Phospholipase action of platelet-activating factor acetylhydrolase, but not paraoxonase-1, on long fatty acyl chain phospholipid hydroperoxides.
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.
Kriska
Tamas
T
Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA.
Marathe
Gopal K
GK
Schmidt
Jacob C
JC
McIntyre
Thomas M
TM
Girotti
Albert W
AW
D043203:1-Alkyl-2-acetylglycerophosphocholine Esterase / Q000502:physiology*; D000818:Animals; D043303:Aryldialkylphosphatase / Q000378:metabolism*; D002384:Catalysis; D004305:Dose-Response Relationship, Drug; D004950:Esterases / Q000737:chemistry; D005227:Fatty Acids / Q000737:chemistry; D006801:Humans; D006861:Hydrogen Peroxide / Q000737:chemistry; D006868:Hydrolysis; D007700:Kinetics; D010743:Phospholipids / Q000737:chemistry*; D011485:Protein Binding; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry
D005227:Fatty Acids; D010743:Phospholipids; D011994:Recombinant Proteins; D006861:Hydrogen Peroxide; D004950:Esterases; D043203:1-Alkyl-2-acetylglycerophosphocholine Esterase; D043303:Aryldialkylphosphatase