Paraoxonase Active Site Required for Protection Against LDL Oxidation Involves Its Free Sulfhydryl Group and Is Different From That Required for Its Arylesterase/Paraoxonase Activities
Selective Action of Human Paraoxonase Allozymes Q and R
Abstract—Human serum paraoxonase (PON 1) exists in 2 major polymorphic forms (Q and R), which differ in the amino acid at position 191 (glutamine and arginine, respectively). These PON allozymes hydrolyze organophosphates and aromatic esters, and both also protect LDL from copper ion–induced oxidation. We have compared purified serum PONs of both forms and evaluated their effects on LDL oxidation, in respect to their arylesterase/paraoxonase activities. Copper ion–induced LDL oxidation, measured by the production of peroxides and aldehydes after 4 hours of incubation, were reduced up to 61% and 58%, respectively, by PON Q, but only up to 46% and 38%, respectively, by an equivalent concentration of PON R. These phenomena were PON-concentration dependent. Recombinant PON Q and PON R demonstrated similar patterns to that shown for the purified serum allozymes. PON Q and PON R differences in protection of LDL against oxidation were further evaluated in the presence of glutathione peroxidase (GPx). GPx (0.1 U/mL) alone reduced copper ion–induced LDL oxidation by 20% after 4 hours of incubation. The addition of PON R to the above system resulted in an additive inhibitory effect on LDL oxidation, whereas PON Q had no such additive effect. The 2 PON allozymes also differed by their ability to inhibit initiation, as well as propagation, of LDL oxidation. PON Q was more efficient in blocking LDL oxidation if added when oxidation was initiated, whereas PON R was more potent when added 1 hour after the initiation of LDL oxidation. These data suggest that the 2 allozymes act on different substrates. Both PON allozymes were also able to reduce the oxidation of phospholipids and cholesteryl ester. PON Q arylesterase activity was reduced after 4 hours of LDL oxidation by only 28%, whereas the arylesterase activity of PON R was reduced by up to 55%. Inactivation of the calcium-dependent PON arylesterase activity by using the metal chelator EDTA, or by calcium ion removal on a Chelex column, did not alter PON’s ability to inhibit LDL oxidation. However, blockage of the PON free sulfhydryl group at position 283 with p-hydroxymercuribenzoate inhibited both its arylesterase activity and its protection of LDL from oxidation. Recombinant PON mutants in which the PON free sulfhydryl group was replaced by either alanine or serine were no longer able to protect against LDL oxidation, even though they retained paraoxonase and arylesterase activities. Overall, these studies demonstrate that PON’s arylesterase/paraoxonase activities and the protection against LDL oxidation do not involve the active site on the enzyme in exactly the same way, and PON’s ability to protect LDL from oxidation requires the cysteine residue at position 283.
- Received January 28, 1998.
- Accepted April 15, 1998.
Although the natural substrates for serum PON are unknown, recent studies suggest that PON prevents LDL oxidation by hydrolyzing lipid peroxides in the lipoprotein.6 7 8 9 10 11 12 The inverse relationship between serum PON activity and the risk for atherosclerotic diseases13 14 15 16 17 18 suggests that PON hydrolytic activity on oxidized LDL may be related to its antiatherogenicity.
The gene for human serum PON shows 2 common polymorphisms: Q or R at position 191 (glutamine or arginine, respectively) and M or L at position 54 (methionine or leucine, respectively).19 20 21 22 PON Q and PON R qualitatively differ in their abilities to hydrolyze various organophosphates.2 5 23 It has been suggested that the Q allele, which is more abundant than the R allele, is responsible for the protective effect of PON against atherosclerosis, whereas the R allele has been reported to be related to the risk for coronary heart disease.24 25 Recently, Mackness et al26 reported in a group of normolipidemic subjects that the protective effect of HDL from individuals with PON RR genotype against LDL oxidation was lower than that of HDL from subjects with the PON QQ genotype.
In the present study, we have compared the effects of purified human serum PON and recombinant PON Q and PON R on LDL oxidation and analyzed the active site requirements for PON arylesterase/paraoxonase activities and for PON’s abilities to protect against LDL oxidation. Unexpectedly, we found that the protective role of PON against LDL oxidation is not identical to that for its hydrolytic activity with aromatic esters or organophosphates.
Serum PON Purification
PONs were purified from the sera of healthy humans previously identified as being homozygous for PON Q or PON R. Phenotyping was done as previously described.3 PONs were purified using blue agarose and DEAE chromatography as described elsewhere,21 27 28 with some modifications. Briefly, to purify serum PON, 1 mol/L CaCl2 (50 mL) was added to 1 L of serum and centrifuged at 8000g for 30 minutes at 4°C to remove the fibrin clot. The supernatant was mixed with blue agarose (Cibacron Blue 3 GA, Sigma Chemical Co) in a solution containing 3 mol/L NaCl, 50 mmol/L Tris/HCl (pH 8.0) with 1 mmol/L CaCl2 and 5 μmol/L EDTA.21 27 28 PON was eluted with 0.1% deoxycholate. The eluted PON was further purified by DEAE BioGel anion exchange chromatography using a linear NaCl gradient as previously described,27 28 except that the nonionic detergent tergitol (NP-10, Sigma) replaced Emulgen 911. To remove residual contamination of albumin, LCAT, and PAF-AH, a concanavalin A column was used, together with the detergent, with a 0 to 0.15 mol/L linear gradient of methyl-α-d-mannopyranoside. Concanavalin A protein fragments were removed by a Centricon 100 microconcentrator (Amicon). The purity of the enzyme was verified by SDS-polyacrylamide gel electrophoresis.27 28 PON Q and PON R hydrolytic activities include both arylesterase (with phenyl acetate) and paraoxonase(with paraoxon). PON arylesterase activity is substantially higher than its paraoxonase activity (micromoles versus nanomoles of substrate hydrolyzed per milliliter per minute, respectively).
Arylesterase Activity Measurements
Arylesterase activity was determined by using phenyl acetate as the substrate. The initial rates of hydrolysis were determined spectrophotometrically at 270 nm. The assay mixture included 1.0 mmol/L phenyl acetate and 0.9 mmol/L CaCl2 in 20 mmol/L Tris HCl, pH 8.0, at 25°C. Nonenzymatic hydrolysis of phenyl acetate was subtracted from the total rate of hydrolysis. The E270 for the reaction is 1310 mol · L−1 · cm−1, and 1 unit of arylesterase activity is equal to 1 μmol of phenyl acetate hydrolyzed per milliliter per minute.27
Paraoxonase Activity Measurements
Paraoxonase activity was assessed by measuring the initial rate of paraoxon hydrolysis to yield p-nitrophenol at 412 nm at 25°C. The basal assay mixture included 1.0 mmol/L paraoxon and 1.0 mmol/L CaCl2 in 50 mmol/L glycine/NaOH buffer, pH 10.5. Nonenzymatic hydrolysis of paraoxon was subtracted from the total rate of hydrolysis. The E412 for the reaction is 18 290 mol · L−1 · cm−1, and 1 unit of paraoxonase activity produced 1 nmol of p-nitrophenol per milliliter per minute.27
Inactivation of PON Arylesterase Activity
Removal of Calcium Ions by a Chelex 100 Column
One gram of Chelex 100 (200 mesh) was washed once with double-distilled water and packed into a 3.0-mL polystyrene column. The packed column was equilibrated with 50 mmol/L Tris/HCl buffer, pH 8.0. Subsequently, 1.0 mL of purified PON Q or PON R was passed through the column at a rate of 0.3 mL/min. Sequential fractions were collected and assayed for arylesterase activity.
Inhibition With EDTA
The purified human serum PON solution containing 1.0 mmol/L Ca2+ in Tris/HCl buffer, pH 8.0, was diluted with equal volume of 1 mmol/L Na2 EDTA. Arylesterase activities were essentially zero after 18 hours of incubation at room temperature.
PON Q or PON R was incubated in PBS at 60°C for 15 minutes. PON 1 was not precipitated by this treatment.
Blockage of Free Sulfhydryl Groups
p-Hydroxymercuribenzoate (PHMB) or iodoacetate (1 to 10 mmol/L) was incubated with PON Q or PON R for 1 hour at 37°C in PBS. Excess sulfhydryl agent was removed before incubation, by dialysis, using a Centricon 100 microconcentrator (Amicon).
Human serum LDL was obtained from PerImmune, Inc. Briefly, serum LDL was isolated from fasted normolipidemic human volunteers. Lipoproteins were prepared by discontinuous density gradient ultracentrifugation.29 The LDL was washed at d=1.063 g/mL and dialyzed overnight against 150 mmol/L NaCl (pH 7.4) at 4°C. The lipoproteins were then sterilized by filtration (0.45 μm), stored at a concentration of 5 mg of protein per milliliter under nitrogen in the dark at 4°C, and used within 2 weeks. The lipoproteins were found to be free of lipopolysaccharide contamination, as analyzed by the Limulus amebocyte lysate assay (Associated of Cape Cod Inc). Before LDL oxidation, the lipoprotein was dialyzed against PBS, EDTA-free solution, pH 7.4, under nitrogen at 4°C. Then the lipoproteins (100 μg of protein per milliliter) were incubated with 10 μmol/L CuSO4 in the air, in the absence or presence of the indicated concentrations of PON allozymes (Q or R) for up to 4 hours at 37°C. The kinetics of lipoprotein oxidation (conjugated diene formation) was analyzed by monitoring the absorbance change at 234 nm.30 The extent of LDL oxidation was measured directly in the medium by the thiobarbituric acid–reactive substances (TBARS) assay at 532 nm, using malondialdehyde (MDA) for the standard curve.31 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.32
Palmitoyl arachidonyl phosphatidylcholine (PAPC) or cholesteryl arachidonate (Sigma Co) were first completely dried from their chloroform solvent using a heating block in air. The lipids were then suspended in PBS to a concentration of 1 mg/mL and sonicated in an ultrasonic processor (3×30 seconds). Then, CuSO4 (10 μmol/L) was added to the lipid solution and incubation was carried out at 37°C for 3 hours. At the various time points, lipid peroxides and associated TBARS were assayed.31 32
Site-Directed Mutagenesis, Transfection, and Expression of Recombinant Enzymes
The procedures for the production of recombinant PONs (ie, production of wild types of PON Q and PON R, as well as mutants with alanine or serine in place of cysteine-283 of PON Q) has been described elsewhere in detail.33 As another control, we also used cells transfected with the pGS expression vector alone, without any PON cDNA insert. LDL oxidation assays using recombinant PONs were carried in 1 mL Ultra Culture (BioWhittaker) medium containing equal activities (0.2 arylesterase units per milliliter) of the various PON preparations.
We have used 40 arylesterase units per milliliter of purified serum enzyme in order to have physiological serum levels. However, it is typical for the recombinant enzyme to have very low activity, which makes it not possible to get similar concentrations as for the serum enzyme. Nevertheless, the recombinant wild-type enzymes were still potent protectors against LDL oxidation.
Selective Reduction in LDL Oxidation by PON Q Versus PON R
LDL oxidation was achieved by lipoprotein incubation with copper ions (10 μmol/L CuSO4) in the absence or presence of purified serum PON Q or PON R (40 arylesterase units per milliliter) for 4 hours at 37°C. PON Q or PON R significantly reduced LDL oxidation, as assessed by the formation of lipid peroxides (61% and 46% reduction, respectively, Figure 1A⇓) and TBARS (58% and 38% reduction, respectively, Figure 1B⇓). A similar pattern of inhibition was obtained by kinetic monitoring of conjugated diene formation at 234 nm (data not shown).
The PON Q allozyme’s greater ability than the PON R allozyme’s in protecting LDL from oxidation was apparent at several enzyme concentrations/activities (Figure 2⇓).
Similar patterns to those shown for serum purified PONs were obtained with recombinant PON Q or PON R (0.3 arylesterase units per milliliter). Copper ion–induced LDL (100 μg of protein per milliliter) oxidation produced 388±15 nmol of peroxides per milligram LDL protein (n=3) in the presence of control medium. Recombinant PON Q caused a 33% reduction (259±12, n=3), whereas recombinant PON R caused only 20% reduction (311±14, n=3) in LDL oxidation.
Next we analyzed the differences between PON Q and PON R in regards to their inhibitory effects on LDL oxidation, under various experimental conditions. Glutathione peroxidase (GPx, 0.1 U/mL) was able to inhibit LDL oxidation by 20%, as measured by the formation of peroxides after 4 hours of incubation with copper ions (10 μmol/L CuSO4) at 37°C (Figure 3A⇓). When LDL oxidation was carried out in the presence of GPx and serum purified PON Q or PON R (40 arylesterase units per milliliter), the protective effect of PON R on LDL oxidation was further increased by an additional 37% (Figure 3A⇓), whereas PON Q did not show any additive effect with GPx (Figure 3A⇓). Assessment of LDL oxidation by the TBARS assay (Figure 3B⇓) under these conditions showed essentially identical results to those obtained by measurement of peroxides (Figure 3A⇓).
To determine whether the PON allozymes showed divergence in their mechanism of inhibiting LDL oxidation, experiments were carried out under conditions in which the allozymic forms were added either at the initiation of oxidation or later on, during the propagation phase of LDL oxidation. When added at the initiation of LDL oxidation, both allozymic forms of PON protected LDL from oxidation; however, PON Q was markedly more protective than PON R, as determined by analysis of lipid peroxides (Figure 4A⇓) and TBARS (Figure 4B⇓) after 4 hours of oxidation. In contrast, when the allozymes were added 1.5 hours after the initiation of copper ion–induced LDL oxidation, PON R was more protective than PON Q (Figure 4A⇓ versus 4B). If added still later on (more than 3 hours), after extensive oxidation of LDL had already taken place, both PON Q and PON R were ineffective (data not shown).
Because the major sources of oxidized polyunsaturated fatty acids in LDL are lipoprotein cholesteryl ester and phospholipid moieties, we also studied PON’s effect on the peroxidation of these components. PAPC (1 mg/mL) was incubated with PON Q or with PON R for 3 hours at 37°C in the presence of copper ions (10 μmol/L CuSO4). A dose-dependent reduction in the extent of PAPC oxidation could be shown (Figure 5⇓), as determined by the peroxides (Figure 5A⇓) and by the TBARS assays (Figure 5B⇓). Under these conditions, PON Q was only a minimally better protector against PAPC oxidation than PON R (Figure 5⇓).
Similar studies were performed with cholesteryl ester. Cholesteryl arachidonate (1 mg/mL) was incubated for 3 hours at 37°C alone, with PON Q, or with PON R (40 arylesterase units per milliliter), in the presence of 10 μmol/L CuSO4. Analysis of lipid peroxidation revealed a significant (P<0.01) reduction, from 375±35 nmol MDA equivalents per milligram cholesteryl ester in the absence of PONs to 300±19 (−20%) or 323±13 (−14%) in the presence of PON Q or PON R, respectively (n=3).
PON Arylesterase Activity During Its Protection Against LDL Oxidation
Analysis of the enzyme’s arylesterase activity as a function of the duration of the LDL oxidation process revealed that PON Q was more stable than PON R (Figure 6⇓). Whereas arylesterase activity of PON Q was reduced by only 28%, PON R lost about 55% of its activity during the 4 hours of incubation, and most of this loss occurred gradually during the first hour of LDL oxidation (Figure 6⇓). After 4 hours of LDL oxidation, PON R paraoxonase activity, like its arylesterase activity, was also reduced to a greater extent (by 51%, from 35±5 to 17±3 U/mL) than that of PON Q (by only 26%, from 15±3 to 11±3 U/mL, n=3).
Because the selective reduction in PON Q and PON R activities may be related to different sensitivity of these allozymes to peroxides produced during LDL oxidation, we compared the sensitivity of PON Q and PON R to H2O2. On incubation of the enzymes with 50 μg/mL H2O2 for 30 minutes at 37°C, PON Q arylesterase activity was not affected (values of 32±3 and 35±5 U · mL−1 · min−1 were obtained in the absence and presence of H2O2, respectively, n=3). Under similar conditions, PON R arylesterase activity was significantly reduced (P<0.01, n=3) by H2O2 treatment (from 36±5 to 24±5 U · mL−1 · min−1).
To assess whether the PON Q and PON R arylesterase activities were directly related to their relative protective potency against LDL oxidation, experiments were carried out with both PON preparations previously inactivated for their arylesterase activities. Because the arylesterase activity of PON is calcium dependent, each allozymic form was treated by either preincubation with 1 mmol/L Na2 EDTA for 30 minutes (resulting in a reduction of PON activity from 40 to 4 arylesterase units per milliliter) or removal of calcium ions with Chelex (activity was reduced to 1 arylesterase unit per milliliter). Under these conditions, paraoxonase activity was also essentially abolished. Surprisingly, inactivation of the arylesterase and paraoxonase activities did not reduce the PON’s inhibitory effect against LDL oxidation (Figure 7⇓). In contrast, complete inactivation of the enzyme arylesterase/paraoxonase activities by heating (60°C, 15 minutes) resulted in a complete loss of protective activity of PON Q or PON R against LDL oxidation (Figure 7⇓).
Possible Role of the PON’s Free Sulfhydryl Group in the Protection Against LDL Oxidation
PON contains 3 cysteines; 2 of them form an intramolecular disulfide bond, while the third, at position 283, is free.33 Cys283 was hypothesized to play a role in PON esterase activity, but earlier site-directed mutagenesis from this laboratory showed that substitution with either serine or alanine resulted in retention of PON arylesterase activity.33 In the current study, reaction of the PON Cys283 with the sulfhydryl reagent PHMB caused a dose-dependent reduction in PON arylesterase activity, by 21%, 42%, or 91% for PON Q, and by 16%, 31%, or 97% for PON R, using PHMB concentrations of 0.1, 1.0, or 10.0 mmol/L, respectively. In parallel, a PHMB dose-dependent reduction in the inhibitory effect of PON on LDL oxidation was also observed (Figure 8⇓).
Preincubation of PON (40 arylesterase units of PON Q per milliliter) with 10 mmol/L PHMB for 1 hour at 37°C also reduced the ability of PON to protect against lipid peroxidation of PAPC (1 mg/mL). Values for lipid peroxidation obtained for control PAPC, PON-treated PAPC, and PHMB-inactivated PON-treated PAPC were 255±27, 128±18, and 237±31 nmol MDA equivalents per milligram PAPC, respectively (n=3). A similar pattern was obtained for the oxidation of cholesteryl arachidonate (1 mg/mL) with 10 mmol/L PHMB-treated PONs under similar conditions as described for PAPC (data not shown).
Iodoacetate, a smaller molecule than PHMB, was also used to block the PON free sulfhydryl group. On incubation of PON Q with 0 mmol/L, 1 mmol/L, or 10 mmol/L iodoacetate, PON arylesterase activities were 36+4, 32±3, or 28±3 U/mL, respectively (n=3). Even though PON arylesterase activity was minimally affected by this iodoacetate treatment, PON’s ability to protect against LDL oxidation was significantly reduced in an iodoacetate concentration–dependent manner, as shown by analysis of LDL-associated peroxides (Figure 9A⇓) and TBARS (Figure 9B⇓).
To address the possible role of the PON Cys283 residue in the protection against LDL oxidation, we tested recombinant PON Q mutants, in which the cysteine residue (Cys283) had been replaced with either serine (Cys283Ser) or alanine (Cys283Ala).33 The media obtained from the cells that were transfected with the wild-type PON Q, the Cys283Ser, and the Cys283Ala contained 0.39±0.05, 0.20±0.05, and 0.28±0.04 arylesterase units per milliliter, respectively (n=3). As a control we have used medium obtained from the same number of cells, which were transfected with the same expression vector (pGS) with the same selective genes, and the only difference between this control and the PON recombinants was that the control lacked the PON’s cDNA. The medium (0.2 arylesterase units per milliliter) from the PON wild type reduced copper ion–induced LDL oxidation by 29%, whereas both PON mutants were unable to inhibit LDL oxidation, as shown by determination of LDL-associated peroxides (Figure 10A⇓) and TBARS (Figure 10B⇓).
The present study indicates that the PON active site requirements for its protective role against LDL oxidation are not identical to those needed for its arylesterase/paraoxonase activities. In addition, this study demonstrates that PON Q has a greater capacity to protect against copper ion–induced LDL oxidation than PON R.
We would like to suggest that PON Q and PON R may act on different substrates generated during LDL oxidation and may possess different sensitivities to the action of peroxides formed during LDL oxidation. These differences may contribute to the divergence in the possible antiatherosclerotic roles of the PON allozymes.
Inactivation of PON arylesterase activity by the addition of EDTA or the removal of calcium ions did not reduce the abilities of the PON allozymic forms to protect LDL from oxidation. These results suggest that the active site requirements for protection against LDL oxidation and for its arylesterase activity differ to some degree.
We have no evidence to suggest that PON has 2 active sites, and it might be that there is an overlapping of the sites required for arylesterase/paraoxonase activities and the protection against LDL oxidation activity. Recently, a PON-independent inhibition of LDL oxidation by HDL was suggested.34 This suggestion was based on the observation that inactivation of PON arylesterase activity (by using EDTA-containing plasma) did not compromise the ability of HDL to inhibit LDL oxidation. The present study, however, clearly shows that arylesterase activity is not a quantitative measure of PON’s ability to protect against LDL oxidation.
At one time, Cys-283 was believed to be the active center nucleophile, because organic mercurial compounds inactivated PON.33 35 We now know that this amino acid is not specifically required for PON arylesterase activity, because site-directed mutagenesis of this residue did not eliminate its arylesterase activity.33 The Cys-283 residue, however, may be located close to the active center of the enzyme and may be required for binding some substrates. The inhibitory effects of the sulfhydryl group agents PHMB and iodoacetate on the ability of PON to protect against LDL oxidation suggest that Cys-283 is essential for substrate orientation, or binding.35 36 The inability of Cys-283 PON mutants to protect LDL against oxidation further indicates the importance of this residue. PHMB inhibition of PON arylesterase activity is probably due to steric hindrance resulting from the introduction of a large substituent near a region of the molecule critical for substrate binding.33 Presumably, this is the reason that a smaller sulfhydryl binding agent, iodoacetate, has much lower inhibitory effect on PON arylesterase activity than PHMB. Interestingly, it has been recently shown37 that cigarette smoke extract can also inhibit PON paraoxonase activity by a modification of the enzyme’s free thiol group, but as this research was performed in plasma, we cannot evaluate the preservation of PON activity in this study.
PON R has about 8-fold higher paraoxonase activity than PON Q,1 21 23 but the 2 allozymes are very similar in their ability to hydrolyze phenyl acetate (ie, arylesterase activity). In contrast, PON R has been shown to be far less efficient than PON Q in the hydrolysis of the organophosphates diazoxon, sarin, and soman, which is just the opposite of the findings for the activities of the PON allozymes with paraoxon.5 Thus, it is not unexpected that the relative activities of PON allozymes for protection against LDL oxidation are not the same for the 2 allozymes.
The polymorphic evolution of PON may have increased its enzymatic permissiveness and capacity to protect against LDL oxidation. Taken together, this hypothesis and the observed increased protection of PON Q versus PON R against LDL oxidation suggest that the 2 PON allozymes differ in their affinities for, and abilities to hydrolyze, various substrates. This evolutionary speculation is supported by our finding of the divergent protective characteristics of PON Q and PON R against LDL oxidation. The differences between PON Q and PON R in the protection against LDL oxidation were demonstrated when the PON’s allozymes were added in the presence of GPx or during various time points of LDL oxidation. These differences may be related to increased sensitivity of PON R to short-lived lipid peroxides that are produced at the initiation of LDL oxidation and are also substrates to GPx.38 Indeed, in the present study, low H2O2 concentrations were shown to preferentially inactivate PON R but not PON Q arylesterase activity during a short incubation of H2O2 with the allozymes. Of interest along this line is the recent observation that human HDL from QQ/MM homozygotes was the most effective among all other PON allozyme-associated HDLs in protecting LDL from oxidation.39
Oxidative stress leads to a reduction in PON arylesterase activity, as shown in the present study for PON that was incubated with LDL and copper ions, as well as in serum from atherosclerotic apolipoprotein E–deficient mice40 or after enzyme incubation with oxidized LDL.41 Oxidized LDL inactivated PON arylesterase and paraoxonase activities, and this effect is shared by the oxidized polyunsaturated arachidonic fatty acids in phospholipids and in cholesteryl ester.41A Intervention to reduce oxidative stress, such as dietary polyphenolic flavonoids from red wine40 or licorice, or hypolipidemic therapy42 can preserve PON activities. Such intervention may enhance PON’s hydrolytic action on specific oxidized lipids and hence lead to increased PON potency against oxidized LDL and oxidized HDL43 and against lipid-peroxidized arterial wall cells.44
We conclude that the structural requirements for PON’s arylesterase and paraoxonase activities are not the same as those required for its protective effect against LDL oxidation. In addition, PON Q and PON R may show different affinities or preferences for the lipid peroxides that are produced in LDL during its oxidation and therefore may contribute in different ways, perhaps even synergistically, to reduce LDL oxidation and possibly impede atherogenesis.
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Aviram M, Rosenblat M, Bidlecke S, Erogul J, Sorenson R, Bisgaier CL, Newton RS, La Du BN. Human serum paraoxonase (PON1) inactivation by oxidized LDL: role for PON1’s free sulfhydryl group and effect of antioxidants. Free Radic Biol Med. In press.