Human paraoxonases (PON1, PON2, and PON3) are lactonases with overlapping and distinct substrate specificities
2005; Elsevier BV; Volume: 46; Issue: 6 Linguagem: Inglês
10.1194/jlr.m400511-jlr200
ISSN1539-7262
AutoresDragomir Draganov, John F. Teiber, Audrey Speelman, Yoichi Osawa, Roger K. Sunahara, Bert N. La Du,
Tópico(s)Cynara cardunculus studies
ResumoThe paraoxonase (PON) gene family in humans has three members, PON1, PON2, and PON3. Their physiological role(s) and natural substrates are uncertain. We developed a baculovirus-mediated expression system, suitable for all three human PONs, and optimized procedures for their purification. The recombinant PONs are glycosylated with high-mannose-type sugars, which are important for protein stability but are not essential for their enzymatic activities. Enzymatic characterization of the purified PONs has revealed them to be lactonases/lactonizing enzymes, with some overlapping substrates (e.g., aromatic lactones), but also to have distinctive substrate specificities. All three PONs metabolized very efficiently 5-hydroxy-eicosatetraenoic acid 1,5-lactone and 4-hydroxy-docosahexaenoic acid, which are products of both enzymatic and nonenzymatic oxidation of arachidonic acid and docosahexaenoic acid, respectively, and may represent the PONs' endogenous substrates. Organophosphates are hydrolyzed almost exclusively by PON1, whereas bulky drug substrates such as lovastatin and spironolactone are hydrolyzed only by PON3. Of special interest is the ability of the human PONs, especially PON2, to hydrolyze and thereby inactivate N-acyl-homoserine lactones, which are quorum-sensing signals of pathogenic bacteria.None of the recombinant PONs protected low density lipoprotein against copper-induced oxidation in vitro. The paraoxonase (PON) gene family in humans has three members, PON1, PON2, and PON3. Their physiological role(s) and natural substrates are uncertain. We developed a baculovirus-mediated expression system, suitable for all three human PONs, and optimized procedures for their purification. The recombinant PONs are glycosylated with high-mannose-type sugars, which are important for protein stability but are not essential for their enzymatic activities. Enzymatic characterization of the purified PONs has revealed them to be lactonases/lactonizing enzymes, with some overlapping substrates (e.g., aromatic lactones), but also to have distinctive substrate specificities. All three PONs metabolized very efficiently 5-hydroxy-eicosatetraenoic acid 1,5-lactone and 4-hydroxy-docosahexaenoic acid, which are products of both enzymatic and nonenzymatic oxidation of arachidonic acid and docosahexaenoic acid, respectively, and may represent the PONs' endogenous substrates. Organophosphates are hydrolyzed almost exclusively by PON1, whereas bulky drug substrates such as lovastatin and spironolactone are hydrolyzed only by PON3. Of special interest is the ability of the human PONs, especially PON2, to hydrolyze and thereby inactivate N-acyl-homoserine lactones, which are quorum-sensing signals of pathogenic bacteria. None of the recombinant PONs protected low density lipoprotein against copper-induced oxidation in vitro. The paraoxonase (PON) gene family in humans has three members, PON1, PON2, and PON3, aligned next to each other on the long arm of chromosome 7q21.3-22.1 (1Primo-Parmo S.L. Sorenson R.C. Teiber J. La Du B.N. The human serum paraoxonase/arylesterase gene (PON1) is one member of a multigene family.Genomics. 1996; 33: 498-507Crossref PubMed Scopus (575) Google Scholar). They show high similarity in their structural characteristics and have ∼65% identity at the amino acid level (1Primo-Parmo S.L. Sorenson R.C. Teiber J. La Du B.N. The human serum paraoxonase/arylesterase gene (PON1) is one member of a multigene family.Genomics. 1996; 33: 498-507Crossref PubMed Scopus (575) Google Scholar). The three genes are well conserved in mammals, sharing 79–95% identity at the amino acid level and 81–95% identity at the nucleotide level between different species (1Primo-Parmo S.L. Sorenson R.C. Teiber J. La Du B.N. The human serum paraoxonase/arylesterase gene (PON1) is one member of a multigene family.Genomics. 1996; 33: 498-507Crossref PubMed Scopus (575) Google Scholar, 2La Du B.N. Aviram M. Billecke S. Navab M. Primo-Parmo S. Sorenson R.C. Standiford T.J. On the physiological role(s) of the paraoxonases.Chem. Biol. Interact. 1999; 119–120: 379-388Crossref PubMed Scopus (172) Google Scholar, 3Draganov D.I. La Du B.N. Pharmacogenetics of paraoxonases: a brief review.Naunyn-Schmiedeberg's Arch. Pharmacol. 2004; 369: 78-88Crossref PubMed Scopus (359) Google Scholar). Phylogenetic analysis reveals PON2 to be the oldest member of the family (3Draganov D.I. La Du B.N. Pharmacogenetics of paraoxonases: a brief review.Naunyn-Schmiedeberg's Arch. Pharmacol. 2004; 369: 78-88Crossref PubMed Scopus (359) Google Scholar). All PON2 and PON3 cDNAs sequenced to date lack the three nucleotides of codon 106, which are present in PON1 (1Primo-Parmo S.L. Sorenson R.C. Teiber J. La Du B.N. The human serum paraoxonase/arylesterase gene (PON1) is one member of a multigene family.Genomics. 1996; 33: 498-507Crossref PubMed Scopus (575) Google Scholar, 3Draganov D.I. La Du B.N. Pharmacogenetics of paraoxonases: a brief review.Naunyn-Schmiedeberg's Arch. Pharmacol. 2004; 369: 78-88Crossref PubMed Scopus (359) Google Scholar). The latter is by far the best studied member of the family. PON1 hydrolyzes the toxic oxon metabolites of a number of organophosphorous insecticides such as parathion, diazinon, and chlorpyrifos (4N. La Du, B. Human serum paraoxonase/arylesterase.in: Kalow W. Genetic Factors Influencing the Metabolism of Foreign Compounds: International Encyclopedia of Pharmacology and Therapeutics. Pergamon Press, New York1992: 51-91Google Scholar, 5Davies H.G. Richter R.J. Keifer M. Broomfield C.A. Sowalla J. Furlong C.E. The effect of the human serum paraoxonase polymorphism is reversed with diazoxon, soman and sarin.Nat. Genet. 1996; 14: 334-336Crossref PubMed Scopus (540) Google Scholar) and even nerve agents such as sarin and soman (5Davies H.G. Richter R.J. Keifer M. Broomfield C.A. Sowalla J. Furlong C.E. The effect of the human serum paraoxonase polymorphism is reversed with diazoxon, soman and sarin.Nat. Genet. 1996; 14: 334-336Crossref PubMed Scopus (540) Google Scholar, 6Broomfield, C. A., and K. W. Ford. 1991. Hydrolysis of nerve gases by plasma enzymes. In Proceedings of the 3rd International Meeting on Cholinesterases, La Grande-Motte, France. 161.Google Scholar). PON1 also hydrolyzes aromatic esters, preferably those of acetic acid (4N. La Du, B. Human serum paraoxonase/arylesterase.in: Kalow W. Genetic Factors Influencing the Metabolism of Foreign Compounds: International Encyclopedia of Pharmacology and Therapeutics. Pergamon Press, New York1992: 51-91Google Scholar). Phenyl acetate is one of the most commonly used substrates for following PON1's enzymatic activity in serum. More recently, PON1 has been shown to catalyze the hydrolysis of a variety of aromatic and aliphatic lactones (7Billecke S. Draganov D. Counsell R. Stetson P. Watson C. Hsu C. La Du B.N. Human serum paraoxonase (PON1) isozymes Q and R hydrolyze lactones and cyclic carbonate esters.Drug Metab. Dispos. 2000; 28: 1335-1342PubMed Google Scholar, 8Biggadike K. Angell R.M. Burgess C.M. Farrell R.M. Hancock A.P. Harker A.J. Irving W.R. Ioannou C. Procopiou P.A. Shaw R.E. et al.Selective plasma hydrolysis of glucocorticoid gamma-lactones and cyclic carbonates by the enzyme paraoxonase: an ideal plasma inactivation mechanism.J. Med. Chem. 2000; 43: 19-21Crossref PubMed Scopus (169) Google Scholar) as well as the reverse reaction, lactonization, of γ- and δ-hydroxycarboxylic acids (9Teiber J.F. Draganov D.I. La Du B.N. Lactonase and lactonizing activities of human paraoxonase (PON1) and rabbit serum PON3.Biochem. Pharmacol. 2003; 66: 887-896Crossref PubMed Scopus (129) Google Scholar). PON3s purified from rabbit serum, rat liver microsomes, and stably transfected HEK 293 cells all have low arylesterase and almost no PON activities, but they share some lactone substrates with PON1 (e.g., dihydrocoumarin) (10Draganov D.I. Stetson P.L. Watson C.E. Billecke S.S. La Du B.N. Rabbit serum paraoxonase 3 (PON3) is a high density lipoprotein-associated lactonase and protects low density lipoprotein against oxidation.J. Biol. Chem. 2000; 275: 33435-33442Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar, 11Rodrigo L. Gil F. Hernandez A.F. Lopez O. Pla A. Identification of paraoxonase 3 in rat liver microsomes. Purification and biochemical properties.Biochem. J. 2003; 376: 261-268Crossref PubMed Google Scholar, 12Rosenblat M. Draganov D. Watson C.E. Bisgaier C.L. La Du B.N. Aviram M. Mouse macrophage paraoxonase 2 activity is increased whereas cellular paraoxonase 3 activity is decreased under oxidative stress.Arterioscler. Thromb. Vasc. Biol. 2003; 23: 468-474Crossref PubMed Scopus (140) Google Scholar). Dihydrocoumarin is the only substrate reported to date for PON2 (12Rosenblat M. Draganov D. Watson C.E. Bisgaier C.L. La Du B.N. Aviram M. Mouse macrophage paraoxonase 2 activity is increased whereas cellular paraoxonase 3 activity is decreased under oxidative stress.Arterioscler. Thromb. Vasc. Biol. 2003; 23: 468-474Crossref PubMed Scopus (140) Google Scholar). Human PON1 is synthesized in the liver and secreted into the blood, where it is associated exclusively with HDLs (13Mackness M.I. Possible medical significance of human serum ‘A-’ esterases.in: Rainer E. Aldridge W.N. Hoskin F.C.G. Enzymes Hydrolysing Organophosphorus Compounds. Ellis Horwood, Chichester, UK1989: 202-213Google Scholar, 14Hassett C. Richter R.J. Humbert R. Chapline C. Crabb J.W. Omiecinski C.J. Furlong C.E. 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Similar to PON1, PON3 is expressed mostly in the liver and at low levels in the kidney (16Reddy S.T. Wadleigh D.J. Grijalva V. Ng C. Hama S. Gangopadhyay A. Shih D.M. Lusis A.J. Navab M. Fogelman A.M. Human paraoxonase-3 is an HDL-associated enzyme with biological activity similar to paraoxonase-1 protein but is not regulated by oxidized lipids.Arterioscler. Thromb. Vasc. Biol. 2001; 21: 542-547Crossref PubMed Scopus (302) Google Scholar). Rabbit and human PON3 are found in serum associated with HDL (10Draganov D.I. Stetson P.L. Watson C.E. Billecke S.S. La Du B.N. Rabbit serum paraoxonase 3 (PON3) is a high density lipoprotein-associated lactonase and protects low density lipoprotein against oxidation.J. Biol. Chem. 2000; 275: 33435-33442Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar, 16Reddy S.T. Wadleigh D.J. Grijalva V. Ng C. Hama S. Gangopadhyay A. Shih D.M. Lusis A.J. Navab M. Fogelman A.M. Human paraoxonase-3 is an HDL-associated enzyme with biological activity similar to paraoxonase-1 protein but is not regulated by oxidized lipids.Arterioscler. Thromb. Vasc. Biol. 2001; 21: 542-547Crossref PubMed Scopus (302) Google Scholar) but are ∼2 orders of magnitude less abundant than PON1 (10Draganov D.I. Stetson P.L. Watson C.E. Billecke S.S. La Du B.N. Rabbit serum paraoxonase 3 (PON3) is a high density lipoprotein-associated lactonase and protects low density lipoprotein against oxidation.J. Biol. Chem. 2000; 275: 33435-33442Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar). PON3 mRNA and protein have been identified in murine but not in human macrophages (12Rosenblat M. Draganov D. Watson C.E. Bisgaier C.L. La Du B.N. Aviram M. Mouse macrophage paraoxonase 2 activity is increased whereas cellular paraoxonase 3 activity is decreased under oxidative stress.Arterioscler. Thromb. Vasc. Biol. 2003; 23: 468-474Crossref PubMed Scopus (140) Google Scholar). PON2 is not detectable in serum, although it is expressed in many tissues, including brain, liver, kidney, and testis (17Ng C.J. Wadleigh D.J. Gangopadhyay A. Hama S. Grijalva V.R. Navab M. Fogelman A.M. Reddy S.T. Paraoxonase-2 is a ubiquitously expressed protein with antioxidant properties and is capable of preventing cell-mediated oxidative modification of low density lipoprotein.J. Biol. Chem. 2001; 276: 44444-44449Abstract Full Text Full Text PDF PubMed Scopus (397) Google Scholar), and may have multiple mRNA forms (18Mochizuki H. Scherer S.W. Xi T. Nickle D.C. Majer M. Huizenga J.J. Tsui L.C. Prochazka M. Human PON2 gene at 7q21.3: cloning, multiple mRNA forms, and missense polymorphisms in the coding sequence.Gene. 1998; 213: 149-157Crossref PubMed Scopus (150) Google Scholar). The physiological roles of the PONs are not known. PON1's association with HDL in serum led to the suggestion by Mackness (13Mackness M.I. Possible medical significance of human serum ‘A-’ esterases.in: Rainer E. Aldridge W.N. Hoskin F.C.G. Enzymes Hydrolysing Organophosphorus Compounds. Ellis Horwood, Chichester, UK1989: 202-213Google Scholar) that the enzyme might have a role in lipid metabolism and protect against the development of atherosclerosis. HDL's ability to prevent oxidative modifications of LDL has been attributed to PON1 (19Mackness M.I. Arrol S. Durrington P.N. Paraoxonase prevents accumulation of lipoperoxides in low-density lipoprotein.FEBS Lett. 1991; 286: 152-154Crossref PubMed Scopus (831) Google Scholar, 20Aviram M. Rosenblat M. Bisgaier C.L. Newton R.S. Primo-Parmo S.L. La Du B.N. Paraoxonase inhibits high-density lipoprotein oxidation and preserves its functions. A possible peroxidative role for paraoxonase.J. Clin. Invest. 1998; 101: 1581-1590Crossref PubMed Scopus (1027) Google Scholar, 21Aviram M. Rosenblat M. Billecke S. Erogul J. Sorenson R. Bisgaier C.L. Newton R.S. Du B. 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Med. 2003; 54: 371-392Crossref PubMed Scopus (233) Google Scholar). The antiatherogenic role of PON1 is further supported by studies in transgenic mice lacking or overexpressing the enzyme (25Shih D.M. Gu L. Xia Y.R. Navab M. Li W.F. Hama S. Castellani L.W. Furlong C.E. Costa L.G. Fogelman A.M. Mice lacking serum paraoxonase are susceptible to organophosphate toxicity and atherosclerosis.Nature. 1998; 394: 284-287Crossref PubMed Scopus (957) Google Scholar, 26Shih D.M. Reddy S.T. Lusis A.J. CHD and atherosclerosis: human epidemiological studies and transgenic mouse models.in: Costa L.G. Furlong C.E. Paraoxonase (PON1) in Health and Disease. Kluwer Academic Publishers, Norwell, MA2002: 93-124Crossref Google Scholar). PON1-knockout mice are more susceptible to developing atherosclerosis than are wild-type mice, and their HDL, in contrast to wild-type HDL, fails to prevent LDL oxidation in cultured artery wall cells (25Shih D.M. Gu L. Xia Y.R. Navab M. Li W.F. Hama S. Castellani L.W. Furlong C.E. Costa L.G. Fogelman A.M. Mice lacking serum paraoxonase are susceptible to organophosphate toxicity and atherosclerosis.Nature. 1998; 394: 284-287Crossref PubMed Scopus (957) Google Scholar). Like PON1, both human PON2 and PON3 have been shown to prevent cell-mediated oxidative modification of LDL (16Reddy S.T. Wadleigh D.J. Grijalva V. Ng C. Hama S. Gangopadhyay A. Shih D.M. Lusis A.J. Navab M. Fogelman A.M. Human paraoxonase-3 is an HDL-associated enzyme with biological activity similar to paraoxonase-1 protein but is not regulated by oxidized lipids.Arterioscler. Thromb. Vasc. Biol. 2001; 21: 542-547Crossref PubMed Scopus (302) Google Scholar, 17Ng C.J. Wadleigh D.J. Gangopadhyay A. Hama S. Grijalva V.R. Navab M. Fogelman A.M. Reddy S.T. Paraoxonase-2 is a ubiquitously expressed protein with antioxidant properties and is capable of preventing cell-mediated oxidative modification of low density lipoprotein.J. Biol. Chem. 2001; 276: 44444-44449Abstract Full Text Full Text PDF PubMed Scopus (397) Google Scholar). However, the exact endogenous substrates and mechanism of the PONs' protective activities remain to be elucidated. Recently, human PON1 was expressed in insect cells and the purified recombinant protein was shown to exhibit similar kinetic properties to PON1 purified from serum (27Brushia R.J. Forte T.M. Oda M.N. La Du B.N. Bielicki J.K. Baculovirus-mediated expression and purification of human serum paraoxonase 1A.J. Lipid Res. 2001; 42: 951-958Abstract Full Text Full Text PDF PubMed Google Scholar). Here, we describe a similar expression system for all three human PONs in insect cells and optimized procedures for their purification. We characterized the enzymatic activities of the recombinant PONs with a wide range of organophosphate, aromatic ester, lactone, and hydroxycarboxylic acid substrates and tested their ability to protect LDL against copper-induced oxidation. Enzymes for molecular biological procedures were purchased from Promega and New England Biolabs. pcDNA3.1(+) and pFastBac1(+) vectors, DH10Bac™ Escherichia coli cells, penicillin-streptomycin (10,000 U/ml penicillin, 10 mg/ml streptomycin), Geneticin® (G-418 sulfate), Cellfectin® reagent, Pluronic® F-68, Sf-900 II, and Grace's insect media were obtained from Invitrogen. Insect Xpress™ medium and fetal bovine serum were purchased from BioWhittaker, Inc. (Walkersville, MD). DEAE-Microprep and low molecular weight prestained Precision Plus protein standards were obtained from Bio-Rad. Concanavalin A-Sepharose was purchased from Amersham. Chlorpyrifos oxon and diazoxon were purchased from Chem Service, Inc. (±)4-hydroxy-5E,7Z,10Z,13Z,16Z,19Z-docosahexaenoic acid (4-HDoHE), (±)5-hydroxy-6E,8Z,11Z,14Z-eicosatetraenoic acid 1,5-lactone (5-HETEL), and 2-thio platelet-activating factor (thio-PAF) were obtained from Cayman Chemical (Ann Arbor, MI). Lovastatin was provided by Merck. All other chemicals were of analytical grade or better and purchased from Sigma-Aldrich or Fisher. The generation of recombinant baculoviruses and the purification procedure for the individual PONs are described in detail in the supplementary data online. Briefly, the recombinant PONs were expressed in Trichoplusia ni High Five™ insect cells (Invitrogen) and extracted from the crude membrane fraction with a detergent [n-dodecyl-β-d-maltoside (DDM)]. PON1 and PON2 were purified to homogeneity using Microprep-DEAE and Concanavalin A-Sepharose chromatography, but for PON3 purification an additional third step (Superdex 200) was necessary. Representative purifications of the recombinant human PONs are shown in supplementary Figs. I–III and are summarized in Table 1.TABLE 1Summary of the purification of recombinant human PONs from HiFive insect cellsPONVolumeProteinActivitySpecific ActivityYieldFold PurificationmlmgunitsU/mg%nHuman PON1Phenyl acetate hydrolysisCell homogenate 100234.29,000 38.41.0Solubilized membranes 2054.611,600 212.31005.6100,000 g supernatant 1938.010,925 287.5947.5DEAE pool 125.96,070 1021.05226.6Concanavalin A pool 71.82,230 1225.01931.9Human PON24-HDoHE lactonizationCell homogenate 80148.01.043 0.0071.0Solubilized membranes 20100.01.316 0.0131.9100,000 g supernatant 1831.71.437 0.0451006.5DEAE pool 60.90.411 0.4662965.6Concanavalin A pool 40.40.212 0.5301375.7Human PON3Lovastatin hydrolysisCell homogenate 100123.00.418 0.0031.0Solubilized membranes 2080.20.618 0.0082.3100,000 g supernatant 1936.20.724 0.0201005.9DEAE pool 104.50.219 0.0493014.4Concanavalin A pool 61.00.065 0.065919.0Superdex 200 pool 3.50.20.054 0.2707.579.44-HDoHE, (±)4-hydroxy-5E,7Z,10Z,13Z,16Z,19Z-docosahexaenoic acid; PON, paraoxonase. Results are given for typical purifications. The enzymatic activities were measured as described in Materials and Methods. Yield = (activity of the fractions combined for the next step)/(activity of the solubilized membranes or the 100,000 g supernatant) × 100 and does not include all of the activity actually recovered. One unit of enzymatic activity is defined as 1 μmol of substrate metabolized per minute. Open table in a new tab 4-HDoHE, (±)4-hydroxy-5E,7Z,10Z,13Z,16Z,19Z-docosahexaenoic acid; PON, paraoxonase. Results are given for typical purifications. The enzymatic activities were measured as described in Materials and Methods. Yield = (activity of the fractions combined for the next step)/(activity of the solubilized membranes or the 100,000 g supernatant) × 100 and does not include all of the activity actually recovered. One unit of enzymatic activity is defined as 1 μmol of substrate metabolized per minute. Nondenaturing and SDS-PAGE were performed according to Laemmli (28Laemmli U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature. 1970; 227: 680-685Crossref PubMed Scopus (206658) Google Scholar). The nondenaturing gels contained 0.05% DDM and no SDS. The proteins were visualized by Coomassie Blue or silver staining. For activity staining (zymogram), gels were immersed in substrate solution, prepared by dissolving 80 mg of β-naphthyl acetate and 40 mg of Fast Blue RR salt in 20 ml of ethylene glycol monomethyl ether and then diluted to 50 ml with 25 mM Tris-HCl, pH 8.0, and 5 mM CaCl2. Red bands representing catalytically active protein developed within seconds. The gels were fixed (20% isopropyl alcohol and 10% acetic acid), scanned, and stained with Coomassie Blue. For Western blotting, gels run under denaturing or nondenaturing conditions were transferred to polyvinylidene difluoride membranes. The primary antibodies used were as follows: for PON1, mouse monoclonal antibody 3C6.33 (3.3 mg/ml, affinity purified from ascites; University of Michigan Hybridoma Core); for PON2, affinity-purified polyclonal rabbit antibody raised against human PON2-specific peptide (HLKEEKPRARELRISRGFDLA); and for PON3, rabbit anti-human PON3 serum (provided by Dr. S. T. Reddy, University of California Los Angeles). Secondary antibodies coupled with alkaline phosphatase (Sigma) were used at 1:5,000 dilutions. The molecular masses of the native PONs were determined by a Ferguson plot (29Ferguson K.A. Starch-gel electrophoresis—application to the classification of pituitary proteins and polypeptides.Metabolism. 1964; 13: 985-1002Abstract Full Text PDF PubMed Scopus (781) Google Scholar, 30Bryan J.K. Molecular weights of protein multimers from polyacrylamide gel electrophoresis.Anal. Biochem. 1977; 78: 513-519Crossref PubMed Scopus (155) Google Scholar) using BSA (monomer, 66 kDa; dimer, 132 kDa), alcohol dehydrogenase (150 kDa), and ferritin (443 kDa) as standards. Purified recombinant PONs were enzymatically deglycosylated with endoglycosidase H (EndoH) or peptide:N-glycosidase F according to the manufacturer's protocols (New England Biolabs, Inc.). EndoH deglycosylation of the recombinant PONs under nondenaturing conditions was carried out overnight at room temperature in 50 mM sodium citrate buffer, pH 5.5, supplemented with 50 mM CaCl2. Ultraviolet/visible spectrophotometric assays were performed on a Cary 3 instrument (Varian) equipped with a temperature block adjusted to 25°C. The initial rates were calculated from the curve slopes using the Cary WinUV Bio software package. Substrate stock solutions (100 mM) were prepared in methanol except phenyl acetate and paraoxon (water suspensions of 20 and 4 mM, respectively) and unless specified otherwise were used at 1 mM final concentration (1% methanol final concentration in the reaction mixture). Arylesterase activity with the substrates phenyl acetate, p-NO2-phenyl acetate, p-NO2-phenyl propionate, and p-NO2-phenyl butyrate was measured in 50 mM Tris-HCl, pH 8.0, and 1 mM CaCl2 (31Kuo C.L. La Du B.N. Comparison of purified human and rabbit serum paraoxonases.Drug Metab. Dispos. 1995; 23: 935-944PubMed Google Scholar, 32Eckerson H.W. Wyte C.M. La Du B.N. The human serum paraoxonase/arylesterase polymorphism.Am. J. Hum. Genet. 1983; 35: 1126-1138PubMed Google Scholar). Organophosphatase activity with the substrates paraoxon, chlorpyrifos oxon (0.32 mM final concentration), and diazoxon was measured in 100 mM Tris-HCl buffer, pH 8.5, 2 mM CaCl2, and 2 M NaCl (5Davies H.G. Richter R.J. Keifer M. Broomfield C.A. Sowalla J. Furlong C.E. The effect of the human serum paraoxonase polymorphism is reversed with diazoxon, soman and sarin.Nat. Genet. 1996; 14: 334-336Crossref PubMed Scopus (540) Google Scholar, 33Furlong C.E. Richter R.J. Seidel S.L. Costa L.G. Motulsky A.G. Spectrophotometric assays for the enzymatic hydrolysis of the active metabolites of chlorpyrifos and parathion by plasma paraoxonase/arylesterase.Anal. Biochem. 1989; 180: 242-247Crossref PubMed Scopus (215) Google Scholar). Lactonase activity with the substrates dihydrocoumarin, 2-coumaronone, and homogentisic acid lactone was assayed in 50 mM Tris-HCl buffer, pH 7.4, and 1 mM CaCl2 (10Draganov D.I. Stetson P.L. Watson C.E. Billecke S.S. La Du B.N. Rabbit serum paraoxonase 3 (PON3) is a high density lipoprotein-associated lactonase and protects low density lipoprotein against oxidation.J. Biol. Chem. 2000; 275: 33435-33442Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar). Lactonase activity with aliphatic lactones was determined by a continuous pH-sensitive colorimetric assay modified from Billecke et al. (7Billecke S. Draganov D. Counsell R. Stetson P. Watson C. Hsu C. La Du B.N. Human serum paraoxonase (PON1) isozymes Q and R hydrolyze lactones and cyclic carbonate esters.Drug Metab. Dispos. 2000; 28: 1335-1342PubMed Google Scholar) using a SPECTRAmax® PLUS microplate reader (Molecular Devices, Sunnyvale, CA). The reactions (200 μl final volume) containing 2 mM HEPES, pH 8.0, 1 mM CaCl2, 0.004% (w/v) Phenol Red, and 2–10 μl of purified enzyme were initiated with 2 μl of 100 mM substrate solution in methanol and were carried out at 37°C for 3–10 min. The rates were calculated from the slopes of the absorbance decrease at 558 nm with correction at 475 nm (isosbestic point) using a rate factor (mOD/μmol H+) estimated from a standard curve generated with known amounts of HCl. The spontaneous hydrolysis of the lactones and acidification by atmospheric CO2 were corrected for by carrying out parallel reactions with the same volume of storage buffer instead of enzyme. The lactonization of 4-HDoHE (10 μM) and coumaric acid (100 μM) as well as the lactone hydrolysis of 5-HETEL (10 μM), lovastatin, spironolactone, and canrenone (25 μM) at the final substrate concentrations indicated in parentheses were analyzed by HPLC using a Supelco Discovery C-18 column (250 × 4.6 mm, 5 μm particles) and a Beckman System Gold 126 equipped with a Beckman 128 diode array detector as described (7Billecke S. Draganov D. Counsell R. Stetson P. Watson C. Hsu C. La Du B.N. Human serum paraoxonase (PON1) isozymes Q and R hydrolyze lactones and cyclic carbonate esters.Drug Metab. Dispos. 2000; 28: 1335-1342PubMed Google Scholar, 9Teiber J.F. Draganov D.I. La Du B.N. Lactonase and lactonizing activities of human paraoxonase (PON1) and rabbit serum PON3.Biochem. Pharmacol. 2003; 66: 887-896Crossref PubMed Scopus (129) Google Scholar, 10Draganov D.I. Stetson P.L. Watson C.E. Billecke S.S. La Du B.N. Rabbit serum paraoxonase 3 (PON3) is a high density lipoprotein-associated lactonase and protects low density lipoprotein against oxidation.J. Biol. Chem. 2000; 275: 33435-33442Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar). The hydrolysis of acylhomoserine lactones (AHLs) was analyzed by HPLC on a Waters 2695 system equipped with Waters 2996 photodiode array detector set at 197 nm using a Supelco Discovery C-18 column (250 × 4.6 mm, 5 μm particles). Enzymatic reactions were carried out at room temperature in 50 μl volume of 25 mM Tris-HCl, pH 7.4, 1 mM CaCl2, 25 μM AHL (from 2 mM stock solution in methanol, except for 3-oxo-hexanoyl homoserine lactone, which was 25 mM stock and 250 μM final), and 2–5 μl of purified enzyme. Reactions were stopped with 50 μl of acetonitrile (ACN) and centrifuged to remove the protein. Supernatants (40 μl) were loaded onto the HPLC system and eluted isocratically with 85% ACN/0.2% acetic acid (tetradeca-homoserine lactone), 75% ACN/0.2% acetic acid (dodeca-homoserine lactone), 50% ACN/0.2% acetic acid (hepta-homoserine lactone), or 20% ACN/0.2% acetic acid (3-oxo-hexanoyl homoserine lactone). The retention times for the open acid forms and the lactones under these conditions were as follows: 5.8/7.5 min for tetradeca-homoserine lactone, 4.9/6.9 min for dodeca-homoserine lacton
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