Dynamics of Xanthine Oxidase- and Fe3+-ADP-Dependent Lipid Peroxidation in Negatively Charged Phospholipid Vesicles

Superoxide (O−2)-dependent lipid peroxidation on addition of xanthine oxidase (XO) and Fe3+-ADP was induced in egg phosphatidylcholine (PC) liposomes containing dicetylphosphate (DCP), which are negatively charged like biological membranes, but not in uncharged egg PC liposomes. Positively charged Fe3+ ADP interacted more with negatively charged egg PC-DCP liposomes than with uncharged egg PC liposomes. The activities of Fe3+-chelates for initiating O−2-dependent lipid peroxidation were in the order Fe3+-ADP > Fe3+-citrate > Fe3+-oxalate = Fe3+-malonate > Fe3+-EDTA = 0. This order was the same as that for the reduction rates of these Fe3+-chelates to Fe2+-chelates by O−2-generated by XO. Lineweaver-Burk plots showed that the chelators inhibited XO by different mechanisms: uncompetitively by ADP and adenosine and noncompetitively by organic acid chelators (citrate and oxalate) and EDTA, These results suggest that ADP interacts with XO in a manner different from the other chelators. Lipid peroxidation by XO-xanthine and Fe3+-ADP was induced in egg PC liposomes containing a trace (0.31-0.35 mol%) of peroxidized egg PC (PC-OOH), but not in PC-OOH-free liposomes of egg PC obtained by their pretreatment with triphenylphosphine. PC-OOH incorporated into dimyristoyl phosphatidylcholine (DMPC) liposomes was degraded on addition of both XO-xanthine and Fe3+-chelate, but not of either one alone. α-Tocopherol in DMPC liposomes was oxidized on addition of XO-xanthine and Fe3+-chelates in the presence, but not in the absence of PC-OOH, Furthermore, PC-OOH was required for decrease of the ESR spectrum of the spin probe 12-(N-oxyl-4,4′-dimethyloxazolidin-2-yl)stearic acid, which labels the hydrophobic region of egg PC liposome membranes, on addition of XO-xanthine and Fe3+-chelates. These results indicate that the "induction message of lipid peroxidation," which is associated with reduction of Fe3+ ADP by O−2 and concurrent degradation of PC-OOH, must be transferred from the membrane surface to the inner hydrophobic region of the membranes.