catalytic properties of the purified rat hepatic cytosolic cholesteryl ester hydrolase

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 225, 413–419 (1996)ARTICLE NO. 1188

Catalytic Properties of the Purified Rat Hepatic CytosolicCholesteryl Ester Hydrolase

Ramesh Natarajan, Shobha Ghosh, and W. McLean Grogan1

Department of Biochemistry and Molecular Biophysics, Virginia Commonwealth University,Richmond, Virginia 23298-0614

Received July 1, 1996

The purified enzyme hydrolyzed cholesteryl oleate, cholesteryl linoleate, and triolein at similar rates overa broad range of concentrations. Hydrolytic activity was relatively low with p-nitrophenyl acetate, but muchhigher with PNP-esters of the more lipophilic C4-C18 fatty acids, in sharp contrast to microsomal esteraseswhich hydrolyze PNP-acetate more efficiently. Zn2/, Cu2/, Cd2/, Hg2/, and phenylmethylsulfonyl fluorideinhibited, whereas N-ethyl maleimide and iodoacetamide stimulated activity of the pure enzyme. Limitedtrypsin digestion selectively inhibited cholesteryl esterase activity with retention of activity toward PNP-octanoate, suggesting involvement of a trypsin-labile loop in the lipophilic substrate binding pocket.q 1996 Academic Press, Inc.

Cholesteryl ester hydrolases are a diverse group of serine esterases which catalyze thereversible hydrolysis of cholesteryl esters to free cholesterol and fatty acids. Classification ofthese enzymes is problematic, inasmuch as they have broad substrate specificities and cholest-eryl ester, the defining substrate, is insoluble in water. Specific CEH isoenzymes which havebeen characterized at the molecular level include the pancreatic CEH (1); the hormone-sensitivelipase of adipose tissue and steroidogenic organs (2,3); the lysosomal acid CEH (4); and thehepatic cytosolic CEH (5). These isoenzymes have distinctly different interorgan/intracellulardistributions, amino acid sequences and regulatory patterns. Although rat liver has lysosomaland microsomal CEH activities, the major regulated activity is associated with a unique cyto-solic esterase (5–10). This enzyme is appropriately located, rationally regulated and able torapidly mobilize free cholesterol from hepatic cholesteryl ester stores (5–11). Although thecDNA for cytosolic CEH has been cloned, sequenced and expressed (5), little has been reportedconcerning the properties of the purified enzyme. Inasmuch as the cDNA for the cytosolicCEH has high homology, though not identity, with the microsomal pI 6.1 esterase (12), andmicrosomal proteins are immunoreactive with antibodies to the purified cytosolic CEH (13),it is important to identify properties which distinguish the CEH from other esterases.

MATERIALS AND METHODSChemicals and supplies. Cholesterol [1-14C]oleate (56.6 mCi/mmol), glyceryl [1-14C]trioleate (112 mCi/mmol) and

cholesterol [1-14C]linoleate (54.9 mCi/mmol) were purchased from New England Nuclear (Boston, MA); solvents,from Fisher Scientific (Columbia, MD); ZnSO4, CuCl2 , CdSO4, sodium taurocholate, PMSF, iodoacetamide, N-ethylmaleimide, mercury benzoate, p-nitrophenyl esters and trypsin agarose beads, from Sigma Chemical Co. (St.Louis, MO); adult male Sprague-Dawley rats and frozen rat livers, from Zivic Miller Laboratories (Zellenople, PA).

Preparation of rat liver cytosol and microsomes. Rats were decapitated and livers excised. Subcellular fractionswere prepared from liver homogenates by differential centrifugation, as described elsewhere (13). Microsomes werewashed with homogenizing buffer, recentrifuged and resuspended for CEH assay.

1 To whom correspondence should be addressed. Fax: (804) 828-1473.Abbreviations: CEH, cholesteryl ester hydrolase; PMSF, phenylmethylsulfonyl fluoride; PNP, p-nitrophenyl.

0006-291X/96 $18.00Copyright q 1996 by Academic Press, Inc.All rights of reproduction in any form reserved.

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Vol. 225, No. 2, 1996 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

FIG. 1. Effect of divalent cations on the activity of purified cholesteryl ester hydrolase. CEH was assayed with75mM cholesteryl oleate (A) or triolein (B) substrate, using indicated concentrations of ZnSO4, CuCl2 or CdSO4, asdescribed in Materials and Methods. Values are means { SEM of 3 experiments, each in triplicate.

Purification of CEH. Hepatic CEH was purified from rat liver cytosol as described earlier (8).Enzyme assays. Hydrolysis of lipophilic substrates, cholesteryl oleate, cholesteryl linoleate and triolein was measured

by the radiometric method described previously (6,7) in 200mM phosphate buffer containing 0.5–2.0mg CEH, 80mMKCl, 5mM b-mercaptoethanol and 5mM taurocholate. The reaction was started by addition of 75mM substrate (40,000–50,000 dpm) and incubated for 30min at 377C. Effects of metal ions on CEH activity were determined by preincubatingthe reaction mixture with ZnSO4, CuCl2 or CdSO4 (1mM, 500mM, 1mM, respectively) for 1–2min at 257C, prior toaddition of substrate. Effects of inhibitors were determined by preincubating the reaction mixture with 3mM iodoacet-amide, N-ethylmaleimide or mercury benzoate, or 0.05 and 0.1mM phenylmethylsulfonyl fluoride, for 5min at 257C,prior to addition of substrate. Hydrolysis of PNP-esters was determined spectrophotometrically by monitoring ab-sorbance of p-nitrophenol at 400nm. 200nmols PNP-ester was added and the reaction incubated for 15min at 377C.

Inactivation of CEH by trypsin. Purified CEH was incubated with trypsin agarose beads (2U) in 10mM Tris-HClbuffer, pH 8.5 at 257C. The reaction was terminated by centrifugation at 3000 1 g. CEH was measured in supernatantsas described above.

Protein estimation. Protein was TCA precipitated and estimated with the Pierce BCA kit.Structural analysis. The amino acid sequence translated from the cDNA (5) was analyzed by GCG PEPTIDESTRUC-

TURE in the Academic Computing Center of Virginia Commonwealth University, using algorithms for hydrophilicity(Kyte-Doolittle), surface probability (Emini), chain flexibility (Karplus-Schulz) and secondary structure (Chou-Fasmanand Garnier-Osguthorpe-Robson).

RESULTS AND DISCUSSION

As shown in Fig 1, the purified cytosolic CEH hydrolyzed oleoyl esters of both cholesteroland glycerol, although activity was consistently higher with cholesteryl oleate (Fig 1A) thanwith triolein (Fig 1B), with or without added metal ions. No divalent cation tested increasedactivity and all were mildly inhibitory at one or more concentrations from 1–1000 mM.Hydrolysis of cholesteryl oleate and triolein were both inhibited to the same degree by eachmetal ion at each concentration, consistent with association of both activities with the same

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FIG. 2. Effect of taurocholate on the activity of purified cholesteryl ester hydrolase. CEH was assayed with 75mMcholesteryl oleate or triolein substrate with indicated concentrations of taurocholate, as described in Materials andMethods. Each data point represents the mean { SEM of 3 experiments, each in triplicate. Where not visible, errorbars are within symbols.

purified enzyme. This parallel inhibition is most apparent in the progressive inhibition of bothactivities by Cu2/.

As seen in Figure 2, 5 mM taurocholate activated hydrolysis of both cholesteryl oleate andtriolein by the purified protein, whereas higher concentrations inhibited both activities, furtherevidence that both activities are attributable to the same enzyme. Cholesterol oleate hydrolysisby the recombinant CEH showed similar bile acid concentration dependency in an earlierstudy (5). Activity with cholesteryl oleate was consistently higher than that with triolein at alltaurocholate concentrations.

As depicted in Fig 3, the purified protein was probed with various group specific reagents,in order to indicate potentially important amino acid residues and for comparison with othersimilarly characterized esterases. Consistent with the identification of a consensus serine ester-ase active site (5), the purified CEH was inhibited by PMSF, which inhibits other cholesterolesterases (14,15). Inhibition was 50% at 0.1 mM PMSF, similar to that reported for themicrosomal pI 6.1 esterase, at the same concentration (16). Of the sulfhydryl specific reagents,

FIG. 3. Effect of enzyme inhibitors on the activity of purified cholesteryl ester hydrolase. CEH was assayed withcholesteryl oleate substrate in the presence of indicated concentrations of PMSF, iodoacetamide, N-ethylmaleimideor mercury benzoate, as described in Materials and Methods. Values are means { SEM of 3 experiments, each intriplicate.

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FIG. 4. Concentration dependency of hydrolysis of cholesteryl oleate, cholesteryl linoleate and triolein by purifiedcholesteryl ester hydrolase. CEH was assayed with indicated substrate concentrations, as described in Materials andMethods. Each data point represents the mean { SEM of 3 experiments, each in triplicate. Where not visible, errorbars are within symbols.

only mercury benzoate inhibited the enzyme (30% at 3 mM). Inasmuch as iodoacetamide andN-ethylmaleimide effected a modest activation, inhibition by Hg2/ probably reflects a generaleffect of divalent cations, rather than interaction with an essential sulfhydryl group (17).Whereas the hepatic cytosolic CEH catalyzes hydrolysis of uncharged esters and is inhibitedby Cu2/ and Hg2/ but not by iodoacetamide, this enzyme has the properties of a class A-esterase, as described by Aldridge (18).

As an indication of physiological function, hydrolytic activity of the purified CEH wasdetermined with a range of different substrates. As shown in Fig 4, CEH hydrolyzed bothcholesteryl oleate and triolein at similar rates with substrate concentrations up to 20 mM.However, activity was greater with cholesterol oleate than with triolein at 75 mM, the substrateconcentration routinely used in most experiments. Activity with cholesteryl linoleate wassomewhat lower than that with either of the oleate esters but approached that of triolein at75 mM.

Catalytic activity was also measured with PNP-esters of fatty acids of different chain lengths,comprising a seris of synthetic substrates with decreasing water solubility. As seen in Fig 5,activity was lowest with the water soluble PNP-acetate, which is efficiently hydrolyzed byother esterases, including triglyceride lipase, pancreatic CEH and the microsomal class Aesterases; e.g., pI 6.1 esterase (16,19). Activity increased with chain length, peaked with themore lipophilic PNP-caprylate and then declined gradually with increasing chain length úC8.Nevertheless, activity was consistently higher with the more lipophilic esters than with PNP-acetate.

The purified CEH requires a minimal level of taurocholate (0.25 mM) to prevent aggregationof the enzyme into an inactive state (8), although bile salts can also modify the physical stateof the substrate. As seen in Fig 5, increasing concentrations of taurocholate had little or noeffect on hydrolysis of PNP-acetate, whereas optimal taurocholate (5 mM) stimulated activitywith the more lipophilic substrates as much as 3-fold. In contrast, 0.5 mM taurocholate hadno effect on hydrolysis of PNP-butyrate or octanoate, but stimulated activity with the longerchained esters. These data indicate that this enzyme is sensitive to physical properties of thesubstrate, particularly lipophilic character and/or aggregation state. Nevertheless, taurocholatehad relatively little effect on the substrate specificity of this enzyme over a 20-fold rangeof concentrations, suggesting that the enzyme has a significant level of intrinsic molecularspecificity.

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FIG. 5. Hydrolysis of p-nitrophenyl fatty acyl esters of various chain lengths by the purified cholesteryl esterhydrolase. Hydrolytic activity (mmols hydrolyzed/hr/mg CEH) was measured with 200 nmold PNP-ester in the presenceof indicated concentrations of taurocholate, as described in Materials and Methods. Each data point represents themean { SEM of 3 experiments, each in triplicate. Where not visible, error bars are within symbols.

Serine esterases, such as lipoprotein lipase and hepatic lipase have highly variable trypsin-labile loop structures formed by disulfide bridges, which confer substrate binding specificity(20,21). Trypsin cleavage at a single site in this loop abolishes lipoprotein lipase activity withtriolein, but not with more hydrophilic tributyrin (22). As seen in Fig 6, mild trypsin digestionof the purified CEH resulted in progressive and selective loss of activity with cholesteryl oleateand triolein, but not with more hydrophilic PNP-octanoate. Analysis of CEH by SDS-PAGEafter 24 hrs of trypsin digestion revealed only a single band at 66 kDa, indistinguishable fromthe mobility of the unmodified protein (data not shown). By analogy with lipoprotein lipase,this is consistent with cleavage of a single peptide bond within a loop domain which conferssubstrate specificity on CEH. Analysis of secondary structure for CEH (see Materials andMethods) predicts such a loop between Cys87 and Cys116, containing a highly exposed trypsincleavage site at Arg104. This site has a high hydrophilicity index (2.4), a high surface probability(5.8) and a predicted turn.

Inasmuch as cytosolic CEH exhibits substantial sequence homology with microsomal ester-

FIG. 6. Effect of limited trypsin digestion on activity and substrate specificity of the purified cholesteryl esterhydrolase. CEH was assayed with cholesteryl oleate, triolein or PNP-caprylate after incubation with trypsin-agarosebeads for times indicated, as described in Materials and Methods. Each data point represents the mean { SEM for 3experiments, each in triplicate. Where not visible, error bars are within symbols.

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FIG. 7. Differential activity of rat liver cytosolic and microsomal esterases with PNP-acetate, PNP-caprylate andcholesteryl oleate. Hydrolytic activity (mmols hydrolyzed/hr/mg protein) was measured with each substrate in cytosoland microsomes, as described in Materials and Methods. Values shown are mean { SEM for 3 rats, each assayed intriplicate with 2 protein concentrations.

ases (5), the substrate specificities of cytosolic and microsomal esterases were compared. Asseen in Fig 7, both cytosol and microsomes contain substantial hydrolytic activity toward PNP-acetate, PNP-octanoate and cholesteryl oleate. Whereas the relative activities of cytosol towardPNP-octanoate and cholesteryl oleate are consistent with those of purified CEH (Fig 5), highactivity toward PNP-acetate suggests the presence in cytosol of an additional esterase withgreater specificity for water soluble substrates. In contrast, the microsomes exhibit dispropor-tionately higher activity with both of the PNP-esters than with cholesteryl oleate, reflectingthe reported substrate specificities of microsomal esterases, which include low levels of micro-somal CEH (õ10% of total cellular CEH activity and immunoreactive protein) (13). This isconsistent with earlier work by Deykin and Goodman, who reported that hepatic CEH activitywas predominantly cytosolic (23).

In view of the high sequence homology of CEH with microsomal esterases (5), the structuralbasis for the differential distributions and specificities of these enzymes is unclear. Althoughthe cDNAs for hepatic CEH and pI 6.1 esterase differ in 14 nucleotides, only 3 of these arein the coding region and none occur in the loop structure described above (5). Nevertheless,pI 6.1 esterase is reported to have 2-fold higher activity with PNP-acetate than with PNP-butyrate (24), whereas hepatic CEH has 10-fold higher activity with PNP-butyrate than withPNP-acetate (Fig 3). Thus, these studies differentiate the substrate specificities of these enzymesand provide additional evidence that the cytosolic CEH is functionally different from themicrosomal homologs.

ACKNOWLEDGMENTThis work was supported by NIH Grant DK44613.

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catalytic properties of the purified rat hepatic cytosolic cholesteryl ester hydrolase

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