Beneficial effect of oleoylated lipids on paraoxonase 1: protection against oxidative inactivation and stabilization

Abstract

The effect of lipids on PON1 (paraoxonase 1), one of antioxidant proteins in high-density lipoprotein, was investigated in respect to inhibition, protection against oxidative inactivation, and stabilization. When the effect of lipids on the PON1 activity was examined, a remarkable inhibition was expressed by polyenoic fatty acids (C18:2-C20:5). Linoleic acid, the most potent ( K(i), 3.8 microM), showed competitive inhibition. Next, various lipids were examined for prevention against the inactivation of PON1 by ascorbate/Cu2+, which caused a remarkable (>or =90%) inactivation of PON1. Compared with saturated fatty acids (C6-C18), exhibiting a modest protection (9-40%), monoenoic acids (C16:1-C20:1) showed a greater maximal protective effect (Emax, 70-82%), with oleic acid being the most effective (EC50, 2.7 microM). In contrast, polyenoic acids showed no protection. Noteworthy, linoleic acid prohibited the protective action of oleic acid non-competitively. In the structure-activity relationship, a negatively charged group seems to be required for the protective action. Consistent with this, dioleoylphosphatidylglycerol, negatively charged, was more protective than dioleoylphosphatidylcholine. These data, together with requirement of Ca2+ (EC50, 0.6 microM) for the protective action, may support the existence of a specific site responsible for the protective action. A similar protective action of lipids was also observed in the inactivation of PON1 by ascorbate/Fe2+, peroxides or p -hydroxymercuribenzoate. Separately, PON1 was stabilized by oleic acid or oleoylated phospholipids, in combination with Ca2+, but not linoleic acid. These results suggest that in contrast to an adverse action of linoleic acid, monoenoic acids or their phospholipid derivatives play a beneficial role in protecting PON1 from oxidative inactivation as well as in stabilizing PON1.

Preferential inhibition of paraoxonase activity of human paraoxonase 1 by negatively charged lipids

“…PON1 was purified from human plasma by chromatographic procedures according to a slight modification of the published procedures (5,34,39). The purified PON1 was kept in 25 mM Tris buffer containing 1 mM Ca 2 ϩ at 4 Њ C. The purified enzyme possessed a specific activity of ‫ف‬ 1,018 and 0.552 mol/min/mg protein in the hydrolysis of phenyl acetate and paraoxon, respectively.…”

“…Arylesterase activity of PON1 was measured by adding enzyme solution to 0.5 ml of 50 mM Tris buffer, pH 7.4, containing 1 mM CaCl 2 and 10 mM phenyl acetate, and the rate of generation of phenol was monitored at 270 nm as described before (5,34). For the inhibition study, the concentration of phenyl acetate was adjusted to 1 mM unless otherwise described.…”

“…PON1 (5 arylesterase units/ml) was preincubated with ascorbate (0.5 mM) in the presence of 1 M Cu 2 ϩ or 2 M Fe 2 ϩ in 0.1 ml of 10 mM PBS buffer (pH 7.4) containing 1 mM Ca 2 ϩ at 38 Њ C as described (34), and then the aliquot (20 l) was subjected to arylesterase or paraoxonase activities. Separately, PON1 was preincubated with H 2 O 2 or NaOCl in 0.1 ml of 10 mM PBS buffer (pH 7.4) containing 1 mM Ca 2 ϩ at 38 Њ C.…”

“…Inhibition of HDL-PON1 or plasma-PON1 by oleic acid, lysoPG, or their combination HDL-PON1 (1 arylesterase unit/ml), prepared as previously described (34), was incubated with paraoxon or phenyl acetate in the presence of oleic acid, monooleoyl-lysoPG (1 or 3 M), or their combination in 0.5 ml of 50 mM Tris buffer (pH 7.4) containing 1 mM Ca 2 ϩ . Separately, plasma (10 or 25 l) was incubated with paraoxon or phenyl acetate in the presence of lipid inhibitor as described above.…”

“…Previously, some lipids such as phosphatidylcholines were reported to stimulate PON1 arylesterase activity (33). Our recent studies showed that the arylesterase activity was inhibited competitively by polyunsaturated fatty acids at low concentrations (32,34). Thus, the effect of lipids on PON1 arylesterase activity seems to differ according to the type of lipid.…”