Mutation of F417 but not of L418 or L420 in the lipid binding domain decreases the activity of triacylglycerol hydrolase

Human triacylglycerol hydrolase (hTGH) has been shown to play a role in hepatic lipid metabolism. Triacylglycerol hydrolase (TGH) hydrolyzes insoluble carboxylic esters at lipid/water interfaces, although the mechanism by which the enzyme adsorbs to lipid droplets is unclear. Three-dimensional modeling of hTGH predicts that catalytic residues are adjacent to an α-helix that may mediate TGH/lipid interaction. The helix contains a putative neutral lipid binding domain consisting of the octapeptide FLDLIADV (amino acid residues 417–424) with the consensus sequence FLXLXXXn (where n is a nonpolar residue and X is any amino acid except proline) identified in several other proteins that bind or metabolize neutral lipids. Deletion of this α-helix abolished the lipolytic activity of hTGH. Replacement of F417 with alanine reduced activity by 40% toward both insoluble and soluble esters, whereas replacement of L418 and L420 with alanine did not. Another potential mechanism of increasing TGH affinity for lipid is via reversible acylation. Molecular modeling predicts that C390 is available for covalent acylation. However, neither chemical modification of C390 nor mutation to alanine affected activity. Our findings indicate that F417 but not L418, L420, or C390 participates in substrate hydrolysis by hTGH. Human triacylglycerol hydrolase (hTGH) has been shown to play a role in hepatic lipid metabolism. Triacylglycerol hydrolase (TGH) hydrolyzes insoluble carboxylic esters at lipid/water interfaces, although the mechanism by which the enzyme adsorbs to lipid droplets is unclear. Three-dimensional modeling of hTGH predicts that catalytic residues are adjacent to an α-helix that may mediate TGH/lipid interaction. The helix contains a putative neutral lipid binding domain consisting of the octapeptide FLDLIADV (amino acid residues 417–424) with the consensus sequence FLXLXXXn (where n is a nonpolar residue and X is any amino acid except proline) identified in several other proteins that bind or metabolize neutral lipids. Deletion of this α-helix abolished the lipolytic activity of hTGH. Replacement of F417 with alanine reduced activity by 40% toward both insoluble and soluble esters, whereas replacement of L418 and L420 with alanine did not. Another potential mechanism of increasing TGH affinity for lipid is via reversible acylation. Molecular modeling predicts that C390 is available for covalent acylation. However, neither chemical modification of C390 nor mutation to alanine affected activity. Our findings indicate that F417 but not L418, L420, or C390 participates in substrate hydrolysis by hTGH. Several endoplasmic reticulum-associated carboxylesterases with broad and overlapping substrate specificities have been identified (1Robbi M. Beaufay H. Purification and characterization of various esterases from rat liver.Eur. J. Biochem. 1983; 137: 293-301Google Scholar, 2Mentlein R. Heiland S. Heymann E. Simultaneous purification and comparative characterization of six serine hydrolases from rat liver microsomes.Arch. Biochem. Biophys. 1980; 200: 547-559Google Scholar, 3Hosokawa M. Maki T. Satoh T. Characterization of molecular species of liver microsomal carboxylesterases of several animal species and humans.Arch. Biochem. Biophys. 1990; 277: 219-227Google Scholar, 4Harano T. Miyata T. Lee S. Aoyagi H. Omura T. Biosynthesis and localization of rat liver microsomal carboxyesterase E1.J. Biochem. (Tokyo). 1988; 103: 149-155Google Scholar, 5Morgan E.W. Yan B. Greenway D. Parkinson A. Regulation of two rat liver microsomal carboxylesterase isozymes: species differences, tissue distribution, and the effects of age, sex, and xenobiotic treatment of rats.Arch. Biochem. Biophys. 1994; 315: 513-526Google Scholar). Mammalian carboxylesterases are known to metabolize numerous analgesic and narcotic compounds, including aspirin, cocaine, heroin, procaine, and meperidine (6Joly J.M. Brown T.M. Metabolism of aspirin and procaine in mice pretreated with O-4-nitrophenyl methyl(phenyl)phosphinate or O-4-nitrophenyl diphenylphosphinate.Toxicol. Appl. Pharmacol. 1986; 84: 523-532Google Scholar, 7Brzezinski M.R. Spink B.J. Dean R.A. Berkman C.E. Cashman J.R. Bosron W.F. Human liver carboxylesterase hCE-1: binding specificity for cocaine, heroin, and their metabolites and analogs.Drug Metab. Dispos. 1997; 25: 1089-1096Google Scholar, 8Kamendulis L.M. Brzezinski M.R. Pindel E.V. Bosron W.F. Dean R.A. Metabolism of cocaine and heroin is catalyzed by the same human liver carboxylesterases.J. Pharmacol. Exp. Ther. 1996; 279: 713-717Google Scholar, 9Lotti M. Ketterman A. Waskell L. Talcott R.E. Meperidine carboxylesterase in mouse and human livers.Biochem. Pharmacol. 1983; 32: 3735-3738Google Scholar). Hepatic carboxylesterases are involved in the biotransformation of xenobiotics and natural substrates by hydrolyzing compounds containing ester, thioester, or amide bonds (10Mentlein R. Suttorp M. Heymann E. Specificity of purified monoacylglycerol lipase, palmitoyl-CoA hydrolase, palmitoyl-carnitine hydrolase, and nonspecific carboxylesterase from rat liver microsomes.Arch. Biochem. Biophys. 1984; 228: 230-246Google Scholar). Carboxylesterases are also used to activate anticancer prodrugs such as CPT-11 (irinotecan) (11Potter P.M. Pawlik C.A. Morton C.L. Naeve C.W. Danks M.K. Isolation and partial characterization of a cDNA encoding a rabbit liver carboxylesterase that activates the prodrug irinotecan (CPT-11).Cancer Res. 1998; 58: 2646-2651Google Scholar, 12Bencharit S. Morton C.L. Howard-Williams E.L. Danks M.K. Potter P.M. Redinbo M.R. Structural insights into CPT-11 activation by mammalian carboxylesterases.Nat. Struct. Biol. 2002; 9: 337-342Google Scholar). Our laboratory has investigated the participation of hepatic carboxylesterases in lipid metabolism. We have purified a carboxylesterase that catalyzes the lipolysis of triacylglycerols and termed it triacylglycerol hydrolase (TGH) (13Lehner R. Verger R. Purification and characterization of a porcine liver microsomal triacylglycerol hydrolase.Biochemistry. 1997; 36: 1861-1868Google Scholar, 14Lehner R. Vance D.E. Cloning and expression of a cDNA encoding a hepatic microsomal lipase that mobilizes stored triacylglycerol.Biochem. J. 1999; 343: 1-10Google Scholar, 15Dolinsky V.W. Sipione S. Lehner R. Vance D.E. The cloning and expression of a murine triacylglycerol hydrolase cDNA and the structure of its corresponding gene.Biochim. Biophys. Acta. 2001; 1532: 162-172Google Scholar) and have isolated and expressed a cDNA for human triacylglycerol hydrolase (hTGH; GenBank accession number NM_001266) (16Alam M. Ho S. Vance D.E. Lehner R. Heterologous expression, purification, and characterization of human triacylglycerol hydrolase.Protein Expr. Purif. 2002; 24: 33-42Google Scholar). Hepatic TGH participates in the mobilization of stored triacylglycerol (TG) for the assembly of VLDL (14Lehner R. Vance D.E. Cloning and expression of a cDNA encoding a hepatic microsomal lipase that mobilizes stored triacylglycerol.Biochem. J. 1999; 343: 1-10Google Scholar,17Lehner R. Cui Z. Vance D.E. Subcellullar localization, developmental expression and characterization of a liver triacylglycerol hydrolase.Biochem. J. 1999; 338: 761-768Google Scholar, 18Dolinsky V.W. Douglas D.N. Lehner R. Vance D.E. Regulation of the enzymes of hepatic microsomal triacylglycerol lipolysis and re-esterification by the glucocorticoid dexamethasone.Biochem. J. 2004; 378: 967-974Google Scholar, 19Gilham D. Ho S. Rasouli M. Martres P. Vance D.E. Lehner R. Inhibitors of hepatic microsomal triacylglycerol hydrolase decrease very low density lipoprotein secretion.FASEB J. 2003; 17: 1685-1687Google Scholar, 20Gilham D. Alam M. Gao W. Vance D.E. Lehner R. Triacylglycerol hydrolase is localized to the endoplasmic reticulum by an unusual retrieval sequence where it participates in VLDL assembly without utilizing VLDL lipids as substrates.Mol. Biol. Cell. 2005; 16: 984-996Google Scholar). Because VLDL is the precursor for plasma LDL and a high level of plasma LDL is a major for the of and and the 1997; Scholar, and the of of the mechanism of of enzymes and proteins involved in the assembly and of VLDL are of Hepatic VLDL assembly is by the of lipid S. of from by of B.J. Biol. Scholar, R.A. R.A. assembly of very low density of of the endoplasmic reticulum of Biol. Scholar, J. J. of from rat of the of to lipoprotein assembly and Biol. Scholar). has been by several that the of in VLDL from and mobilization of this lipolysis by to D. The of hepatic role in the of lipoprotein and its to and J. Scholar, A. of acid to very low density lipoprotein from of from Res. 1996; Scholar, A. H. of from from reduced of to a Res. 1996; Scholar, Brown of triacylglycerol hydrolysis and of acid for the of triacylglycerol by rat Res. 1998; Scholar, of triacylglycerol Biophys. Acta. Scholar). TGH is a to decrease plasma lipid and of the in hTGH have been in proteins that with or metabolize lipids. TGH from the to (13Lehner R. Verger R. Purification and characterization of a porcine liver microsomal triacylglycerol hydrolase.Biochemistry. 1997; 36: 1861-1868Google Scholar) and has been with lipid droplets R. Cui Z. Vance D.E. Subcellullar localization, developmental expression and characterization of a liver triacylglycerol hydrolase.Biochem. J. 1999; 338: 761-768Google the the that TGH affinity for lipids have not been and J. and of and human plasma in the is for both lipoprotein binding and of Scholar) a neutral lipid binding domain in human plasma putative lipid binding octapeptide an α-helix with the consensus sequence FLXLXXXn (where n is a nonpolar residue and X is any amino acid except sequence identified a putative in porcine (13Lehner R. Verger R. Purification and characterization of a porcine liver microsomal triacylglycerol hydrolase.Biochemistry. 1997; 36: 1861-1868Google Scholar) and human M. Vance D.E. Lehner R. of human triacylglycerol hydrolase by of the catalytic and a 2002; Scholar) that a porcine carboxylesterase to hTGH may S. A. J. A. and of carboxylesterase from porcine and liver.Eur. J. Biochem. 2002; Scholar). Because the of residues is an has that of hTGH to lipids by this The amino acid sequence of hTGH has Our structure that two are and M. Vance D.E. Lehner R. of human triacylglycerol hydrolase by of the catalytic and a 2002; Scholar). The residue at available for modification and to this the of the putative and C390 residues in hTGH and to and from and from and for of from or acid for purification of proteins and the from and molecular from other from and of the for a by the for at the of The used to hTGH are in the in with the for of human triacylglycerol with the are by in a human triacylglycerol with the are by We this with a hTGH cDNA with a in the (16Alam M. Ho S. Vance D.E. Lehner R. Heterologous expression, purification, and characterization of human triacylglycerol hydrolase.Protein Expr. Purif. 2002; 24: 33-42Google Scholar). The in the that it (16Alam M. Ho S. Vance D.E. Lehner R. Heterologous expression, purification, and characterization of human triacylglycerol hydrolase.Protein Expr. Purif. 2002; 24: 33-42Google Scholar). the for to the and the in from as for the and used for the expression of and in as The and also by the in The used a of hTGH the for the sequence D. Alam M. Gao W. Vance D.E. Lehner R. Triacylglycerol hydrolase is localized to the endoplasmic reticulum by an unusual retrieval sequence where it participates in VLDL assembly without utilizing VLDL lipids as substrates.Mol. Biol. Cell. 2005; 16: 984-996Google Scholar) with an to the to The in the mammalian expression The by used for expression in as at with with and of hTGH in as M. Vance D.E. Lehner R. of human triacylglycerol hydrolase by of the catalytic and a 2002; Scholar). for expression of and hTGH with the mutation in tissue with at a of of for in and for with as a and from a as M. Vance D.E. Lehner R. of human triacylglycerol hydrolase by of the catalytic and a 2002; Scholar). of hTGH by the in laboratory (16Alam M. Ho S. Vance D.E. Lehner R. Heterologous expression, purification, and characterization of human triacylglycerol hydrolase.Protein Expr. Purif. 2002; 24: 33-42Google Scholar). The with S. at in in containing and to of containing or and to the the with without and for an and with in of and as of in the of hTGH into the to a of and and the at for a to the hTGH at in a for and the The with binding the of hTGH to the the with to The proteins in and to a The of hTGH (16Alam M. Ho S. Vance D.E. Lehner R. Heterologous expression, purification, and characterization of human triacylglycerol hydrolase.Protein Expr. Purif. 2002; 24: 33-42Google Scholar, M. Vance D.E. Lehner R. of human triacylglycerol hydrolase by of the catalytic and a 2002; Scholar) and by The of at in a containing and with and with in and with (16Alam M. Ho S. Vance D.E. Lehner R. Heterologous expression, purification, and characterization of human triacylglycerol hydrolase.Protein Expr. Purif. 2002; 24: 33-42Google Scholar, M. Vance D.E. Lehner R. of human triacylglycerol hydrolase by of the catalytic and a 2002; Scholar). as (13Lehner R. Verger R. Purification and characterization of a porcine liver microsomal triacylglycerol hydrolase.Biochemistry. 1997; 36: 1861-1868Google Scholar) from hTGH with a the endoplasmic reticulum retrieval sequence D. Alam M. Gao W. Vance D.E. Lehner R. Triacylglycerol hydrolase is localized to the endoplasmic reticulum by an unusual retrieval sequence where it participates in VLDL assembly without utilizing VLDL lipids as substrates.Mol. Biol. Cell. 2005; 16: 984-996Google Scholar). the expressed hTGH is in this of microsomal as R. A. Purification of an hydrolase from rat in Biol. Scholar) with of microsomal with of in in a of at for with and containing and the proteins from insoluble by is the acid is not for and other including H. R. S. J.R. of are at the plasma of mammalian Biol. 1996; Scholar). hTGH from with of to at a The with in the with acid of the of which is Biochem. Scholar). in acid and in by and to a also The for the of hTGH with the to a and the with proteins a Molecular proteins the with the corresponding to hTGH and proteins and the by of and lipase with and as (13Lehner R. Verger R. Purification and characterization of a porcine liver microsomal triacylglycerol hydrolase.Biochemistry. 1997; 36: 1861-1868Google Scholar, M. Ho S. Vance D.E. Lehner R. Heterologous expression, purification, and characterization of human triacylglycerol hydrolase.Protein Expr. Purif. 2002; 24: 33-42Google Scholar). in a containing and an the of as the at a Molecular D. Lehner R. to lipase and activity in 2005; 36: Scholar). is expressed as of of or of a used that as a as D. Alam M. Gao W. Vance D.E. Lehner R. Triacylglycerol hydrolase is localized to the endoplasmic reticulum by an unusual retrieval sequence where it participates in VLDL assembly without utilizing VLDL lipids as substrates.Mol. Biol. Cell. 2005; 16: 984-996Google D. Lehner R. to lipase and activity in 2005; 36: Scholar). is expressed as of of or of as a TGH with both (16Alam M. Ho S. Vance D.E. Lehner R. Heterologous expression, purification, and characterization of human triacylglycerol hydrolase.Protein Expr. Purif. 2002; 24: 33-42Google Scholar) and and the to chemical modification of the residue in purified for with at the of the lipolytic activity. TGH hydrolyzes various carboxylic esters, including lipids (13Lehner R. Verger R. Purification and characterization of a porcine liver microsomal triacylglycerol hydrolase.Biochemistry. 1997; 36: 1861-1868Google Scholar, 14Lehner R. Vance D.E. Cloning and expression of a cDNA encoding a hepatic microsomal lipase that mobilizes stored triacylglycerol.Biochem. J. 1999; 343: 1-10Google Scholar, M. Vance D.E. Lehner R. of human triacylglycerol hydrolase by of the catalytic and a 2002; Scholar). TGH lipid substrates has been J. and of and human plasma in the is for both lipoprotein binding and of Scholar) that a in a of proteins that with or metabolize neutral this interaction. The in human J. and of and human plasma in the is for both lipoprotein binding and of Scholar) but is also in porcine TGH (13Lehner R. Verger R. Purification and characterization of a porcine liver microsomal triacylglycerol hydrolase.Biochemistry. 1997; 36: 1861-1868Google human J. D. H. B. W. W. R. Cloning and expression of human 1986; human C.W. Molecular cloning and expression of cDNA for rat Biophys. Acta. rat lipase S. T. expression, and localization to 1988; and human M. Molecular cloning and sequence of cDNA encoding human 1990; Scholar). consensus sequence has been identified in hTGH and is an α-helix adjacent to a to catalytic residues M. Vance D.E. Lehner R. of human triacylglycerol hydrolase by of the catalytic and a 2002; Scholar, V.W. D. Alam M. Vance D.E. Lehner R. Triacylglycerol role in lipid 2004; Scholar). the may or with such as or substrate such as lipid of putative neutral lipid binding X X X X porcine triacylglycerol human human human rat human residues are shown in a nonpolar residue is with and any amino acid except is with in a porcine triacylglycerol human human human rat human residues are shown in a nonpolar residue is with and any amino acid except is with We used expression to and various of hTGH in (16Alam M. Ho S. Vance D.E. Lehner R. Heterologous expression, purification, and characterization of human triacylglycerol hydrolase.Protein Expr. Purif. 2002; 24: 33-42Google Scholar, M. Vance D.E. Lehner R. of human triacylglycerol hydrolase by of the catalytic and a 2002; Scholar). this and expressed a hTGH that the residues the containing this and used to The hTGH expressed to a level as as by The to with a molecular the as a of the of amino of the expressed hTGH in the of not of lipolytic of with containing hTGH that the not of expression of an of lipolytic activity in the The of lipolytic activity of the to a role of the for catalytic or to a of residues that in the to the expressed by of the from and purification by affinity for a used for the purification of hTGH M. Vance D.E. Lehner R. of human triacylglycerol hydrolase by of the catalytic and a 2002; Scholar). However, did not in the hTGH from that the the of the putative by amino acid The and residues the putative have been to the for with lipids J. and of and human plasma in the is for both lipoprotein binding and of Scholar). residues are L418, and L420 and the for of the in used a hTGH that the in a D. Alam M. Gao W. Vance D.E. Lehner R. Triacylglycerol hydrolase is localized to the endoplasmic reticulum by an unusual retrieval sequence where it participates in VLDL assembly without utilizing VLDL lipids as substrates.Mol. Biol. Cell. 2005; 16: 984-996Google Scholar). a hTGH is that the the of in the endoplasmic reticulum L. M. A. the in the 1999; Scholar, L. A. in the endoplasmic Biol. 2003; Scholar, and in the Biol. 2003; Scholar, S. proteins are by a mechanism of Biol. 2004; Scholar). The also with six residues at the to purification by affinity The into the mammalian expression and expression of the and or in the hTGH proteins from the via the We to the residues with alanine this to the structure of the domain but decrease its which the affinity of the hydrolase for lipids. The of the hTGH proteins from the affinity by with and an of the hTGH proteins in by and lipase activity not activity in the substrates with shown in the and did not any in lipolytic activity with the insoluble lipase substrate and the the same level of activity as However, of the lipolytic activity in the with and of activity by the also with two soluble and whereas and to catalytic activity of substrate We that F417 is for the hydrolysis of including insoluble and of and the into the from of with acid to the binding of expressed proteins as in and the with and proteins by and to by with with activity in of containing the same of and the and as for activity by of from with with are the of and of and the into the from of with acid to the binding of expressed proteins as in and the with and proteins by and to by with with activity in of containing the same of and the and as for activity by of from with with are the of and of and the into the from of with acid to the binding of expressed proteins as in and the with and proteins by and to by with with activity in of containing the same of and the and as for activity by of from with with are the of The hTGH amino acid sequence contains residues and are to and C390 the structure of hTGH M. Vance D.E. Lehner R. of human triacylglycerol hydrolase by of the catalytic and a 2002; Scholar). C390 available for the of However, treatment of purified hTGH with or without did not its that covalent via a TGH and or other proteins that to or of may have been expression in that potential high molecular the expressed in treatment of hTGH with the did not in the of hTGH that C390 is not involved in is known that and a of and of localization, with in of insights into of and Biophys. Acta. 1999; or enzyme activity L. S. Regulation of activity by acylation. role for acid of Biol. 1994; Scholar). is a reversible modification of Biol. 1997; 9: Scholar, M. The role of in Biochem. Scholar). has been that a porcine carboxylesterase with high to hTGH may S. A. J. A. and of carboxylesterase from porcine and liver.Eur. J. Biochem. 2002; Scholar). Because hTGH also contains a investigated the enzyme this shown in of isolated from hTGH with in the of proteins in the of the hTGH not by of the of the containing the hTGH that C390 of hTGH not play a role in or in the with or other substrate via acylation. a hTGH and expressed the in a expression and lipase activity that the hTGH expressed in with in with of the same of in the hydrolysis of the lipid that C390 is not for hTGH is not in proteins from hTGH with as in and by of hTGH with proteins and microsomal proteins to by to a and molecular in the by a in with hTGH proteins and molecular of the of the by and activity of hTGH from with and the hTGH as in and by and to a with of of from and or as are the of Hepatic TGH has been shown to in the mobilization of for VLDL assembly (14Lehner R. Vance D.E. Cloning and expression of a cDNA encoding a hepatic microsomal lipase that mobilizes stored triacylglycerol.Biochem. J. 1999; 343: 1-10Google Scholar, 18Dolinsky V.W. Douglas D.N. Lehner R. Vance D.E. Regulation of the enzymes of hepatic microsomal triacylglycerol lipolysis and re-esterification by the glucocorticoid dexamethasone.Biochem. J. 2004; 378: 967-974Google Scholar, 19Gilham D. Ho S. Rasouli M. Martres P. Vance D.E. Lehner R. Inhibitors of hepatic microsomal triacylglycerol hydrolase decrease very low density lipoprotein secretion.FASEB J. 2003; 17: 1685-1687Google Scholar, 20Gilham D. Alam M. Gao W. Vance D.E. Lehner R. Triacylglycerol hydrolase is localized to the endoplasmic reticulum by an unusual retrieval sequence where it participates in VLDL assembly without utilizing VLDL lipids as substrates.Mol. Biol. Cell. 2005; 16: 984-996Google Scholar). have shown that the amino acid residues and the catalytic M. Vance D.E. Lehner R. of human triacylglycerol hydrolase by of the catalytic and a 2002; Scholar). Three-dimensional modeling of hTGH the structure of M. Vance D.E. Lehner R. of human triacylglycerol hydrolase by of the catalytic and a 2002; Scholar) the of a hydrolase with known of other serine esterases H. The lipase Res. 2002; Scholar). The structure the of a rabbit liver carboxylesterase S. Morton C.L. Howard-Williams E.L. Danks M.K. Potter P.M. Redinbo M.R. Structural insights into CPT-11 activation by mammalian carboxylesterases.Nat. Struct. Biol. 2002; 9: 337-342Google Scholar) and the structure of a human carboxylesterase S. Morton C.L. Potter P.M. Redinbo M.R. Structural of heroin and cocaine by a human Struct. Biol. 2003; Scholar). Structural also indicate that in the of carboxylesterase substrate specificity V.W. D. Alam M. Vance D.E. Lehner R. Triacylglycerol role in lipid 2004; Scholar). with other neutral and proteins to the of a putative that is involved in with lipid substrates J. and of and human plasma in the is for both lipoprotein binding and of Scholar). We that the in hTGH is involved in The of an octapeptide with a FLXLXXXn consensus that is several proteins involved in the or hydrolysis of neutral lipids. that in the residues for binding to and lipid in with other J. and of and human plasma in the is for both lipoprotein binding and of Scholar). We that in the corresponding sequence at play a role in the of this enzyme with We used of amino acid and by expression of the proteins in and and of the lipolytic and Deletion of the in of and the of of the it that the expressed Our mutation that activity reduced toward insoluble and soluble substrates F417 by F417 may an for substrate However, that F417 not play a role in the activation of the mutation of the residue also activity toward soluble is that the reduced activity in to the expressed and to as a of in the of proteins in the endoplasmic Carboxylesterases as and both of which activity S. A. J. A. and of carboxylesterase from porcine and liver.Eur. J. Biochem. 2002; Scholar). F417 involved in a of the of mutation of F417 not to the activity of the are residues hTGH the amino acid sequence of the cDNA M. Vance D.E. Lehner R. of human triacylglycerol hydrolase by of the catalytic and a 2002; Scholar). of the residues are involved in residue investigated several of in of a porcine carboxylesterase and identified by S. A. A. in the in porcine J. Biochem. Scholar). The of the not although as in is that hTGH as a covalent in purified hTGH from and in at a molecular of in both and The porcine carboxylesterase also to S. A. J. A. and of carboxylesterase from porcine and liver.Eur. J. Biochem. 2002; Scholar). We C390 in hTGH the localization of proteins and their with lipid Gao T. E. of of in the localization of the Biol. 1998; Scholar, D. D. M. The domain of is to a to plasma of is for the of in Biol. 1999; Scholar, for in Biol. Cell. 1999; Scholar). The substrate for TGH are the lipid droplets with the endoplasmic reticulum D. Ho S. Rasouli M. Martres P. Vance D.E. Lehner R. Inhibitors of hepatic microsomal triacylglycerol hydrolase decrease very low density lipoprotein secretion.FASEB J. 2003; 17: 1685-1687Google Scholar). TGH has been shown to with lipid droplets from liver R. Cui Z. Vance D.E. Subcellullar localization, developmental expression and characterization of a liver triacylglycerol hydrolase.Biochem. J. 1999; 338: 761-768Google Scholar). with the is known consensus amino acid sequence for of C390 of hTGH a for this However, that C390 in hTGH is not and that of hTGH with lipid via this modification is are also by the that rat (14Lehner R. Vance D.E. Cloning and expression of a cDNA encoding a hepatic microsomal lipase that mobilizes stored triacylglycerol.Biochem. J. 1999; 343: 1-10Google Scholar, R. Cui Z. Vance D.E. Subcellullar localization, developmental expression and characterization of a liver triacylglycerol hydrolase.Biochem. J. 1999; 338: 761-768Google Scholar) and murine V.W. Sipione S. Lehner R. Vance D.E. The cloning and expression of a murine triacylglycerol hydrolase cDNA and the structure of its corresponding gene.Biochim. Biophys. Acta. 2001; 1532: 162-172Google Scholar) TGH not have a corresponding residue but the same insoluble The Alam for by a from the of is by the of and of is a and is a of the for the the Molecular and of human plasma human triacylglycerol hydrolase acid neutral lipid binding domain triacylglycerol triacylglycerol hydrolase

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