Phospholipase Action of Platelet-activating Factor Acetylhydrolase, but Not Paraoxonase-1, on Long Fatty Acyl Chain Phospholipid Hydroperoxides

Tamas Kriska,Gopal K Marathe,Jacob C Schmidt,Thomas M Mcintyre,Albert W Girotti

doi:10.1074/jbc.m608135200

Tamas Kriska, Gopal K Marathe + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m608135200

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Abstract

Phospholipid hydroperoxide (PLOOH) degrading activity of high density lipoprotein (HDL)-derived paraoxonase-1 (PON1) was investigated, using peroxidized 1-palmitoyl-2-oleoyl phosphatidylcholine (PCOOH) as substrate and high performance thin layer chromatography for quantitative peroxide analysis. Incubation of PCOOH with PON1 resulted in decay of the latter and reciprocal buildup of oleic acid hydroperoxide (OAOOH) at rates unaffected by GSH or other reductants. A serine esterase inhibitor blocked this activity and a recombinant PON1 was devoid of it, raising the possibility that the activity represents platelet-activating factor acetylhydrolase (PAF-AH), an esterase that co-purifies with PON1 from HDL. This was verified by showing that a recombinant PAF-AH recapitulates the ability of natural PON1 to hydrolyze PCOOH and release OAOOH while having essentially no effect on parental PC. Furthermore, recombinant PAF-AH and natural PON1 were shown to have similar K(m) values for PCOOH hydrolysis. Finally, we found that recombinant PAF-AH, but not PON1, catalyzes PLOOH hydrolysis in peroxidized low density lipoprotein. We conclude from this study that PON1 is neither a PLOOH peroxidase nor hydrolase and that the phospholipase A(2)-like activity previously attributed to PON1 in natural enzyme preparations was actually due to novel PLOOH hydrolytic activity of contaminating PAF-AH.

Highlights

Like PON1, platelet-activating factor acetylhydrolase (PAF-AH) is a 43-kDa high density lipoprotein (HDL)-associated protein, but unlike PON1, it can be found in low density lipoprotein (LDL) as well as HDL [10]
Recombinant PAF-AH readily hydrolyzed PCOOH, releasing stoichiometric amounts of oleic acid hydroperoxide (OAOOH) in the process. These and related other findings suggest that the PLOOH hydrolyzing/detoxifying role often attributed to PON1 on the basis of in vitro studies with enzyme isolated from HDL [6] reflects a new activity of PAF-AH, which co-purifies with PON1 [5]
There was no obvious loss of CEOOH during incubation with PAF-AH, suggesting that the lipolysis reaction was specific for oxidized LDL PLOOHs and emphasizing the lack of peroxidatic activity

Summary

PAF Acetylhydrolase as a PLOOH Phospholipase

Using 1-palmitoyl-2hydroperoxyoleoyl-sn-glycero-3-phosphocholine (PCOOH) as a substrate, we have found that recombinant PON1 exhibits no significant peroxidatic or hydrolytic activity. Recombinant PAF-AH readily hydrolyzed PCOOH, releasing stoichiometric amounts of oleic acid hydroperoxide (OAOOH) in the process. These and related other findings suggest that the PLOOH hydrolyzing/detoxifying role often attributed to PON1 on the basis of in vitro studies with enzyme isolated from HDL [6] reflects a new activity of PAF-AH, which co-purifies with PON1 [5]

EXPERIMENTAL PROCEDURES

RESULTS

Phenyl acetatea

DISCUSSION