Use of an inhibitor to identify members of the hormone-sensitive lipase family

Hormone-sensitive lipase (HSL) contributes importantly to the mobilization of fatty acids from the triacylglycerols stored in adipocytes, which provide the main source of energy in mammals. On the basis of amino acid sequence alignments and three-dimensional structures, this enzyme was previously found to be a suitable template for defining a family of serine carboxylester hydrolases. In this study, the HSL family members are characterized rather on the basis of their inhibition by 5-methoxy-3-(4-phenoxyphenyl)-3H-[1,3,4]oxadiazol-2-one (compound 7600). This compound inhibits mammalian HSL as well as other HSL family members, such as EST2 from the thermophilic eubacterium Alicyclobacillus acidocaldarius and AFEST from the hyperthermophilic archaeon Archaeoglobus fulgidus. Various carboxylester hydrolases that are not members of the HSL family were found not to be inhibited by compound 7600 under the same experimental conditions. These include nonlipolytic hydrolases such as Torpedo californica acetylcholinesterase and pig liver esterase, as well as lipolytic hydrolases such as human pancreatic lipase, dog gastric lipase, Thermomyces lanuginosus lipase, and Bacillus subtilis LipA. When vinyl esters were used as substrates, the residual activity of HSL, AFEST, and EST2 decreased with an increase in compound 7600 concentration in the incubation mixture. The inhibitor concentration at which the enzyme activity decreased to 50% after incubation for 5 min was 70, 20, and 15 nM with HSL, AFEST, and EST2, respectively. Treating EST2 and AFEST with the inhibitor resulted in an increase in the molecular mass, as established by performing matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis. This increase in the molecular mass, which corresponds approximately to the molecular mass of the inhibitor, indicates that a covalent enzyme-inhibitor complex has been formed. Surface-enhanced laser desorption ionization time-of-flight mass spectrometry analysis of a trypsin digest of AFEST treated with the inhibitor or not treated showed the occurrence of an increase in the molecular masses of the "GESAGG"-containing peptide, which is compatible with the formation of a covalent complex with the inhibitor.     

Potential active-site residues in polyneuridine aldehyde esterase, a central enzyme of indole alkaloid biosynthesis, by modelling and site-directed mutagenesis.

In the biosynthesis of the antiarrhythmic alkaloid ajmaline, polyneuridine aldehyde esterase (PNAE) catalyses a central reaction by transforming polyneuridine aldehyde into epi-vellosimine, which is the immediate precursor for the synthesis of the ajmalane skeleton. The PNAE cDNA was previously heterologously expressed in E. coli. Sequence alignments indicated that PNAE has a 43% identity to a hydroxynitrile lyase from Hevea brasiliensis, which is a member of the alpha/beta hydrolase superfamily. The catalytic triad, which is typical for this family, is conserved. By site-directed mutagenesis, the members of the catalytic triad were identified. For further detection of the active residues, a model of PNAE was constructed based on the X-ray crystallographic structure of hydroxynitrile lyase. The potential active site residues were selected on this model, and were mutated in order to better understand the relationship of PNAE with the alpha/beta hydrolases, and as well its mechanism of action. The results showed that PNAE is a novel member of the alpha/beta hydrolase enzyme superfamily.     

Characterization of an acetyl xylan esterase from the anaerobic fungus Orpinomyces sp. strain PC-2.

A 1,067-bp cDNA, designated axeA, coding for an acetyl xylan esterase (AxeA) was cloned from the anaerobic rumen fungus Orpinomyces sp. strain PC-2. The gene had an open reading frame of 939 bp encoding a polypeptide of 313 amino acid residues with a calculated mass of 34,845 Da. An active esterase using the original start codon of the cDNA was synthesized in Escherichia coli. Two active forms of the esterase were purified from recombinant E. coli cultures. The size difference of 8 amino acids was a result of cleavages at two different sites within the signal peptide. The enzyme released acetate from several acetylated substrates, including acetylated xylan. The activity toward acetylated xylan was tripled in the presence of recombinant xylanase A from the same fungus. Using p-nitrophenyl acetate as a substrate, the enzyme had a K(m) of 0.9 mM and a V(max) of 785 micromol min(-1) mg(-1). It had temperature and pH optima of 30 degrees C and 9.0, respectively. AxeA had 56% amino acid identity with BnaA, an acetyl xylan esterase of Neocallimastix patriciarum, but the Orpinomyces AxeA was devoid of a noncatalytic repeated peptide domain (NCRPD) found at the carboxy terminus of the Neocallimastix BnaA. The NCRPD found in many glycosyl hydrolases and esterases of anaerobic fungi has been postulated to function as a docking domain for cellulase-hemicellulase complexes, similar to the dockerin of the cellulosome of Clostridium thermocellum. The difference in domain structures indicated that the two highly similar esterases of Orpinomyces and Neocallimastix may be differently located, the former being a free enzyme and the latter being a component of a cellulase-hemicellulase complex. Sequence data indicate that AxeA and BnaA might represent a new family of hydrolases.     

Hydrolase activity in the venom of the pupal endoparasitic wasp, Pimpla hypochondriaca

Venom from the pupal endoparasitoid, Pimpla hypochondriaca has previously been shown to contain a mixture of biologically active molecules. Currently, P. hypochondriaca venom was examined for the presence of hydrolase activity. Six hydrolases were consistently detected using the API ZYM semiquantitative colourimetric kit. The main hydrolases detected were; acid phosphatase, beta-glucosidase, esterase, beta-galactosidase, esterase lipase, and lipase. The most rapid and intense colour reaction was detected for acid phosphatase. The pH optimum and the specific activity of venom acid phosphatase was determined using p-nitrophenol phosphate as a substrate and were 4.8 and 0.47 nmol p-nitrophenol/min/microg of venom protein, respectively. The acid phosphatase activity was inhibited in a dose dependent manner by sodium fluoride (IC(50) 4.2 x 10(-4) M), and by cocktail inhibitor 2 (CI 2). P. hypochondriaca venom has previously been shown to display potent cytotoxic activity towards Lacanobia oleracea haemocytes maintained in vitro. The contribution of acid phosphatase in venom to this cytotoxic activity was investigated by titrating venom against CI 2 prior to the addition of L. oleracea haemocytes. The results suggest that, despite the relatively high levels of acid phosphatase activity in venom, venom acid phosphatase plays no role in the antihaemocytic activity of P. hypochondriaca venom in vitro.     

Comparative modeling of the three-dimensional structures of family 3 glycoside hydrolases

There are approximately 100 known members of the family 3 group of glycoside hydrolases, most of which are classified as beta-glucosidases and originate from microorganisms. The only family 3 glycoside hydrolase for which a three-dimensional structure is available is a beta-glucan exohydrolase from barley. The structural coordinates of the barley enzyme is used here to model representatives from distinct phylogenetic clusters within the family. The majority of family 3 hydrolases have an NH(2)-terminal (alpha/beta)(8) barrel connected by a short linker to a second domain, which adopts an (alpha/beta)(6) sandwich fold. In two bacterial beta-glucosidases, the order of the domains is reversed. The catalytic nucleophile, equivalent to D285 of the barley beta-glucan exohydrolase, is absolutely conserved across the family. It is located on domain 1, in a shallow site pocket near the interface of the domains. The likely catalytic acid in the barley enzyme, E491, is on domain 2. Although similarly positioned acidic residues are present in closely related members of the family, the equivalent amino acid in more distantly related members is either too far from the active site or absent. In the latter cases, the role of catalytic acid is probably assumed by other acidic amino acids from domain 1.     

Functional metagenomics unveils a multifunctional glycosyl hydrolase from the family 43 catalysing the breakdown of plant polymers in the calf rumen

Microbial communities from cow rumen are known for their ability to degrade diverse plant polymers at high rates. In this work, we identified 15 hydrolases through an activity-centred metagenome analysis of a fibre-adherent microbial community from dairy cow rumen. Among them, 7 glycosyl hydrolases (GHs) and 1 feruloyl esterase were successfully cloned, expressed, purified and characterised. The most striking result was a protein of GH family 43 (GHF43), hereinafter designated as R_09-02, which had characteristics very distinct from the other proteins in this family with mono-functional β-xylosidase, α-xylanase, α-L-arabinase and α-L-arabinofuranosidase activities. R_09-02 is the first multifunctional enzyme to exhibit β-1,4 xylosidase, α-1,5 arabinofur(pyr)anosidase, β-1,4 lactase, α-1,6 raffinase, α-1,6 stachyase, β-galactosidase and α-1,4 glucosidase activities. The R_09-02 protein appears to originate from the chromosome of a member of Clostridia, a class of phylum Firmicutes, members of which are highly abundant in ruminal environment. The evolution of R_09-02 is suggested to be driven from the xylose- and arabinose-specific activities, typical for GHF43 members, toward a broader specificity to the glucose- and galactose-containing components of lignocellulose. The apparent capability of enzymes from the GHF43 family to utilise xylose-, arabinose-, glucose- and galactose-containing oligosaccharides has thus far been neglected by, or could not be predicted from, genome and metagenome sequencing data analyses. Taking into account the abundance of GHF43-encoding gene sequences in the rumen (up to 7% of all GH-genes) and the multifunctional phenotype herein described, our findings suggest that the ecological role of this GH family in the digestion of ligno-cellulosic matter should be significantly reconsidered.     

Defining Sequence Space and Reaction Products within the Cyanuric Acid Hydrolase (AtzD)/Barbiturase Protein Family

Cyanuric acid hydrolases (AtzD) and barbiturases are homologous, found almost exclusively in bacteria, and comprise a rare protein family with no discernible linkage to other protein families or an X-ray structural class. There has been confusion in the literature and in genome projects regarding the reaction products, the assignment of individual sequences as either cyanuric acid hydrolases or barbiturases, and spurious connection of this family to another protein family. The present study has addressed those issues. First, the published enzyme reaction products of cyanuric acid hydrolase are incorrectly identified as biuret and carbon dioxide. The current study employed (13)C nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry to show that cyanuric acid hydrolase releases carboxybiuret, which spontaneously decarboxylates to biuret. This is significant because it revealed that homologous cyanuric acid hydrolases and barbiturases catalyze completely analogous reactions. Second, enzymes that had been annotated incorrectly in genome projects have been reassigned here by bioinformatics, gene cloning, and protein characterization studies. Third, the AtzD/barbiturase family has previously been suggested to consist of members of the amidohydrolase superfamily, a large class of metallohydrolases. Bioinformatics and the lack of bound metals both argue against a connection to the amidohydrolase superfamily. Lastly, steady-state kinetic measurements and observations of protein stability suggested that the AtzD/barbiturase family might be an undistinguished protein family that has undergone some resurgence with the recent introduction of industrial s-triazine compounds such as atrazine and melamine into the environment.     

Isolation and characterization of a metagenome-derived thermoalkaliphilic esterase with high stability over a broad pH range

A novel alkaliphilic esterase (EstJ) was identified from a soil metagenome of Jeju Island, Korea, using a 96-well plate-based functional assay for determination of pH dependence of activity. The amino acid sequence of EstJ showed low similarity (32–45 %) to putative α/β hydrolases derived from whole-genome sequencing studies. EstJ, although not belonging to any of the known families of bacterial lipolytic enzymes, however, it showed closest sequence identity to the family IV enzymes that are related to the mammalian hormone-sensitive lipases. The highly conserved motifs of family IV enzymes were found in EstJ, but the corresponding sequences of each motif in EstJ were unique; most particularly the –(F/Y)(F/Y/L)HGGG– motif was represented by –WMVSGG–. The purified EstJ was highly active from pH 8.5 to 10.5. More than 90 % of maximum activity was also retained over a wide pH range of 5.5–0.5 after prolonged incubation. EstJ was also moderately thermophilic with an optimum temperature of 55 °C. Therefore, EstJ is the first metagenome-derived bacterial family IV esterase possessing both highly alkaliphilic and moderately thermophilic properties.     

YesT: a new rhamnogalacturonan acetyl esterase from Bacillus subtilis

YesT, a putative protein from Bacillus subtilis ATCC 6633 that has been provisionally classified as a rhamnogalacturonan acetyl esterase (RGAE) in CE-12 family, was cloned, expressed in Escherichiacoli Rosetta (DE3), and purified. The enzyme is monomeric with a molecular mass of 37 kDa and presents thermophilic properties similar to RGAE from Aspergillus aculeatus, although YesT is more alkaliphilic. The study of inhibitors confirmed the importance of the His and the nucleophilic Ser for the esterase activity, apart from the Asp from the catalytic triad. This enzyme also presents broad substrate specificity, and is active toward 7-aminocephalosporanic acid, cephalosporin C, p-nitrophenyl acetate, beta-naphthyl acetate, glucose pentaacetate, and acetylated xylan. Moreover, YesT achieves a synergistic effect together with xylanase A toward acetylated xylan. As a member of the SGNH family, it does not adopt the common alpha/beta hydrolase fold. The primary sequence analysis and multiple sequence alignment revealed the lack of a two beta-stranded antiparallel sheet, which results in a clear change in the structure together with the disappearance of one of the three 3(10)-helices presented in RGAE structure. The similarities found in this article among the topological diagrams of RGAE, YesT, and Esterase A from Streptomyces scabies, Platelet-Activating Factor AcetylHydrolase, isoform Ib, alpha subunit [PAF-AH(Ib)alpha(1)], PAF-AH(Ib)alpha(2), the esterase domain from hemagglutinin esterase fusion glycoprotein (HEF1) from Influenza C virus, the thioesterase I (TAP) from E. coli, the hypothetical protein a1r1529 from Nostoc sp., and the hypothetical YxiM precursor that all belong to the SGNH family could indicate a possible divergence of such proteins from a common ancestor.     

Structure activity studies with xenobiotic substrates using carboxylesterases isolated from Arabidopsis thaliana

Carboxylesterases (CXEs) catalyse the hydrolysis of xenobiotics and natural products radically altering their biological activities. Whereas the substrate selectivity of animal CXEs, such as porcine liver esterase (PLE) have been well studied, the respective enzymes in plants have yet to be defined and their activities determined. Using Arabidopsis thaliana (At) as a source, five representative members of the alpha/beta hydrolase AtCXE family of proteins have been cloned, expressed and the purified recombinant proteins assayed for esterase activity with xenobiotic substrates. Two members, AtCXE5 and AtCXE18 were found to be active carboxylesterases, though AtCXE5 proved to be highly unstable as a soluble protein. AtCXE18 and the previously characterised S-formylglutathione hydrolase from Arabidopsis (AtSFGH) were assayed against a series of esters based on methylumbelliferone in which the acyl moiety was varied with respect to size and conformation. The same series was used to assay crude esterase preparation from Arabidopsis plants and the results compared with those obtained with the commonly used PLE. With straight chain esters, AtCXE18 behaved like PLE, but the Arabidopsis hydrolases proved less tolerant of branched chain acyl components than the mammalian enzyme. While none of the enzyme preparations accurately reflected all the activities determined with crude Arabidopsis protein extracts, the plant enzymes proved more useful than PLE in predicting the hydrolysis of the more sterically constrained esters.     

FLJ14813 missense mutation: a candidate for autosomal dominant thrombocytopenia on human chromosome 10

The gene for a novel nonsyndromic autosomal dominant thrombocytopenia has been previously mapped to a region on human chromosome 10p11-12 (THC2, OMIM number *188000). This disorder is characterized by moderate thrombocytopenia and incomplete differentiation of megakaryocytes. We report here a novel missense mutation in the human gene FLJ14813 that segregates perfectly with thrombocytopenia in our kindred of 51 family members. The mutation is not detected in 94 random unrelated and unaffected individuals, nor is it reported in the Entrez single nucleotide polymorphism (SNP) database. A substitution of cytosine for guanidine (G to C) at nucleotide position 565 was present in all thrombocytopenic family members, causing a predicted substitution of aspartic acid for glutamic acid (E167D) in exon four.     

Analysis of O-acetyl-ADP-ribose as a target for Nudix ADP-ribose hydrolases

The Sir2 family of NAD(+)-dependent histone/protein deacetylases has been implicated in a wide range of biological activities, including gene silencing, life span extension, and chromosomal stability. Recent evidence has indicated that these proteins produce a novel metabolite O-acetyl-ADP-ribose (OAADPr) during deacetylation. Cellular studies have demonstrated that this metabolite exhibits biological effects when microinjected in living cells. However, the molecular targets of OAADPr remain to be identified. Here we have analyzed the ADP-ribose-specific Nudix family of hydrolases as potential in vivo metabolizing enzymes of OAADPr. In vitro, we found that the ADP-ribose hydrolases (yeast YSA1, mouse NudT5, and human NUDT9) cleaved OAADPr to the products AMP and acetylated ribose 5'-phosphate. Steady-state kinetic analyses revealed that YSA1 and NudT5 hydrolyzed OAADPr with similar kinetic constants to those obtained with ADP-ribose as substrate. In dramatic contrast, human NUDT9 was 500-fold less efficient (k(cat)/K(m) values) at hydrolyzing OAADPr compared with ADP-ribose. The inability of OAADPr to inhibit the reaction of NUDT9 with ADP-ribose suggests that NUDT9 binds OAADPr with low affinity, likely due to steric considerations of the additional acetylated-ribose moiety. We next explored whether Nudix hydrolytic activities against OAADPr could be observed in cell extracts from yeast and human. Using a detailed analysis of the products generated during the consumption of OAADPr in extracts, we identified two robust enzymatic activities that were not consistent with the known Nudix hydrolases. Instead, we identified cytoplasmic esterase activities that hydrolyze OAADPr to acetate and ADP-ribose, whereas a distinct activity residing in the nucleus is consistent with an OAADPr-specific acetyltransferase. These findings establish for the first time that select members of the ADP-ribose hydrolases are potential targets of OAADPr metabolism. However, the predominate endogenous activities observed from diverse cell extracts represent novel enzymes.     

The wood rot ascomycete Xylaria polymorpha produces a novel GH78 glycoside hydrolase that exhibits α-L-rhamnosidase and feruloyl esterase activities and releases hydroxycinnamic acids from lignocelluloses

Soft rot (type II) fungi belonging to the family Xylariaceae are known to substantially degrade hardwood by means of their poorly understood lignocellulolytic system, which comprises various hydrolases, including feruloyl esterases and laccase. In the present study, several members of the Xylariaceae were found to exhibit high feruloyl esterase activity during growth on lignocellulosic materials such as wheat straw (up to 1,675 mU g(-1)) or beech wood (up to 80 mU g(-1)). Following the ester-cleaving activity toward methyl ferulate, a hydrolase of Xylaria polymorpha was produced in solid-state culture on wheat straw and purified by different steps of anion-exchange and size-exclusion chromatography to apparent homogeneity (specific activity, 2.2 U mg(-1)). The peptide sequence of the purified protein deduced from the gene sequence and verified by de novo peptide sequencing shows high similarity to putative α-L-rhamnosidase sequences belonging to the glycoside hydrolase family 78 (GH78; classified under EC 3.2.1.40). The purified enzyme (98 kDa by SDS-PAGE, 103 kDa by size-exclusion chromatography; pI 3.7) converted diverse glycosides (e.g., α-L-rhamnopyranoside and α-L-arabinofuranoside) but also natural and synthetic esters (e.g., chlorogenic acid, hydroxycinnamic acid glycoside esters, veratric acid esters, or p-nitrophenyl acetate) and released free hydroxycinnamic acids (ferulic and coumaric acid) from arabinoxylan and milled wheat straw. These catalytic properties strongly suggest that X. polymorpha GH78 is a multifunctional enzyme. It is the first fungal enzyme that combines glycosyl hydrolase with esterase activities and may help this soft rot fungus to degrade lignocelluloses.     

High-throughput screening of activity and enantioselectivity of esterases

A procedure for the high-throughput screening of esterases is described. This includes enzyme expression in microtiter plates and the measurement of activity and enantioselectivity (E) of the esterase variants using acetates of secondary alcohols as model substrates. Acetic acid released is converted in an enzyme cascade leading to the stoichiometric formation of NADH, which is quantified in a spectrophotometer. The method allows screening of several thousand mutants per day and has already been successfully applied to identify an esterase mutant with an E>100 toward an important building block for organic synthesis. This protocol can also be used for lipases and possibly other hydrolases that are expressed in soluble form in conventional Escherichia coli strains. This protocol can be completed in 3-4 days.     

Hepatic overexpression of cholesteryl ester hydrolase enhances cholesterol elimination and in vivo reverse cholesterol transport

Neutral cholesteryl ester hydrolase (CEH)-mediated hydrolysis of cellular cholesteryl esters (CEs) is required not only to generate free cholesterol (FC) for efflux from macrophages but also to release FC from lipoprotein-delivered CE in the liver for bile acid synthesis or direct secretion into the bile. We hypothesized that hepatic expression of CEH would regulate the hydrolysis of lipoprotein-derived CE and enhance reverse cholesterol transport (RCT). Adenoviral-mediated CEH overexpression led to a significant increase in bile acid output. To assess the role of hepatic CEH in promoting flux of cholesterol from macrophages to feces, cholesterol-loaded and [(3)H]cholesterol-labeled J774 macrophages were injected intraperitoneally into mice and the appearance of [(3)H]cholesterol in gallbladder bile and feces over 48 h was quantified. Mice overexpressing CEH had significantly higher [(3)H]cholesterol radiolabel in bile and feces, and it was associated with bile acids. This CEH-mediated increased movement of [(3)H]cholesterol from macrophages to bile acids and feces was significantly attenuated in SR-BI(-/-) mice. These studies demonstrate that similar to macrophage CEH that rate-limits the first step, hepatic CEH regulates the last step of RCT by promoting the flux of cholesterol entering the liver via SR-BI and increasing hepatic bile acid output.     

Primary structure and catalytic properties of a cold-active esterase from a psychrotroph,Acinetobacter sp. strain No. 6. isolated from Siberian soil

We cloned a gene coding for a cold-active esterase from a genomic library of Acinetobacter sp. strain No. 6, a psychrotroph isolated from Siberian soil. The gene, aest, encoded a protein of 301 amino acid residues, the deduced sequence of which had less than 17% identity to sequences of known esterases and lipases. However, the esterase seemed to belong to the alpha/beta hydrolase superfamily, because it contained a sequence, Gly-Xaa-Ser-Xaa-Gly (with Xaa an arbitrary amino acid residue), found in most serine hydrolases of this superfamily. Sequence comparison earlier suggested a weak phylogenetic relationship of gene product AEST to the EST group of the esterase-lipase family, which has been found only in eukaryotes. The aest gene was expressed in Escherichia coli BL21(DE3) cells under the control of the T7 promoter, and the expression product was purified to homogeneity and characterized. It catalyzed the hydrolysis of esters with short-chain acyl groups and had lower activation energy and lower thermostability than do mesophilic enzymes, as expected from the cold-adapted nature of this enzyme.     

Screening and identification of a novel esterase EstPE from a metagenomic DNA library

Esterases represent a large family of hydrolases with broad substrate specificity and functional sequence space. Although many attempts to screen new esterases have been conducted, there have been few reports conducted to discriminate unique enzymes from typical ones based on novel structure and function. In this study, we discovered an esterase and a novel family through a successive assay of whole cells and crude lysates (oxidative open condition). The screened putative esterases from the metagenomic DNA of salted shrimp consisted of 753 bp encoding 27 kDa of polypeptide, namely PE esterase. Sequence analyses revealed that an identical gene was reported from whole genome sequencing of Stenotrophomonas maltophilia K279a. However, its biochemical and phylogenetic characteristics have not yet been evaluated. PE esterase was overexpressed only by the MBP fusion state in E. coli and was easily purified using an affinity column. This enzyme showed a typical spectrum of substrate specificity and possessed the consensus motifs, Ser-Asp-His and GXSXG, which are essential for most esterase/lipase superfamilies. Interestingly, the entire organization of the ORF and consensus sequence around the active site were distinct from the related enzymes, and its structure could be affected by a reducing agent, DTT.     

Purification and properties of a cholesteryl ester hydrolase from rat liver microsomes

This report describes a purification procedure for a cholesteryl ester hydrolase (CEH) from female rat liver microsomes, and some structural, immunological, kinetic, and regulatory properties of the enzyme that distinguish the microsomal CEH from other hepatic cholesteryl ester-splitting enzymes. CEH was purified 12.4-fold from reisolated microsomes using sequential solubilization by sonication, polyethylene glycol precipitation, fractionation with hydroxyapatite, anion exchange chromatography, and chromatography on hydroxyapatite, with an overall yield of 3.2%. CEH activity was purified 141-fold over nonspecific esterase activity and 56-fold over triacylglycerol lipase activity. In sharp contrast with most esterases and lipases, CEH did not bind to concanavalin A-Sepharose and heparin-Sepharose. After polyacrylamide gel electrophoresis, the purified enzyme exhibited two silver-stained bands, but only the protein electroeluted from the low mobility band had CEH activity. Affinity-purified polyclonal antibodies raised to electroeluted CEH inhibited 90% of the activity of liver microsomal CEH and reacted with a 106 kDa protein band on Western blot analysis. This 106 kDa CEH contains a unique N-terminal amino acid sequence. The purified enzyme had optimal activity at pH 6 and no taurocholate requirements, and was inhibited by the serine active site inhibitor phenylmethylsulfonyl fluoride and by free sulfhydryl specific reagents. It hydrolyzed cholesteryl oleate much more efficiently than trioleine, and hydrolytic activity with p-nitrophenyl acetate was higher than with p-nitrophenyl butyrate. These results indicate that rat liver microsomes contain a bile salt-independent catalytic protein that is relatively specific for cholesteryl ester hydrolysis.     

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.