Rabbit Serum Paraoxonase 3 (PON3) Is a High Density Lipoprotein-associated Lactonase and Protects Low Density Lipoprotein against Oxidation

The paraoxonase gene family contains at least three members: PON1, PON2, andPON3. The physiological roles of the corresponding gene products are still uncertain. Until recently, only the serum paraoxonase/arylesterase (PON1) had been purified and characterized. Here we report the purification, cloning, and characterization of rabbit serum PON3. PON3 is a 40-kDa protein associated with the high density lipoprotein fraction of serum. In contrast to PON1, PON3 has very limited arylesterase and no paraoxonase activities but rapidly hydrolyzes lactones such as statin prodrugs (e.g.lovastatin). These differences facilitated the complete separation of PON3 from PON1 during purification. PON3 hydrolyzes aromatic lactones and 5- or 6-member ring lactones with aliphatic substituents but not simple lactones or those with polar substituents. We cloned PON3 from total rabbit liver RNA and expressed it in mammalian 293T/17 cells. The recombinant PON3 has the same apparent molecular mass and substrate specificity as the enzyme purified from serum. Rabbit serum PON3 is more efficient than rabbit PON1 in protecting low density lipoprotein from copper-induced oxidation. This is the first report that identifies a second PON enzyme in mammalian serum and the first to describe an enzymatic activity for PON3. The paraoxonase gene family contains at least three members: PON1, PON2, andPON3. The physiological roles of the corresponding gene products are still uncertain. Until recently, only the serum paraoxonase/arylesterase (PON1) had been purified and characterized. Here we report the purification, cloning, and characterization of rabbit serum PON3. PON3 is a 40-kDa protein associated with the high density lipoprotein fraction of serum. In contrast to PON1, PON3 has very limited arylesterase and no paraoxonase activities but rapidly hydrolyzes lactones such as statin prodrugs (e.g.lovastatin). These differences facilitated the complete separation of PON3 from PON1 during purification. PON3 hydrolyzes aromatic lactones and 5- or 6-member ring lactones with aliphatic substituents but not simple lactones or those with polar substituents. We cloned PON3 from total rabbit liver RNA and expressed it in mammalian 293T/17 cells. The recombinant PON3 has the same apparent molecular mass and substrate specificity as the enzyme purified from serum. Rabbit serum PON3 is more efficient than rabbit PON1 in protecting low density lipoprotein from copper-induced oxidation. This is the first report that identifies a second PON enzyme in mammalian serum and the first to describe an enzymatic activity for PON3. high density lipoprotein low density lipoprotein rabbit liver microsomal paraoxonase polyacrylamide gel electrophoresis high performance liquid chromatography very low density lipoproteins high performance gel filtration chromatography apolipoprotein platelet-activated factor acetyl hydrolase reverse transcriptase-polymerase chain reaction The paraoxonase gene family in mammals includes at least three members: PON1, PON2 and PON3 (1Primo-Parmo S.L. Sorenson R.C. Teiber J. La Du B.N. Genomics. 1996; 33: 498-507Crossref PubMed Scopus (589) Google Scholar). The PON genes appear to have arisen by gene duplication of a common evolutionary precursor because they share considerable structural homology and are located adjacently on chromosome 7 in humans and on chromosome 6 in mice (1Primo-Parmo S.L. Sorenson R.C. Teiber J. La Du B.N. Genomics. 1996; 33: 498-507Crossref PubMed Scopus (589) Google Scholar). PON1, also known as serum paraoxonase/arylesterase (EC 3.1.8.1), is closely associated with high density lipoproteins (HDL)1 and catalyzes the hydrolysis of a variety of aromatic carboxylic acid esters and several organophosphates (2La Du B.N. Kalow W. Pharmacogenetics of Drug Metabolism. Pergamon Press, Inc., New York, NY1992: 51-91Google Scholar, 3Davies H.G. Richter R.J. Keifer M. Broomfield C.A. Sowalla J. Furlong C.E. Nat. Genet. 1996; 14: 334-336Crossref PubMed Scopus (553) Google Scholar). Recently, we reported that purified human and rabbit serum PON1 also hydrolyze of a variety of lactones and cyclic carbonate esters, including naturally occurring lactones and pharmacological agents (4Billecke S. Draganov D. Counsell R. La Du B.N. FASEB J. 1999; 13 (Abstr. 764): A1013Google Scholar, 5Billecke, S., Draganov, D., Counsell, R., Stetson, P., Watson, C., Hsu, C., and La Du, B. N. (2000) Drug Metab. Dispos.,28, in press.Google Scholar). PON1 requires Ca2+ for both its stability and hydrolytic activity (2La Du B.N. Kalow W. Pharmacogenetics of Drug Metabolism. Pergamon Press, Inc., New York, NY1992: 51-91Google Scholar), and the latter is stimulated by several phospholipids (6Kuo C.-L. La Du B.N. Drug Metab. Dispos. 1995; 23: 935-944PubMed Google Scholar). Any physiological role of PON1 is still speculative; however, several lines of evidence suggest that this enzyme protects against atherosclerosis by preventing the oxidation of low density lipoproteins (LDL) (see Refs. 7Mackness M.I. Mackness B. Durrington P.N. Connelly P.W. Hegele R.A. Curr. Opin. Lipidol. 1996; 7: 69-76Crossref PubMed Scopus (424) Google Scholar, 8Navab M. Hama S.Y. Hough G.P. Hedrick C.C. Sorenson R. La Du B.N. Kobashigawa J.A. Fonarow G.C. Berliner J.A. Laks H. Fogelman A.M. Curr. Opin. Lipidol. 1998; 9: 449-456Crossref PubMed Scopus (76) Google Scholar, 9Aviram M. Mol. Med. Today. 1999; 5: 381-386Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar for reviews). Oxidized phospholipids and/or their degradation/metabolic products may be physiological substrates of PON1 (10Watson A.D. Berliner J. Hama S.Y. La Du B.N. Faull K.F. Fogelman A.M. Navab M. J. Clin. Invest. 1995; 96: 2882-2891Crossref PubMed Scopus (1047) Google Scholar). PON1 knockout mice are more susceptible to organophosphate toxicity as well as to atherosclerosis produced by an atherogenic diet (11Shih 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. Lusis A.J. Nature. 1998; 394: 284-287Crossref PubMed Scopus (974) Google Scholar). PON1 also protects against bacterial endotoxin-induced toxicity (12La Du B.N. Aviram M. Billecke S. Navab M. Primo-Parmo S. Sorenson R.C. Standiford T.J. Chem. Biol. Interact. 1999; 119–120: 379-388Crossref PubMed Scopus (178) Google Scholar). The roles of PON2 and PON3 are much less well understood than are those for PON1. Within a given species, PON1, PON2, and PON3 share about 70% identity at the nucleotide level and about 60% identity at the amino acid level, whereas between the mammalian species particular PONs (1, 2, or 3) share 81–91% identity on the nucleotide level and 79–90% identity at the amino acid level (1Primo-Parmo S.L. Sorenson R.C. Teiber J. La Du B.N. Genomics. 1996; 33: 498-507Crossref PubMed Scopus (589) Google Scholar, 12La Du B.N. Aviram M. Billecke S. Navab M. Primo-Parmo S. Sorenson R.C. Standiford T.J. Chem. Biol. Interact. 1999; 119–120: 379-388Crossref PubMed Scopus (178) Google Scholar). To date, all PON2 and PON3 cDNAs sequenced lack the three nucleotide residues of codon 106 in PON1 (1Primo-Parmo S.L. Sorenson R.C. Teiber J. La Du B.N. Genomics. 1996; 33: 498-507Crossref PubMed Scopus (589) Google Scholar, 12La Du B.N. Aviram M. Billecke S. Navab M. Primo-Parmo S. Sorenson R.C. Standiford T.J. Chem. Biol. Interact. 1999; 119–120: 379-388Crossref PubMed Scopus (178) Google Scholar). Some lactone hydrolases previously described in bacteria (13Kanagasundaram V. Scopes R. Biochim. Biophys. Acta. 1992; 1171: 198-200Crossref PubMed Scopus (35) Google Scholar) and in fungi (14Kobayashi M. Shinohara M. Sakoh C. Kataoka M. Shimizu S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12787-12792Crossref PubMed Scopus (61) Google Scholar) share remarkable structural homology with the PON family members, and we hypothesized that the lactonase activity rather than arylesterase or organophosphatase activities could be a common feature of the PON proteins. In the present study, a rabbit serum lactonase distinct from PON1 was purified and characterized. This is a 40-kDa protein that has an N-terminal sequence (25 amino acids) that exactly matches the deduced amino acid sequence predicted by the rabbit PON3 cDNA we have cloned and that agrees closely with the sequence reported recently for rabbit liver microsomal paraoxonase (MsPON) (15Ozols J. Biochem. J. 1999; 338: 265-272Crossref PubMed Google Scholar). We defined the substrate specificity of the purified rabbit serum PON3 over a number of esters and lactones and confirmed it with transiently expressed recombinant rabbit PON3. Of considerable interest is the close HDL association of serum PON3 and its enhanced ability compared with rabbit serum PON1 to protect LDL against oxidation. Rabbit blood was collected by ear vein bleeding and cardiac puncture of anesthetized (ketamine, 40 mg/kg intramuscular, and acetopromazine, 1 mg/kg intramuscular) New Zealand White female rabbits. The blood was allowed to clot for 30 min at room temperature, and the serum was obtained after centrifugation for 15 min at 2,000 × g. DEAE Bio-Gel A and SDS-PAGE low range protein molecular weight standards were obtained from Bio-Rad. Sephacryl 200 was purchased from Amersham Pharmacia Biotech. Cibacron Blue 3GA-agarose type 3000, concanavalin A-Sepharose 4B, Tergitol NP-10, and mevastatin (Compactin) were obtained from Sigma. Lovastatin (Mevacor®) and simvastatin (Zocor®) were purchased as 20-mg tablets from Merck, from which the lactones were extracted with chloroform, evaporated to dryness, and redissolved in methanol. ε-Caprolactone was obtained from Acros Chimica (Fair Lawn, NJ). All other lactones and esters used were purchased from Sigma-Aldrich. Acetonitrile high performance liquid chromatography (HPLC) grade was purchased from Mallinckrodt Chemical Works. Glacial acetic acid, methanol, and water (HPLC grade) were obtained from Fisher. Centicons and Centriprep concentrators were purchased from Amicon, Inc. (Beverly, MA). The initial steps in the purification of rabbit serum PON3 essentially followed the procedure for purification of serum PON1 previously developed in this laboratory (6Kuo C.-L. La Du B.N. Drug Metab. Dispos. 1995; 23: 935-944PubMed Google Scholar,16Gan K.N. Smolen A. Eckerson H.W. La Du B.N. Drug Metab. Dispos. 1991; 19: 100-106PubMed Google Scholar). Rabbit serum was mixed batchwise with Cibacron Blue 3 GA-agarose in 3 m NaCl, 50 mm Tris/HCl buffer (pH 8.0), 1 mm CaCl2, and 5 μm EDTA, poured into a column, and washed with the same buffer until theA 280 was below 0.3 A units and further with two column volumes of 25 mm Tris/HCl buffer (pH 8.0), 1 mm CaCl2. The column was developed with 25 mm Tris/HCl buffer (pH 8.0), 1 mmCaCl2, 20% glycerol, and 0.1% sodium deoxycholate; 10-ml fractions were collected at 1 ml/min. Fractions were monitored for both arylesterase activity (phenyl acetate hydrolysis) and lactonase activity (lovastatin hydrolysis) to localize PON1 and PON3, respectively. The two activities co-eluted after Blue-agarose chromatography of serum but were almost completely separated on a DEAE anion exchange column by elution with a linear 300-ml of 0–175 mm NaCl gradient in TCGT buffer (25 mm Tris/HCl (pH 8.0), 1 mm CaCl2, 20% glycerol, and 0.2% non-ionic detergent Tergitol NP-10). Fractions (5 ml) were collected at 0.55 ml/min. Fractions with arylesterase or lactonase activities were purified further separately on a second DEAE column with a NaCl gradient of 0–120 mm for PON1 and 0–175 mmfor PON3. Fractions with lactonase activity from the second DEAE column were pooled, concentrated 10-fold using a 10-kDa Centricon ultrafiltration unit, and loaded onto a Sephacryl 200 gel filtration column (100 cm × 1 cm). Proteins were eluted with TCGT buffer and 1 ml fractions were collected at 17 ml/h. Fractions with the highest lactonase activities and the lowest contamination with other proteins, as determined by SDS-polyacrylamide gel electrophoresis (PAGE), were pooled and passed through a concanavalin A-Sepharose 4B column. PON3 did not bind to this column and was collected in 25 mmTris/HCl buffer (pH 7.2), 1 mm CaCl2. PON1-containing fractions from a second DEAE column were pooled, loaded on a concanavalin A column, and eluted with a 0–200 mmα-d-mannopyranoside gradient in 25 mmTris/HCl buffer (pH 7.2), 1 mm CaCl2. Finally, the PON1 and PON3 preparations were washed with TCGT buffer using a 50-kDa cut-off Centricon ultrafiltration unit to adjust the buffer and to remove residual concanavalin A fragments. The enzyme preparations were stored at 4 °C without substantial loss of activity for more than 3 months. Protein was determined by its UV absorption at 280 nm or in samples containing Tergitol NP-10 by the BCA protein assay (Pierce), according to the manufacturer's protocol. SDS-PAGE was performed as described previously (17Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (218068) Google Scholar), and the gels were stained with Coomassie Blue. The N-terminal peptide sequencing was performed at the University of Michigan Protein Sequencing Core Laboratory using the Model 494 HT Protein Sequencer (Applied Biosystems Inc., Foster City, CA). Total rabbit liver RNA was isolated using the RNeasy Kit (Qiagen, Valencia, CA) following the supplier's protocol. RT-PCR was performed with the Titan One-tube RT-PCR system (Roche Molecular Biochemicals) according to the manufacturer's instructions. Primers for human PON3 (1Primo-Parmo S.L. Sorenson R.C. Teiber J. La Du B.N. Genomics. 1996; 33: 498-507Crossref PubMed Scopus (589) Google Scholar), Px3–6 (GGCATAGAACTGTTCTGGTCCAAGAACC) and Px3–17 (GCTTCTGAAGATATTGATATACTCCCCAGTGGGC) were purchased from Midland Certified Reagent Co. (Midland, TX). The RT-PCR products were separated on a 1% agarose gel. Bands of the expected size (as deduced by their similarity to the human PON3) were excised and submitted for sequencing in our DNA Sequencing Core Laboratory. Based on the obtained sequence, primers RPx3–4 (CTCATCTGGTGCAAAGTTTGG) and RPx3–1 (ACAACAACGCTCTCTTGTAC) were designed (these and all of the primers below were purchased from Life Technologies, Inc.) and used for amplification of the 5′- and 3′-ends of rabbit PON3 cDNA using a 5′,3′-rapid amplification of cDNA ends (RACE) kit (Roche Molecular Biochemicals) according to the manufacturer's instructions. Based on the sequence of the new primers of these fragments, RPx3–5 (ATCGGAA TTCCATGGCGAAGCTCCTGC) and RPx3–6 (AGGCCTCGAGCTGGAGACTAGAGCAC) were designed and used to amplify the full-length cDNA. The PCR product (∼1200 base pairs) was cloned in the pCR®II vector with a TOPO®TA cloning kit (Invitrogen, Carlsbad, CA) and sequenced. PON3 clones from at least five animals were sequenced in both directions, and the consensus sequence was submitted to the GenBankTM (accession number AF220944). The rabbit PON3 clone in pCR®II vector was digested withEcoRI (Promega) and XhoI (Life Technologies, Inc.) and ligated into a pcDNA3.1(+) expression vector (Invitrogen). The plasmid was propagated in Escherichia coliTOP10 cells (Invitrogen) and then purified using the Qiagen plasmid maxi kit. The insert was sequenced to verify its structure. The highly transfectable 293T/17 human embryonic kidney cell line (ATCC CRL 11268) was used with the permission of David Baltimore (Rockefeller University, New York). Cells were grown in minimum essential medium (Celox Laboratories, Inc., St. Paul, MN) supplemented with 10% heat-deactivated fetal bovine serum (Life Technologies, Inc.) and transfected with 10 μg of plasmid DNA as described (18Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989: 16.30-16.37Google Scholar). The medium containing the recombinant enzyme was collected after allowing transfected cells to grow for 4–5 days. The recombinant PON3 was partly purified using a small Blue-agarose column as described above. The lactonase activity (lovastatin hydrolysis) was used to follow the level of expression/secretion in the culture medium and to localize PON3 in the column fractions. Esterase activities with substrates phenyl acetate, S-phenyl thioacetate, α-naphtyl acetate, and paraoxon were measured as described elsewhere (6Kuo C.-L. La Du B.N. Drug Metab. Dispos. 1995; 23: 935-944PubMed Google Scholar, 16Gan K.N. Smolen A. Eckerson H.W. La Du B.N. Drug Metab. Dispos. 1991; 19: 100-106PubMed Google Scholar). Hydrolysis of aromatic lactones was monitored by the increase in UV absorbance at 270 nm (dihydrocoumarin), 274 nm (2-coumaranone), and 290 nm (homogentisic acid lactone). In a typical experiment a cuvette contained 1 mm substrate (from a 100 mm stock, dissolved in methanol) in 50 mm Tris/HCl (pH 8.0), 1 mm CaCl2, and 5–20 μl of enzyme in a total volume of 1 ml. The molar difference extinction coefficients (difference between the absorption coefficients of the acid formed and the lactone) used to calculate the rate of hydrolysis were 1295, 876, and 816 m−1cm−1 for dihydrocoumarin, 2-coumaranone, and homogentisic acid lactone, respectively (5Billecke, S., Draganov, D., Counsell, R., Stetson, P., Watson, C., Hsu, C., and La Du, B. N. (2000) Drug Metab. Dispos.,28, in press.Google Scholar). The hydrolysis of other lactones was followed by a colorimetric assay with phenol red (5Billecke, S., Draganov, D., Counsell, R., Stetson, P., Watson, C., Hsu, C., and La Du, B. N. (2000) Drug Metab. Dispos.,28, in press.Google Scholar, 19Billecke S.S. Primo-Parmo S.L. Dunlop C.S. Dorn J.A. La Du B.N. Broomfield C.A. Chem. Biol. Interact. 1999; 119–120: 251-256Crossref PubMed Scopus (35) Google Scholar). The reaction cuvette contained 1 mm substrate (from a 100 mm stock solution, dissolved in methanol) in 2 mm HEPES (pH 8.0), 1 mm CaCl2, 0.004% (106 μm) phenol red, 0.005% bovine albumin, and 5–20 μl of enzyme in a total volume of 1 ml. The rate of acid production was followed by monitoring the increase in absorbance at 422 nm. The rate of hydrolysis was derived from a calibration curve obtained using known amounts of HCl as described previously (19Billecke S.S. Primo-Parmo S.L. Dunlop C.S. Dorn J.A. La Du B.N. Broomfield C.A. Chem. Biol. Interact. 1999; 119–120: 251-256Crossref PubMed Scopus (35) Google Scholar). The enzymatic assays were performed at 25 °C using a dual beam Cary 3E UV/visible spectrophotometer (Varian, Australia). Reference cuvettes containing the appropriate buffer plus substrate were used in each assay to correct for any spontaneous hydrolysis. The hydrolysis of the statin lactones (mevastatin, lovastatin, and simvastatin) was analyzed by HPLC using a Beckman System Gold HPLC with a model 126 programmable solvent module, a model 168 diode array detector set at 238 nm, a model 7125 rheodyne manual injector valve with a 20-μl loop, and a Beckman ODS Ultrasphere column (C18, 250 × 4.6 mm, 5 μm). In a final volume of 1 ml, 10–200 μl of enzyme and 10 μl of substrate solution in methanol (0.5 mg/ml) were incubated at 25 °C in 25 mm Tris/HCl (pH 7.6), 1 mm CaCl2. Aliquots (100 μl) were removed at specified times and added to acetonitrile (100 μl), vortexed, and centrifuged for 1 min at maximum speed (Beckman Microfuge). The supernatants were poured into new tubes, capped, and stored on ice until HPLC analysis. Samples were eluted isocratically at a flow rate of 1.0 ml/min with a mobile phase consisting of the following: A = acetic acid/acetonitrile/water (2:249:249, v/v/v) and B = acetonitrile, in A/B ratios of 50/50, 45/55, and 40/60 for mevastatin, lovastatin, and simvastatin, respectively. Under the above conditions the retention times for the carboxylic acid formed and the lactone substrate were as follows: 4.5/6.4 min (mevastatin), 4.4/6.6 min (lovastatin), and 4.8/6.6 min (simvastatin). Response factors for the acid products were calculated from the peak heights after complete alkaline hydrolysis of the lactones in 0.02 m NaOH. Serum was loaded onto a Bio-Gel A-15m column (36 × 1.8 cm) and eluted with 50 mm Tris HCl (pH 8.0), 1 mm CaCl2. Fractions were collected and analyzed on a Hitachi 912 autoanalyzer (Roche Molecular Biochemicals) for phospholipids, triglycerides, and total and esterified cholesterol using commercially available kits from Wako Bioproducts (Richmond, VA). Representative fractions were combined, concentrated, and analyzed by high performance gel filtration chromatography (HPGC) to determine the lipoprotein cholesterol distribution as described elsewhere (20Kieft K.A. Bocan T.M. Krause B.R. J. Lipid Res. 1991; 32: 859-866Abstract Full Text PDF PubMed Google Scholar). Fractions were prepared by sequential flotation ultracentrifugation to isolate very low density lipoproteins (VLDL) (d <1.019 g/ml), LDL (d = 1.019 to 1.063 g/ml), and HDL (d = 1.063 to 1.2 g/ml) (21Hatch F.T. Adv. Lipid Res. 1968; 6: 1-68Crossref PubMed Google Scholar). Lipoprotein fractions were dialyzed against phosphate-buffered saline (pH 7.4) containing 1 mm CaCl2 at 4 °C for 24 h, then kept at 4 °C in the dark. The isolated fractions were analyzed within the next 3–4 days for enzymatic activities and by HPGC to confirm their identity and purity as described (20Kieft K.A. Bocan T.M. Krause B.R. J. Lipid Res. 1991; 32: 859-866Abstract Full Text PDF PubMed Google Scholar). Human LDL was isolated by ultracentrifugation as described above and used within the next 3–4 days. LDL (0.1 mg protein/ml) oxidation was induced with 2–10 μm CuSO4 in phosphate-buffered saline (pH 7.4) in the absence or presence of PON3 or PON1 for up to 5 h at 37 °C. The kinetics of lipoprotein oxidation was followed by monitoring conjugated diene formation at 234 nm (22Esterbauer H. Striegl G. Puhl H. Rotheneder M. Free Radic. Res. Commun. 1989; 6: 67-75Crossref PubMed Scopus (1716) Google Scholar) using a quartz microtiter plate in a SPECTRAmax® 190 plate reader (Molecular Devices, Sunnyvale, CA). The lag time was estimated by drawing a perpendicular line to the x axis from the intersection of straight lines drawn through the absorption curves during the lag phase and the propagation phase, as illustrated on Fig.4 A. Lipoprotein oxidation was also determined by the lipid peroxides test, which analyzes lipid peroxides by their capacity to convert iodide to iodine, as measured photometrically at 365 nm (23El-Saadani M. Esterbauer H. El-Sayed M. Goher M. Nassar A.Y. Jurgens G.A. J. Lipid Res. 1989; 30: 627-630Abstract Full Text PDF PubMed Google Scholar). The purification of PON3 from rabbit serum is summarized in TableI. We used lovastatin hydrolysis (lactonase activity) and phenyl acetate hydrolysis (arylesterase activity) to follow purification of PON3 and PON1, respectively. During the Blue-agarose chromatography of serum, both activities co-eluted (Fig. 1 A) but were resolved by DEAE anion exchange chromatography (Fig. 1 B). Fractions with peak arylesterase activity were eluted at 87 mm NaCl and fractions with peak lactonase activity were eluted at 105 mm NaCl, respectively. The lactonase activity was further purified through a second DEAE and a Sephacryl 200 gel filtration column and correlated with the intensity of a 40-kDa protein on SDS-PAGE. Two other proteins with molecular masses of 50 and 63 kDa co-purified through all steps and became enriched during the purification (Fig. 1 C, 2nd lane). The three proteins were transferred to polyvinylidene difluoride membrane, and each was submitted for N-terminal sequencing. The resolved peptide sequences are presented on TableII. The N terminus of the 40-kDa protein is identical with the deduced amino acids sequence of the rabbit PON3 cDNA we cloned (see below) and 96% identical with the amino acid sequence of MsPON (15Ozols J. Biochem. J. 1999; 338: 265-272Crossref PubMed Google Scholar). The N terminus of the 50-kDa protein is 73% identical with the deduced sequence (residues 22 to 36) of the human platelet-activated factor acetyl hydrolase (PAF-AH) precursor (25Tjoelker L.W. Wilder C. Eberhardt C. Staffarini D.M. Dietsch G. Schimpf B. Hooper S. Le Trong H. Cousens L.S. Zimmerman G.A Yamada Y. McIntyre T.M. Prescott S.M. Gray P.W. Nature. 1995; 374: 549-553Crossref PubMed Scopus (487) Google Scholar). The N-terminal amino acid of the purified human PAF-AH is Ile-42, and the estimated size of the enzyme on SDS-PAGE is 44–45 kDa (25Tjoelker L.W. Wilder C. Eberhardt C. Staffarini D.M. Dietsch G. Schimpf B. Hooper S. Le Trong H. Cousens L.S. Zimmerman G.A Yamada Y. McIntyre T.M. Prescott S.M. Gray P.W. Nature. 1995; 374: 549-553Crossref PubMed Scopus (487) Google Scholar). Thus, the rabbit analogue of PAF-AH has 20 additional N-terminal amino acids, which could explain the observed difference in the molecular mass. The N terminus of the 63-kDa protein is 93% identical with both human and mouse vanin 1 (26Granjeaud S. Naquet P. Galland F. Immunogenetics. 1999; 49: 964-972Crossref PubMed Scopus (37) Google Scholar). Both PAF-AH and vanin proteins areN-glycosylated (25Tjoelker L.W. Wilder C. Eberhardt C. Staffarini D.M. Dietsch G. Schimpf B. Hooper S. Le Trong H. Cousens L.S. Zimmerman G.A Yamada Y. McIntyre T.M. Prescott S.M. Gray P.W. Nature. 1995; 374: 549-553Crossref PubMed Scopus (487) Google Scholar, 26Granjeaud S. Naquet P. Galland F. Immunogenetics. 1999; 49: 964-972Crossref PubMed Scopus (37) Google Scholar) and were retained by the concanavalin A column we used as a final step in the purification of PON3. The lactonase activity did not bind to the column, and the washout fractions showed a single band on SDS-PAGE with trace contamination of albumin and concanavalin A fragments (Fig.1 C, lanes 3–5).Table IPurification of rabbit serum PON3VolumeProteinActivitySpecific activityYieldFold purificationmlmgnmol/minnmol/min/mg%nSerum1557,0002,2940.331001Blue-agarose721088357.837241st DEAE401032632.614992nd DEAE217.132145.814139Sephacryl 20060.7592125.84382Concanavalin A60.2431130.01.4395Results are given for a typical purification run. Enzyme activity with lovastatin was measured as described under “Experimental Procedures”; yield = (activity of the fractions combined for the next step)/(total activity in serum) × 100 and does not include all of the activity actually recovered. Open table in a new tab Table IIN-terminal sequence analysis of the proteins present in the Sephacryl gel filtration column poolMolecular massSequenceProteinkDa40AKL LL LTLLG AS LAFVGE RLLAFRNPON350LDWQDVNPVAHIKSSPAF-AH63XDTFIAAVYEHAVILPVanin-1The 40-kDa protein has 3 more amino acids at the beginning and an arginine instead of a valine (italicized) in comparison with the rabbit MsPON (15Ozols J. Biochem. J. 1999; 338: 265-272Crossref PubMed Google Scholar). The identical amino acids with the deduced sequence of the human PON3 (1Primo-Parmo S.L. Sorenson R.C. Teiber J. La Du B.N. Genomics. 1996; 33: 498-507Crossref PubMed Scopus (589) Google Scholar, 12La Du B.N. Aviram M. Billecke S. Navab M. Primo-Parmo S. Sorenson R.C. Standiford T.J. Chem. Biol. Interact. 1999; 119–120: 379-388Crossref PubMed Scopus (178) Google Scholar) are in bold, and those identical with the rabbit serum PON1 (24Hassett C. Richter R. Humbert R. Chapline C. Crabb J.W. Omiecinski C.J. Furlong C.E. Biochemistry. 1991; 30: 10141-10149Crossref PubMed Scopus (213) Google Scholar) are underlined. The bold letters in the 50- and 63-kDa proteins represent identical amino acids in human PAF-AH precursor (25Tjoelker L.W. Wilder C. Eberhardt C. Staffarini D.M. Dietsch G. Schimpf B. Hooper S. Le Trong H. Cousens L.S. Zimmerman G.A Yamada Y. McIntyre T.M. Prescott S.M. Gray P.W. Nature. 1995; 374: 549-553Crossref PubMed Scopus (487) Google Scholar) and vanin 1 (26Granjeaud S. Naquet P. Galland F. Immunogenetics. 1999; 49: 964-972Crossref PubMed Scopus (37) Google Scholar), respectively; X, undetermined amino acid. Open table in a new tab Results are given for a typical purification run. Enzyme activity with lovastatin was measured as described under “Experimental Procedures”; yield = (activity of the fractions combined for the next step)/(total activity in serum) × 100 and does not include all of the activity actually recovered. The 40-kDa protein has 3 more amino acids at the beginning and an arginine instead of a valine (italicized) in comparison with the rabbit MsPON (15Ozols J. Biochem. J. 1999; 338: 265-272Crossref PubMed Google Scholar). The identical amino acids with the deduced sequence of the human PON3 (1Primo-Parmo S.L. Sorenson R.C. Teiber J. La Du B.N. Genomics. 1996; 33: 498-507Crossref PubMed Scopus (589) Google Scholar, 12La Du B.N. Aviram M. Billecke S. Navab M. Primo-Parmo S. Sorenson R.C. Standiford T.J. Chem. Biol. Interact. 1999; 119–120: 379-388Crossref PubMed Scopus (178) Google Scholar) are in bold, and those identical with the rabbit serum PON1 (24Hassett C. Richter R. Humbert R. Chapline C. Crabb J.W. Omiecinski C.J. Furlong C.E. Biochemistry. 1991; 30: 10141-10149Crossref PubMed Scopus (213) Google Scholar) are underlined. The bold letters in the 50- and 63-kDa proteins represent identical amino acids in human PAF-AH precursor (25Tjoelker L.W. Wilder C. Eberhardt C. Staffarini D.M. Dietsch G. Schimpf B. Hooper S. Le Trong H. Cousens L.S. Zimmerman G.A Yamada Y. McIntyre T.M. Prescott S.M. Gray P.W. Nature. 1995; 374: 549-553Crossref PubMed Scopus (487) Google Scholar) and vanin 1 (26Granjeaud S. Naquet P. Galland F. Immunogenetics. 1999; 49: 964-972Crossref PubMed Scopus (37) Google Scholar), respectively; X, undetermined amino acid. The isolation of PON3 from its natural environment and the use of detergents, which was unavoidable because of the tight association of PON3 with other proteins, led to a decrease in its specific activity. Phospholipids, which were shown to stimulate the hydrolytic activity of pu