Hydrolase Activity Measurement.

OM-hydrolyzing activity was determined as follows. Animal and human plasma for the species difference examination and each eluent fraction obtained from successive column chromatography for purification of our target hydrolase were appropriately diluted (final dilutions, 50–400-fold and 10–100-fold, respectively) with 10 mM potassium phosphate buffer. The diluted protein solutions were incubated at 37°C for 5 to 10 min with OM as a substrate (final solvent concentration, 2% acetonitrile). After reaction termination through addition of ice-cold acetonitrile, the concentration of the active metabolite olmesartan was determined with a high-performance liquid chromatography system (SLC-10A system; Shimadzu, Kyoto, Japan). Olmesartan was separated from the OM peak with a reverse-phase C18 column (YMC-Pack ODS-A A-312, C18, 5 μm, 6.0 i.d. × 150 mm; YMC, Kyoto, Japan) and a mobile phase of 40% acetonitrile containing 2% paired-ion chromatography reagent A (Waters, Milford, MA), at a flow rate of 1.0 ml/min, and was detected with UV detection at 254 nm. The lower limit of quantitation was set at 0.2 μM. In the other in vitro experiments, the OM-hydrolyzing activities of diluted human plasma (final dilution, 500-fold), recombinant PON1 proteins (final protein concentration, 0.01 mg/ml), and purified human serum albumin (final protein concentration, 5 mg/ml) were measured at 37°C with OM as a substrate, in 100 mM Tris-HCl buffer (pH 7.5) containing 1 mM CaCl₂ (final solvent concentration, 2.5% acetonitrile). After incubation at 37°C for a designated reaction time, the reaction was terminated through addition of ice-cold acetonitrile containing RNH-6272, as an internal standard for determination of the metabolite concentration, and 0.25% trifluoroacetic acid, for prevention of the nonenzymatic degradation of OM. After filtration and addition of 50% methanol containing 1% formic acid, the concentration of the active metabolite olmesartan was determined with a liquid chromatography-tandem mass spectrometry system consisting of a Prominence LC-20A system (Shimadzu) and an API 3200 mass spectrometer (AB Sciex, Foster City, CA). Olmesartan was separated with a reverse-phase C18 column (Atlantis T3, 5 μm, 2.1 mm i.d. × 150 mm; Waters) and a mobile phase of 64% methanol containing 0.2% formic acid, at a flow rate of 0.2 ml/min, and was determined through monitoring of the ion transition of m/z 447 to m/z 207 with multiple-reaction monitoring in the positive electrospray ionization mode. The lower limit of quantitation was set at 20 nM. The enzymatic activity was expressed as a metabolite formation rate (in nanomoles per minute per milligram of protein) on the basis of the production of olmesartan for the reaction by the recombinant protein, which was subtracted from that in the buffer control (nonenzymatic hydrolysis).

Phenyl acetate-hydrolyzing activity was determined as follows. Over the process of protein purification from human serum, 20 to 40 μl of each fraction was incubated at 37°C for 10 to 20 min with 0.2 ml of 10 mM phenyl acetate solution in 50 mM Tris-HCl buffer (pH 8). After reaction termination through addition of 1 ml of 0.04% 4-aminoantipyrine in acetonitrile and then color development with addition of 2 ml of 0.08% potassium ferricyanide with 50 mM Tris-HCl buffer (pH 8) in acetonitrile, the absorbance at 510 nm was measured. After column purification, the activity of the purified protein was measured as reported previously (Gan et al., 1991), with slight modification. Twenty microliters of the purified esterase solution were incubated at room temperature with 1.5 ml of 1 mM phenyl acetate solution in 50 mM Tris-HCl buffer (pH 8) containing 1 mM CaCl₂. Formation of the metabolite phenol was determined through monitoring of absorbance changes at 270 nm. The activity was expressed with units of change in absorbance at 270 nm per minute per milligram of protein.

Paraoxon-hydrolyzing activity was measured as reported previously (Gan et al., 1991), with slight modification. Twenty microliters of the appropriately diluted purified protein solution were incubated at room temperature with 0.8 ml of paraoxon solution (1 mM) in 50 mM Tris-HCl buffer (pH 8) containing 1 mM CaCl₂ and 1 M NaCl. Formation of the metabolite p-nitrophenol was determined through monitoring of absorbance changes at 412 nm. The activity was expressed with units of change in absorbance at 412 nm per minute per milligram of protein.

Benzoyl choline-hydrolyzing activity was measured as follows. One hundred twenty microliters of the appropriately diluted purified protein solution were incubated at 37°C for 10 min with benzoyl choline (final concentration, 1 mM). After reaction termination through addition of ice-cold acetonitrile, the concentration of the metabolite benzoic acid was determined with a high-performance liquid chromatography system (SLC-10A system; Shimadzu). The metabolite was separated from the benzoyl choline peak with a reverse-phase C18 column (YMC-Pack ODS-A A-312, C18, 5 μm, 6.0 i.d. × 150 mm; YMC) and a mobile phase of 55% acetonitrile containing phosphoric acid for pH adjustment (pH 3.0), at a flow rate of 1.0 ml/min. UV detection was performed at 225 nm. The activity was expressed with units of nanomoles per minute per milligram of protein.

Purification of OM Hydrolase from Human Plasma.

Because there were findings of species differences in plasma OM-hydrolyzing activities (Fig. 2) and chemical inhibition properties were reported previously (Ishizuka et al., 2010) that resembled the enzymatic characteristics of human PON1, all column purification steps were performed according to the plasma PON1 purification method reported previously (Gan et al., 1991), with slight modification. The peak fraction was determined on the basis of phenylacetate-hydrolyzing activity with absorbance at 510 nm, because OM-hydrolyzing activity and phenylacetate-hydrolyzing activity were confirmed to behave quite similarly in the preliminary column chromatography. Over the purification process, each fraction was analyzed with SDS-PAGE performed according to the method described by Laemmli (1970), with 8% or 10% SDS-polyacrylamide gels (Bio-Rad Laboratories, Hercules, CA). Human plasma was added to 2 volumes of column buffer A (50 mM Tris-HCl buffer, pH 8, containing 1 mM CaCl₂, 5 μM EDTA, and 5% glycerol) containing 3 M NaCl. The mixture was allowed to stand for ∼1 h. After centrifugation, the supernatant was loaded onto a Blue Sepharose column (5 × 7 cm; GE Healthcare Japan, Tokyo, Japan). The column was pre-equilibrated with column buffer A containing 3 M NaCl, washed with column buffer A containing 3 M NaCl and then with the same buffer without NaCl, and then eluted with column buffer A containing 0.1% sodium deoxycholate. The active fractions were dialyzed against column buffer A, concentrated, and loaded onto an ion-exchange column (DEAE-Sephacel column, 2.5 × 8 cm; GE Healthcare Japan), which had been pre-equilibrated with 25 mM Tris-HCl buffer (pH 8) containing 1 mM CaCl₂, 5 μM EDTA, and 5% glycerol (referred to as column buffer B), was washed with column buffer B containing 1% Emulgen 911 (Kao Corp., Tokyo, Japan) and 5% dimethylacetamide and then buffer containing 0.1% Emulgen, and then was eluted with a linear gradient of 0 to 350 mM NaCl in column buffer B. The active fractions were again loaded onto a DEAE-Sephacel column, which was washed with column buffer B containing 1% Emulgen and 5% dimethylacetamide, buffer containing 0.1% Emulgen, and buffer containing 100 mM NaCl and then was eluted with a linear gradient of 100 to 350 mM NaCl in column buffer B. The resultant active fractions were dialyzed against column buffer B containing 0.1% Emulgen, concentrated, and stored frozen at −80°C until use as the final purified esterase, which was subsequently determined to be human PON1. A portion of the active fraction from each column purification step was loaded onto a SDS-polyacrylamide gel (8% gel; Bio-Rad Laboratories) according to the method described by Laemmli (1970), and the gel was stained with Coomassie Brilliant Blue. The protein concentration was determined by using a detergent-compatible protein assay (Bio-Rad Laboratories), with bovine serum albumin as the standard.

• Download figure
• Open in new tab
• Download powerpoint

Fig. 2.

Cross-species differences in the activity of OM hydrolysis in human and animal plasma. Data represent the results of single determinations with pooled plasma from three individuals.

N-Terminal Amino Acid Sequencing.

The N-terminal sequence of the target protein was determined from the purified protein transferred electrophoretically onto a PVDF membrane (Immun-Blot PVDF membrane; Bio-Rad Laboratories) after SDS-PAGE. Amino acids were sequenced through automated Edman degradation by using a gas-phase protein sequencer (model PPSQ-10; Shimadzu), according to the manufacturer's procedure.

Immunoblotting and Immunoprecipitation Analysis.

Specific antiserum against the purified protein, which was later identified as human PON1, was raised in female Japanese white rabbits. The animals received three boosts with equal volumes of the purified protein as an antigen in complete Freund's adjuvant, with 2-week intervals. The antiserum was obtained from the animals, and the IgG was purified from the antiserum by using 50% saturated ammonium sulfate precipitation, followed by DEAE-Sephacel column chromatography. The IgG was further purified with an Econo-Pac serum IgG purification column kit (Bio-Rad Laboratories), to remove components with OM-hydrolyzing activity.

The plasma proteins in each purification step that were separated with SDS-PAGE and then blotted on a PVDF membrane were detected with the purified anti-PON1 IgG described above, followed by enhanced chemiluminescence, horseradish peroxidase-linked, donkey anti-rabbit IgG (GE Healthcare, Little Chalfont, UK), as primary and secondary antibodies, respectively. These immunoblots were observed through chemiluminescence with an enhanced chemiluminescence detecting reagent (GE Healthcare).

Inhibitory effects of the purified IgG against PON1 and albumin on the OM-hydrolyzing activity in human plasma were investigated to estimate the contribution of each protein. Diluted human plasma at an appropriate dilution ratio was incubated overnight at 4°C with the respective IgG fractions at various IgG fraction/plasma volume ratios. After separation of the antigen-antibody complex through centrifugation, the supernatant was used as an enzyme source for OM-hydrolyzing activity measurements.

Expression of Human PON1 in Mammalian Cell Line.

The open reading frame of the full-length human PON1 (amino acids 1–355) and that of the Q192R mutant were subcloned into a vector plasmid (pcDNA6-myc-His; Invitrogen, Carlsbad, CA) providing a C-terminal myc-polyhistidine-epitope tag, confirmed with DNA sequencing, and expressed in mammalian FreeStyle 293-F cells (Invitrogen). The transfected cells were cultured for 7 days in FreeStyle 293 expression medium (Invitrogen), and the conditioned media from the cells overexpressing human PON1_192QQ and PON1_192RR were filtered with polyethersulfone membrane filters (0.45 μm; Thermo Fisher Scientific, Waltham, MA). After dialysis against 20 mM Tris-HCl buffer (pH 7.5), the overexpressed histidine-tagged proteins were purified with a two-step purification process involving anion chromatography with a HiTrap Q-XL column (GE Healthcare) and Ni-affinity chromatography with a HisTrap FF column (GE Healthcare). The eluates were collected and desalted with PD-10 columns (GE Healthcare), and then the resultant proteins were stored frozen at −80°C until use. The protein concentration was determined by using a micro-bicinchoninic acid Pierce protein assay (Thermo Fisher Scientific), with bovine serum albumin as the standard.

Kinetic Analysis.

The enzyme kinetics for OM hydrolysis by human plasma, recombinant PON1 proteins, and purified human serum albumin were evaluated with substrate concentrations ranging from 3.125 to 400 μM. For reactions with purified serum albumin, 100 mM potassium phosphate buffer (KPB) (pH 7.4) was used instead of 100 mM Tris-HCl buffer (pH 7.5) containing 1 mM CaCl₂. Kinetic parameters, namely, the Michaelis constant (K_m) and the maximal velocity (V_max), were estimated from the data on substrate concentrations ([S]) and initial velocities (v) with WinNonlin Professional (version 5.2.1; Pharsight, Mountain View, CA), by using nonlinear least-squares regression analysis fitted to the Michaelis-Menten equation, v = V_max × [S]/(K_m + [S]).

Distribution of OM-Hydrolyzing Activity in Serum Lipoprotein Fractions.

The lipoprotein fractions, namely, very-low-density lipoprotein (VLDL) including chylomicron, low-density lipoprotein (LDL), and HDL, were separated from human serum through sequential ultracentrifugation in continuous-density gradients (Havel et al., 1955) (CS150GXL ultracentrifuge with S120AT2 rotor; Hitachi Koki, Tokyo, Japan), desalted (PD-10 desalting columns; GE Healthcare), and concentrated through centrifugation (Amicon Ultra filters; molecular weight cutoff, 10,000; Millipore Corporation, Billerica, MA). The lipoprotein-deficient serum (LPDS) fraction obtained after the lipoprotein separation, which was thought to include serum albumin, was also desalted with PD-10 columns and was used for activity measurements. The OM-hydrolyzing activity was measured at a substrate concentration of 10 μM, as described above. Incubations with the LPDS fraction in 100 mM KPB (pH 7.4) also were performed, because purified serum albumin showed higher OM-hydrolyzing activity in KPB than in Tris-HCl buffer containing CaCl₂.

Other Methods.

Protein concentrations were determined by using the Bradford (Bradford, 1976) protein assay (Bio-Rad Laboratories), with bovine serum albumin as the standard, unless indicated otherwise.