavalin A-Sepharose, and finally by isoelectric focussing. At each step of the purification, both transfer activities coelute. The purified protein(s), molecular weight 150,000, transfer cholesteryl esters and phosphatidylcholine with a 1:1 stoichiometry and at equal rates of flux. Rat plasma contains a protein(s) which facilitates the transfer of phosphatidylcholine as effectively as human plasma. However, the rat plasma protein(s) does not facilitate the transfer of cholesteryl esters. These results suggest that human plasma contains one or more proteins which transfer both lipids, possibly as a 1:1 complex, whereas rat plasma lacks the cholesteryl ester transfer protein.     

Properties of the mouse embryo conditioned medium factor(s) stimulationg colony formation by mouse bone marrow cells grown in vitro

The properties of the mouse embryo cell conditioned medium (ECM) colony stimulating factor(s) from six day mouse embryo cultures have been examined. The general properties were similar to those described previously for the human urine colony stimulating factor. The ECM colony stimulating activity (CSA) was not lost following treatment with nucleases, glycosidases, phospholipases and proteolytic enzymes with the exception of α-chymotrypsin. ECM CSA was lost following mild periodate treatment. Fractionation of ECM CSA revealed a slight size heterogeneity on gel-filtration and on zone sedimentation in sucrose gradients. There was a discrepancy between the apparent molecular weights determined by gel-filtration (70,000–150,000) and by zone sedimentation (64,000) as has been reported previously for other colony stimulating factors. A gross charge heterogeneity of ECM CSA was apparent on electrophoresis and DEAE-cellulose chromatography, and a heterogeneity of the elution profile on stepwise elution from calcium phosphate gel was observed. This heterogeneity was still apparent in the presence of 6M urea and appeared to be unchanged following re-chromatography on DEAE-cellulose under the usual fractionation conditions. These studies suggested that the heterogeneity was not due to easily reversible combinations of active subunits. The electrophoretic heterogeneity of six day ECM CSA was found to develop gradually from an electrophoretically monodisperse band at day 2 of culture. Experiments in which preparations containing concentrated monodisperse ECM CSA were added back to culture dishes during and after ECM production suggested that the development of heterogeneity was related to the production or release of factor(s) from the cells rather than the action on the colony stimulating factor(s) of an extracellular enzyme in the medium. Alteration of the electrophoretic mobility of six day ECM CSA by incubation with purified sialidase suggested the presence of sialic acid on the active molecules. Purification procedures for the ECM factor(s) were not developed to any large extent primarily in view of the charge heterogeneity. The results of this study suggest that the ECM colony stimulating factor(s) is a glycoprotein(s).     

Direct fluorometric analysis of benzo(a)pyrene metabolite formation by mouse liver microsomes

We have examined the metabolites produced by in vitro incubation of benzo(a)pyrene with 3-methylcholanthrene-induced mice liver microsomes. Our objective was to observe directly a possible difference in microsomal enzyme systems of animal models having different susceptibility to chemical carcinogens. The metabolites produced by the two animal models,$\text{C57BL}\text{6J}$ and $\text{DBA}\text{2}$ mice, were analyzed by a highly sensitive, “three-dimensional” fluorescence plotting technique. The fluorescence spectra of the total ethyl acetate-soluble metabolites clearly indicate that the metabolites produced by $\text{DBA}\text{2}$ enzymes were predominantly monohydroxylated benzo(a)pyrene while those produced by the liver microsomes of $\text{C57BL}\text{6J}$ were highly enriched with the 7,8-dihydrodihydroxybenzo(a)pyrene type.     

Inhibition of erythroid differentiation of mouse erythroleukemia cells by a macrophage product(s)

Conditioned media from established murine macrophage cell lines (RAW264.7, P388D1, and WEHI-3) incubated with endotoxin in a serum-free medium contain an erythroid inhibitory activity (EIA) that inhibited dimethylsulfoxide-induced erythroid differentiation of mouse Friend virus-transformed erythroleukemia cells. Endotoxin itself has no EIA activity. Partial purification of EIA demonstrated that it is distinct from other macrophage products such as IL-1, TGF beta, ECGF, FGF, G-CSF, hepatocyte stimulating factor, interferon, PDGF, and cachectin/TNF. These findings indicate that EIA is a macrophage product distinct from other monokines.     

Complex formation of bovine serum albumin with a poly(ethylene glycol) lipid conjugate

In this work, we report the formation of complexes by self-assembly of bovine serum albumin (BSA) with a poly(ethylene glycol) lipid conjugate (PEG2000-PE) in phosphate saline buffer solution (pH 7.4). Three different sets of samples have been studied. The BSA concentration remained fixed (1, 0.01, or 0.001 wt % BSA) within each set of samples, while the PEG2000-PE concentration was varied. Dynamic light scattering (DLS), rheology, and small-angle X-ray scattering (SAXS) were used to study samples with 1 wt % BSA. DLS showed that BSA/PEG2000-PE aggregates have a size intermediate between a BSA monomer and a PEG2000-PE micelle. Rheology suggested that BSA/PEG2000-PE complexes might be surrounded by a relatively compact PEG-lipid shell, while SAXS results showed that depletion forces do not take an important role in the stabilization of the complexes. Samples containing 0.01 wt % BSA were studied by circular dichroism (CD) and ultraviolet fluorescence spectroscopy (UV). UV results showed that at low concentrations of PEG-lipid, PEG2000-PE binds to tryptophan (Trp) groups in BSA, while at high concentrations of PEG-lipid the Trp groups are exposed to water. CD results showed that changes in Trp environment take place with a minimal variation of the BSA secondary structure elements. Finally, samples containing 0.001 wt % BSA were studied by zeta-potential experiments. Results showed that steric interactions might play an important role in the stabilization of the BSA/PEG2000-PE complexes.     

Retinyl ester formation by lecithin: retinol acyltransferase is a key regulator of retinoid homeostasis in mouse embryogenesis

The developing mammalian embryo is entirely dependent on the maternal circulation for its supply of retinoids (vitamin A and its metabolites). The mechanisms through which mammalian developing tissues maintain adequate retinoid levels in the face of suboptimal or excessive maternal dietary vitamin A intake have not been established. We investigated the role of retinyl ester formation catalyzed by lecithin:retinol acyltransferase (LRAT) in regulating retinoid homeostasis during embryogenesis. Dams lacking both LRAT and retinol-binding protein (RBP), the sole specific carrier for retinol in serum, were maintained on diets containing different amounts of vitamin A during pregnancy. We hypothesized that the lack of both proteins would make the embryo more vulnerable to changes in maternal dietary vitamin A intake. Our data demonstrate that maternal dietary vitamin A deprivation during pregnancy generates a severe retinoid-deficient phenotype of the embryo due to the severe retinoid-deficient status of the double mutant dams rather than to the lack of LRAT in the developing tissues. Moreover, in the case of excessive maternal dietary vitamin A intake, LRAT acts together with Cyp26A1, one of the enzymes that catalyze the degradation of retinoic acid, and possibly with STRA6, the recently identified cell surface receptor for retinol-RBP, in maintaining adequate levels of retinoids in embryonic and extraembryonic tissues. In contrast, the pathway of retinoic acid synthesis does not contribute significantly to regulating retinoid homeostasis during mammalian development except under conditions of severe maternal retinoid deficiency.