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Paraoxon-hydrolyzing activity of HDL (HDL-PON1) is known to lose its activity under oxidative stress condition. Here, we attempted to elucidate the possible causes for the oxidative inactivation of HDL-PON1 in vivo system. Of various oxidative systems, ascorbate/Cu2+ system was the most potent in inactivating the paraoxon-hydrolyzing activity of purified PON1 (PON1). The inclusion of Cu2+ (0.5–2.0 μM) remarkably enhanced the ascorbate (0.5 mM)-induced inactivation of purified PON1. A similar inactivation was also obtained with HDL-PON1, although to a less extent. The inactivation of PON1, either purified or HDL-bound, by ascorbate/Cu2+ was prevented by catalase or thiols, but not general hydroxyl radical scavengers, suggesting the involvement of Cu2+-catalyzed oxidation in PON1 inactivation. In addition, some lipids such as oleic acid or dioleoylphophatidylglycerol expressed a partial protection. Noteworthy, HDL-PON1, but not purified PON1, was inactivated significantly in a concentration-dependent manner by Cu2+ alone, and the inactivation of HDL-PON1 by Cu2+ was prevented by catalase, consistent with the intermediacy of H2O2 in Cu2+-induced inactivation of HDL-PON1. Separately, PON1, either purified or HDL-bound, was found to be susceptible to hypochlorite oxidation. While the susceptibility to hypochlorite below 1 mM was similar between purified PON1 and HDL-PON1, the inactivation of HDL-PON1 by hypochlorite at >1 mM seemed to be interfered by the membrane. Moreover, the sequential inclusion of hypochlorite and ascorbate/Cu2+ showed a cooperative action in inactivating HDL-PON1. Based on these results, it is proposed that copper ion-catalyzed oxidation and hypochlorite oxidation may be mainly responsible for the loss of HDL-associated paraoxonase1 activity. [Copyright &y& Elsevier]

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.