Pancreatic cholesterol esterases. 1. Pancreatic cholesterol esterase induction during maturation

The activities of pancreatic cholesterol esterase from calf and cow pancreas were examined in detail. A 1300-fold enhancement of enzymatic activity was found after maturation, even though cholesterol esterase activity levels in other organs did not change from the juvenile to the adult species. Radioimmunoassays also showed that the calf pancreas contained at least 100-fold less cholesterol esterase protein. Decreased amounts of protein were not due to enhanced proteolysis, since cytosol from cow pancreas degrades exogenously added cholesterol esterase faster than that from calf pancreas. Rather, enhancement of pancreatic cholesterol esterase activity associated with bovine maturation was the result of specific, increased synthesis of a 72-kDa enzyme. This labile 72-kDa cholesterol esterase species was purified to homogeneity by a two-step process in 75% yield and is the major form of bovine pancreatic cholesterol esterase (99%). A much less abundant 67-kDa species, accounting for less than 1% of total pancreatic cholesterol esterase activity, was also purified to homogeneity in a similar two-step process. These results demonstrate that a specific form of pancreatic cholesterol esterase is induced during maturation, and they bear importantly on understanding juvenile cholesterol metabolism as related to dietary absorption of this sterol.     

A biochemical study of non-specific esterases from plant cells, employing the histochemical substrate, naphthol AS-D acetate

Synopsis The reagents routinely employed in the histochemical detection of nonspecific esterases, namely naphthol AS-D acetate and Fast Red Violet LB salt, have been used to study these enzymes biochemically with spectrophotometric procedures. A range of parameters that affect the coupling of the hydrolyzed substrate and diazonium salt were examined and their relevance to future histochemical procedures is noted.TheK _m of broad-bean root-tip esterases was estimated to be approximately 0.07 mM, but other kinetic data suggest that the true value is lower. From an analysis of the kinetics of the hydrolytic reaction it appears that they are of a mixed nature over the time course and substrate concentrations used.The effect of pH on root tip esterase activity has been examined and the recorded optimum of 5.5 is similar to that reported by other workers for plant cells. Non-enzymic hydrolysis of the substrate at alkaline pH levels prevented measurement of enzyme activity beyond pH 7.2.Magnesium ions at a final concentration of 5 mM are important in retaining esterases in their natured state during enzyme extraction, although the addition of increasing amounts of the ion to the assay system caused a corresponding increase in esterase inhibition.     

Identity of a cytosolic neutral cholesterol esterase in rat liver with the bile salt stimulated cholesterol esterase in pancreas

A neutral cholesterol esterase has been purified to homogeneity from the cytosolic fraction of rat liver. The 105,000 x g supernatant fraction of rat liver was applied to a DEAE-cellulose column to isolate a partially purified fraction of hepatic cholesterol esterase. Immunoblot analysis of the partially purified liver fraction with the anti-porcine pancreatic cholesterol esterase IgG demonstrated a single band with a molecular weight of 67,000. The hepatic protein was then isolated by immunoaffinity chromatography technique using a column constructed with antibodies prepared against the pancreatic cholesterol esterase. Characterization of the hepatic cholesterol esterase revealed that the hepatic enzyme shared antigenic epitopes with the pancreatic cholesterol esterase and was similarly activated by addition of bile salt such as taurocholate. Moreover, amino-terminal sequencing analysis of the hepatic cholesterol esterase showed an identical sequence with the pancreatic enzyme. Taken together, these results showed that the cholesterol esterases in the liver and the pancreas are very similar and possibly identical proteins.     

Immunological comparison of cholesterol esterases.

Rat pancreas cholesterol esterase has been immunologically compared with rat intestinal cholesterol esterase. Monospecific precipitating antisera against purified rat pancreas cholesterol esterase were produced in rabbits. Immune IgG, isolated from the antisera, crossreacted with the cholesterol esterase of intestine in the immunodiffusion assay with a pattern of complete identity. Titration of the pancreatic and intestinal enzyme with immune IgG revealed a maximum precipitation (99 and 98%) and maximum inhibition of enzyme activity (66 and 65%) when the ratio of enzyme activity (units) to immune IgG (mg) was 4.1 and 4.0, respectively. The immunological identity demonstrated in these studies lend support to the concept that intestinal cholesterol esterase is derived from the pancreatic enzyme. In additional studies, the immune IgG was employed in the immunodiffusion assay to test for cross-reaction with cholesterol esterases prepared from rat aorta, adrenal, and liver and with cholesterol esterases prepared from the pancreas of rabbit, dog, cow, and guinea pig. There was no evidence of cross-reaction in any case. Further, cholesterol esterase prepared from the pancreas of rabbit, dog, and cow retained full enzymatic activity when titrated with immune IgG.     

Interaction of bile salt monomer and cholesterol in the aqueous phase

The purpose of the present study was to evaluate the possible interaction of bile salt monomer and cholesterol in the intermicellar aqueous phase. Cholesterol and taurocholate monomer concentrations in the intermicellar aqueous phase were determined using 0-20 mM taurocholate solutions saturated with cholesterol. Maximal solubilities of cholesterol in aqueous solutions having various concentrations of taurocholate, especially below its intermicellar monomer concentration (critical micellar concentration), were determined and compared with the intermicellar cholesterol concentration. The intermicellar monomer concentration of taurocholate was constant (6 mM) and independent of taurocholate concentrations. The cholesterol concentration in the intermicellar aqueous phase gradually increased, depending upon taurocholate concentrations, and became constant (1,3 microM) above 10 mM taurocholate. The solubility of cholesterol increased linearly with the taurocholate concentration even below the critical micellar concentration, and was 0.3 microM at 6 mM taurocholate, which was approx. 20-times higher than the aqueous solubility of cholesterol, but a fifth of the maximal intermicellar cholesterol concentration. The results indicate that the higher cholesterol concentration in the intermicellar aqueous phase compared to its aqueous solubility can be primarily ascribed to the interaction of cholesterol with bile salt monomers possibly forming bile salt-cholesterol dimers, and partly to the sustaining forces induced by numerous micelles.     

Hydrolysis of chyle cholesterol esters with cell-free preparations of rat liver.

1. The effect of pH on the hydrolysis of chylomicron and chylomicron remnant cholesterol ester with rat liver homogenate was examined. The hydrolysis had three pH optima, at pH 4.5, at pH 6.0-6.5 and at pH 8.5. At the two upper pH optima extensive cholesterol ester hydrolysis occurred without simultaneous degradation of the triacylglycerol portion. 2. Similarly, microsomes (at pH 6.5-8.0) and 100 000 X g supernatant (at pH 7.5-8.5) efficiently hydrolyzed the cholesterol ester but not the triacylglycerol of chylomicron remnants. 3. With the same substrate no enrichment of neutral cholesterol esterase activity was seen in isolated plasma membranes. 4. At pH 4.5 lysosomes efficiently hydrolyzed both the cholesterol ester and the triacylglycerol portion of chylomicron remnants. 5. Three conclusions are drawn: (a) the study provides evidence against the existence of a plasma membrane-bound enzyme-hydrolyzing chylomicron cholesterol ester before or during its penetration into the cell; (b) enzymes of the cell sap and possibly of the endoplasmic reticulum can degrade cholesterol ester of chylomicron remnants without preceeding hydrolysis of the triacylglycerol core; and (c) lysosomal enzymes can degrade both the cholesterol ester and the triacylglycerol portion of chylomicron remnants if these are taken up as whole particles by endocytosis.     

Molecular modeling of the structures of human and rat pancreatic cholesterol esterases.

Structural models have been generated for rat and human cholesterol esterases by molecular modeling. For rat cholesterol esterase, three separate models were generated according to the following procedure: (1) the cholesterol esterase sequence was aligned with those of three template enzymes: Torpedo californica acetylcholinesterase, Geotrichum candidum lipase and Candida rugosa lipase; (2) the X-ray structure coordinates of the three template enzymes were used to construct cholesterol esterase models by amino acid replacements of matched sequence positions and by making sequence insertions and deletions as required; (3) bad contracts in each of the cholesterol esterase models were relaxed by molecular dynamics and mechanics; (4) the three cholesterol esterase models were merged into one by arithmetic averaging of atomic coordinates; (5) Ramachandran analysis indicated that the model generated from the AChE template possessed the best set of phi/psi angles. Therefore, this model was subjected to molecular dynamics, with harmonic constraints imposed on the C(alpha) coordinates to drive them toward the coordinates of the averaged model. (6) Subsequent relaxation by molecular mechanics produced the final rat cholesterol esterase model. A model for human cholesterol esterase was produced by repeating steps 1-3 above, albeit with the rat cholesterol esterase model as the template. Hydrophobic and electrostatic analyses of the rat and human cholesterol esterase models suggest the structural origins of molecular recognition of hydrophobic substrates and interfaces, of charged interfaces, and of bile salt activators.     

Enzyme studies on Epstein-Barr virus-transformed lymphoid cell lines from Wolman's disease. Lipases, cholesterol esterase and 4-methylumbelliferyl acyl ester hydrolases

(1) In lymphoid cell lines established by Epstein-Barr virus transformation of B-lymphocytes from normal subjects there exist two lipases hydrolysing triolein (the first one with acid optimum pH and the other one with alkaline optimum pH) and one cholesterol esterase (with acidic optimum pH). The acid triolein lipase (optimum pH 3.75-4.0) and the acid cholesterol esterase are activated by taurocholate (optimal concentration between 1 and 2.5 g/l) whereas alkaline triolein-lipase is inhibited by crude taurocholate. (2) Acid lipase deficiency is demonstrated in lymphoid cell lines from a Wolman's patient, using natural substrates, triolein and cholesteryl oleate (residual activity 5 and 8%, respectively). Thus, this similar deficiency demonstrates that, in lymphoid cell lines, triolein and cholesteryl esters are hydrolysed (under the conditions used here) by a single enzyme, i.e., lysosomal acid lipase muted in Wolman's disease. (3) pH profiles of synthetic substrate hydrolysis show marked differences between methylumbelliferyl oleate and methylumbelliferyl palmitate, and are greatly dependent on the assay conditions used. In the presence of optimal concentrations of taurocholate (1-2.5 g/l), nonspecific carboxylesterases are inhibited and acid lipase is activated: in this case, methylumbelliferyl oleate can be used to demonstrate the acid lipase deficiency in Wolman's lines (15-20% of residual activity). Methylumbelliferyl palmitate hydrolysis is less dependent on assay conditions and thus can be more accurately used for the diagnosis of Wolman's disease, with lower residual activity (10-15%) than using methylumbelliferyl oleate. Thus, Epstein-Barr virus-transformed lymphoid cell lines represent an accurate model system in culture for experimental studies of Wolman's disease.     

The hydrolysis of cholesterol esters in plasma lipoproteins by hormone-sensitive cholesterol esterase from adipose tissue.

Adipose tissue contains a high level of neutral esterase active against emulsions of cholesteryl oleate. The present studies show that this enzyme can also effectively hydrolyze the cholesterol esters in native rat plasma high density lipoproteins (HDL) and low density lipoproteins (LDL). The hydrolysis of lipoprotein cholesterol esters by a pH 5.2 isoelectric precipitate fraction from the freshly prepared 100,000 X g supernatant of chicken adipose tissue was low, but increased more than 50-fold on activation with cyclic AMP-dependent protein kinase. Rat adipose tissue homogenates were also very active against lipoprotein cholesterol esters, hydrolyzing as much as 60% of the total labeled cholesterol ester in HDL or LDL in 1 h. Activity was optimal at pH 7 and very low at pH 4. No protease activity was detected at pH 7 and, since assays were done in 2 mM EDTA, phospholipase A activity was presumably negligible. The results show that hormone-sensitive cholesterol esterase of adipose tissue has ready access to the neutral lipid core of plasma lipoproteins, either because the enzyme penetrates the polar shell or because the cholesterol ester in the core is exposed, at least intermittently, to allow enzyme-substrate complex formation. Whether or not this enzyme activity plays a role in lipoprotein degradation by adipose tissue remains to be determined.     

Pancreatic cholesterol esterases. 2. Purification and characterization of human pancreatic fatty acid ethyl ester synthase

Human pancreatic fatty acid ethyl ester synthase has been isolated and purified 1200-fold to homogeneity, and its activities, binding properties, and N-terminal amino acid sequence indicate that it is a member of the lipase family. This 52-kDa monomeric protein is present at 0.6-1.2 mg/g of pancreas, and it catalyzes the synthesis and hydrolysis of ethyl oleate at rates of 2400 nmol mg-1 h-1 and 30 nmol mg-1 h-1, respectively. Kinetic analyses reveal a pronounced substrate specificity for unsaturated octadecanoic fatty acids, with ethyl ester synthetic rates of 2400 nmol mg-1 h-1 (linoleic), 2400 nmol mg-1 h-1 (oleic), 400 nmol mg-1 h-1 (arachidonic), 300 nmol mg-1 h-1 (palmitic), and 100 nmol mg-1 h-1 (stearic). Like cholesterol esterase, the enzyme binds to immobilized heparin, and this property was critical for its purification to homogeneity. Its N-terminal amino acid sequence is virtually identical with that reported for human triglyceride lipase, NH2-X-Glu-Val-Cys-5Tyr-Glu-Arg-Leu-Gly-10Cys-Phe-Ser-Asp- Asp-15Ser-Pro-Trp-Ser-Gly-20Ile, and it differs by only four residues from that reported for porcine pancreatic lipase. The synthase purified here also cleaves triglycerides, hydrolyzing triolein at a rate of 30 nmol mg-1 h-1, and this activity is stimulated by colipase and inhibited by sodium chloride. Conversely, commercially available porcine triglyceride lipase exhibits fatty acid ethyl ester synthase activity (1530 nmol mg-1 h-1) and hydrolyzes triolein at a rate of 23 nmol mg-1 h-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Continuous monitoring of cholesterol oleate hydrolysis by hormone-sensitive lipase and other cholesterol esterases

Hormone-sensitive lipase (HSL) contributes importantly to the hydrolysis of cholesteryl ester in steroidogenic tissues, releasing the cholesterol required for adrenal steroidogenesis. HSL has broad substrate specificity, because it hydrolyzes triacylglycerols (TAGs), diacylglycerols, monoacylglycerols, and cholesteryl esters. In this study, we developed a specific cholesterol esterase assay using cholesterol oleate (CO) dispersed in phosphatidylcholine and gum arabic by sonication. To continuously monitor the hydrolysis of CO by HSL, we used the pH-stat technique. For the sake of comparison, the hydrolysis of CO dispersion was also tested using other cholesteryl ester-hydrolyzing enzymes. The specific activities measured on CO were found to be 18, 100, 27, and 3 micromol/min/mg for HSL, cholesterol esterase from Pseudomonas species, Candida rugosa lipase-3, and cholesterol esterase from bovine pancreas, respectively. The activity of HSL on CO is approximately 4- to 5-fold higher than on long-chain TAGs. In contrast, with all other enzymes tested, the rates of TAG hydrolysis were higher than those of CO hydrolysis. The relatively higher turnover of HSL on CO observed in vitro adds further molecular insight on the physiological importance of HSL in cholesteryl ester catabolism in vivo. Thus, HSL could be considered more as a cholesteryl ester hydrolase than as a TAG lipase.     

Purification of pancreatic cholesterol esterase expressed in recombinant baculovirus-infected Sf9 cells.

A cDNA clone encoding the entire coding sequence of rat pancreatic cholesterol esterase (bile salt-stimulated lipase) was subcloned into the Baculovirus transfer vector pVL1392 and used to co-transfect Spodoptera frugiperda (Sf9) insect cells with wild-type Autographa californica nuclear polyhedrosis virus (AcNPV) DNA. Two recombinant proteins (M(r) 74 kDa and 64 kDa) reactive with anti-cholesterol esterase IgG were produced and secreted by the infected Sf9 cells in large quantities in a time-dependent manner. The 74-kDa protein was detectable in the cultured medium at the second day post-infection and increased progressively, reaching a level of 50 micrograms/ml of culture medium after 8 days. Amino-terminal sequencing of this recombinant protein showed that the signal peptide of cholesterol esterase was correctly cleaved, resulting in the production of mature protein. The 64-kDa recombinant protein was not detected in the medium until Day 5 post-infection and accumulated to a level of 25 micrograms/ml at Day 8. Both the 74- and the 64-kDa cholesterol esterases were biologically active and hydrolyzed the artificial substrate p-nitrophenyl butyrate. Results of this study demonstrated that Baculovirus-infected Sf9 cells can be used for high-level expression of pancreatic cholesterol esterase. The recombinant enzyme will be useful for further characterization of this protein.     

A retinyl ester hydrolase activity intrinsic to the brush border membrane of rat small intestine.

Retinol esterified with long-chain fatty acids is a common dietary source of vitamin A. Hydrolysis of these esters in the lumen of the small intestine is required prior to absorption. Bile salt-stimulated retinyl esterase activity was present with purified rat intestinal brush border membrane, with the maximum rate of ester hydrolysis at approximately pH 8, the physiological luminal pH. Taurocholate, a trihydroxy bile salt, stimulated hydrolysis of short-chain fatty acyl retinyl esters more than hydrolysis of long-chain fatty acyl esters. Deoxycholate, a dihydroxy bile salt, primarily stimulated hydrolysis of long-chain esters. Calculated Kms of 0.74 microM for retinyl palmitate (16:0) hydrolysis and 9.6 microM for retinyl caproate (6:0) hydrolysis suggested the presence of two separate activities. Consistent with that, the activity responsible for retinyl caproate hydrolysis could be inactivated to a greater degree than retinyl palmitate hydrolysis by preincubation of the brush border membrane at 37 degrees C for extended times. Brush border membrane from animals who had undergone common duct ligation 48 h prior to tissue collection showed little ability to hydrolyze retinyl caproate but retained 70% of retinyl palmitate hydrolytic activity, compared to sham-operated controls. Thus, two distinguishable retinyl esterase activities were recovered with purified brush border membranes. One apparently originated from the pancreas, was stimulated by trihydroxy bile salts, and preferentially hydrolyzed short-chain retinyl esters, properties similar to cholesterol ester hydrolase, known to bind to the brush border. The other was intrinsic to the brush border, stimulated by both trihydroxy and dihydroxy bile salts, and preferentially hydrolyzed long-chain retinyl esters, providing the majority of activity of the brush border against dietary retinyl esters.     

Synthesis of fatty acid sterol esters using cholesterol esterase from Trichoderma sp. AS59

We recently reported the characterization of novel cholesterol esterase (EC. 3.1.1.13) from Trichoderma sp. and preliminary work on sterol ester synthesis. In the present study, we further examined the enzyme ability to synthesize cholesterol esters from cholesterol and free fatty acids of various chain lengths, and compared the fatty acid specificity in synthesis with that in hydrolysis. The enzyme catalyzed the synthesis of medium- and long-chain fatty acid cholesterol esters, but failed to synthesize short-chain fatty acid esters. The fatty acid specificities in the synthesis and hydrolysis of cholesterol esters were entirely different from each other. Unlike other lipolytic enzymes, the enzyme was largely independent of water content in the synthesis of cholesterol oleate, and it achieved near-complete esterification in the presence of an equimolar excess of oleic acid. Of additional interest is the finding that the addition of n-hexane markedly enhanced the esterification activities on all the medium- and long-chain saturated fatty acids used. Based on these findings, we attempted to synthesize stigmasterol stearate as a food additive to lower cholesterol levels in blood plasma, and found that the enzyme catalyzed effective synthesis of the ester without the need of dehydration during the reaction, indicating the potential utility of the enzyme in the food industry.     

Enzymatic properties of polyethylene glycol-modified cholesterol esterase in organic solvents.

The solvent dependency and substrate specificity of polyethylene glycol (PEG)-modified cholesterol esterase (CEH) catalyzing cholesterol ester synthesis in organic solvents were studied. When cholesterol and linoleic acid were used as the substrates, PEG-modified CEH synthesized cholesterol linoleate only in water-immiscible organic solvents. Among some solvents capable of solubilizing all of the reaction components (PEG-modified CEH, cholesterol, and linoleic acid), chloroform was most suitable for enzymatic cholesterol linoleate synthesis, and the synthetic activity for cholesterol linoleate decreased in the order chloroform, benzene, toluene, and cyclohexane. PEG-modified CEH synthesized various cholesterol esters with significant substrate specificity. The substrate specificity for cholesterol ester synthesis in benzene was analogous to that for cholesterol ester hydrolysis in aqueous solution.     

Esterification of cholesterol and lipid-soluble vitamins by human pancreatic carboxyl ester hydrolase

Human pancreatic carboxyl ester hydrolase is shown to catalyse the esterification of cholesterol and lipid-soluble vitamins A, E and D3 with oleic acid. The acitivity requires the presence of bile salts, and the trihydroxylated or the 3 alpha, 7 alpha dihydroxylated bile salts are better activators than the 3 alpha, 12 alpha dihydroxylated bile salts. The hydrolyzing and synthetizing activities of human pancreatic carboxyl ester hydrolase are separated by a large pH range since the synthesis of cholesterol esters is optimal at pH 5.25 and the hydrolysis of cholesterol and vitamin E esters is optimal at pH 8.0. From the comparison of the catalytic constants determined for the hydrolyzing and synthetizing activities and from the pH dependence of the two activities, it appears that human carboxyl ester hydrolase plays an important part in the intestinal lumen. The role of the enzyme in the esterification of cholesterol and lipid-soluble vitamins is questionable.     

Lipolytic enzymes of the human pancreas. II. Purification and properties of cholesterol ester hydrolase

Cholesterol ester hydrolase (sterol-ester acylhydrolase, EC 3.1.1.13) was purified from human pancreatic tissue by column chromatography and acetone precipitation, leading to a 400-fold enrichment. Isoelectric focusing of this product reveals a double-band at pH 4.5 and 4.6. The molecular weight was estimated at 320 kDa by means of Sephadex filtration on calibrated columns. Obviously these large molecules represent a tetrameric form of the monomeric subunit (molecular mass 76-80 kDa), which is also enzymatically active. It was found together with the dimeric form in pancreatic juice, where the tetrameric enzyme is responsible for the major part of the hydrolytic activity, splitting cholesterol ester as well as synthetic substrates, such as fluorescein or p-nitrophenyl esters. Attempts to split the tetrameric cholesterol ester hydrolase, isolated from pancreatic tissue, into active subunits found additionally in pancreatic juice by the influence of bile acids and proteolytic enzymes failed. The spectral shift method using Rhodamine fluorescence was employed in order to prove that fluorescein dilaurate forms micellar solutions and mixed micelles when bile salts are present.     

Cholesterol esterase--a polymeric enzyme

A direct interaction of cholic acid and pancreatic cholesterol esterase has been shown using labeled bile salt of high specific activity and Sephadex G-50 chromatography. This interaction results in the formation of a protein of M.W. 400,000 which appears to be a hexamer of the protein in pancreatic juice (M.W. 65,000 – 69,000). Rechromatography of the polymer on Sephadex G-200 results in dissociation to the monomeric form. Evidence is provided, suggesting that the polymeric form of cholesterol esterase represents the active enzyme responsible for sterol ester synthesis and hydrolysis.     

Importance of arginines 63 and 423 in modulating the bile salt-dependent and bile salt-independent hydrolytic activities of rat carboxyl ester lipase

Previous studies using chemical modification approach have shown the importance of arginine residues in bile salt activation of carboxyl ester lipase (CEL) activity. However, the x-ray crystal structure of CEL failed to show the involvement of arginine residues in CEL-bile salt interaction. The current study used a site-specific mutagenesis approach to determine the role of arginine residues 63 and 423 in bile salt-dependent and bile salt-independent hydrolytic activities of rat CEL. Mutations of Arg(63) to Ala(63) (R63A) and Arg(423) to Gly(423) (R423G) resulted in enzymes with increased bile salt-independent hydrolytic activity against lysophosphatidylcholine, having 6.5- and 2-fold higher k(cat) values, respectively, in comparison to wild type CEL. In contrast, the R63A and R423A mutant enzymes displayed 5- and 11-fold decreases in k(cat), in comparison with wild type CEL, for bile salt-dependent cholesteryl ester hydrolysis. Although taurocholate induced similar changes in circular dichroism spectra for wild type, R63A, and R423G proteins, this bile salt was less efficient in protecting the mutant enzymes against thermal inactivation in comparison with control CEL. Lipid binding studies revealed less R63A and R423G mutant CEL were bound to 1,2-diolein monolayer at saturation compared with wild type CEL. These results, along with computer modeling of the CEL protein, indicated that Arg(63) and Arg(423) are not involved directly with monomeric bile salt binding. However, these residues participate in micellar bile salt modulation of CEL enzymatic activity through intramolecular hydrogen bonding with the C-terminal domain. These residues are also important, probably through similar intramolecular hydrogen bond formation, in stabilizing the enzyme in solution and at the lipid-water interface.     

Plant sterol and stanol substrate specificity of pancreatic cholesterol esterase

Consumption of plant sterols or stanols (collectively referred to as phytosterols) and their esters results in decreased low-density lipoprotein cholesterol, which is associated with decreased atherosclerotic risk. The mechanisms by which phytosterols impart their effects, however, are incompletely characterized. The objective of the present study is to determine if pancreatic cholesterol esterase (PCE; EC 3.1.1.13), the enzyme primarily responsible for cholesterol ester hydrolysis in the digestive tract, is capable of hydrolyzing various phytosterol esters and to compare the rates of sterol ester hydrolysis in vitro. We found that PCE hydrolyzes palmitate, oleate and stearate esters of cholesterol, stigmasterol, stigmastanol and sitosterol. Furthermore, we found that the rate of hydrolysis was dependent on both the sterol and the fatty acid moieties in the following order of rates of hydrolysis: cholesterol>(sitosterol=stigmastanol)>stigmasterol; oleate>(palmitate=stearate). The addition of free phytosterols to the system did not change hydrolytic activity of PCE, while addition of palmitate, oleate or stearate increased activity. Thus, PCE may play an important but discriminatory role in vivo in the liberation of free phytosterols to compete with cholesterol for micellar solubilization and absorption.