Platelet-activating factor formation during oxidative modification of low-density lipoprotein when PAF-acetylhydrolase has been inactivated.
A PAF aggregating activity corresponding to 427 +/- 91, 668 +/- 111 and 1319 +/- 217 pg/mg protein was detected when LDL was preincubated at pH 3.5 or with 4 mM PMSF or both for 30 min (treatments that inactivate PAF-AH) and then oxidized with 20 microM Cu2+ at 37 degrees C for 24 h. This molecule was characterized as PAF by its chromatographic behavior on TLC and other established methods and was further characterized as 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine (C16: PAF) by its retention time on reverse phase HPLC and by fast atom bombardment-mass spectroscopy. Native LDL incubated under non oxidizing conditions, even when PAF-AH has been inactivated, or oxidized in the absence of PAF-AH inactivating agents or after pretreatment with 0.5 mM pBPB, does not produce detectable amounts of PAF. The kinetics of PAF formation in relation to PAF-AH activity, show that the apparent rate of PAF formation as well as its total amount depends on both the existence of oxidative conditions and the remaining PAF-AH activity the first hours following the onset of oxidation. Peroxidation of the phosphatidylcholine (PC) content of native LDL produces PAF-like aggregating activity much lower than that produced when intact LDL is oxidized and is not inhibited by BN 52021 as effectively as PAF produced by LDL peroxidation. Our results provide evidence that C16: PAF is formed during LDL peroxidation when PAF-AH has been inactivated and it does not result as a product of peroxidation of the LDL-PC content.
Liapikos
T A
TA
Department of Chemistry, University of Ioannina, Greece.
Antonopoulou
S
S
Karabina
S P
SP
Tsoukatos
D C
DC
Demopoulos
C A
CA
Tselepis
A D
AD
D043203:1-Alkyl-2-acetylglycerophosphocholine Esterase; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D015227:Lipid Peroxidation; D008077:Lipoproteins, LDL / Q000302:isolation & purification / Q000378:metabolism*; D013058:Mass Spectrometry; D010084:Oxidation-Reduction; D010741:Phospholipases A / Q000037:antagonists & inhibitors*; D010972:Platelet Activating Factor / Q000032:analysis* / Q000737:chemistry; D014105:Tosyl Compounds / Q000494:pharmacology
D008077:Lipoproteins, LDL; D010972:Platelet Activating Factor; D014105:Tosyl Compounds; C003614:4-toluenesulfonyl fluoride; D010741:Phospholipases A; D043203:1-Alkyl-2-acetylglycerophosphocholine Esterase