Reduced Triphosphopyridine Nucleotide Oxidase-catalyzed Alterations of Membrane Phospholipids

Evidence is given for the generation of a component having the properties of a free radical during the oxidation of TPNH by liver microsomes. The component is apparently responsible for the peroxidative chain scission of polyunsaturated fatty acids in the microsomal membrane which occurs during TPNH-dependent electron transport. The component is also capable of causing rapid lysis of erythrocytes when the latter are added to the reaction system. The hemolysis is prevented (a) by inhibitors of TPNH oxidation, (b) by prior heat denaturation of the microsomes, (c) by including free radical trapping agents in the incubation system, and (d) by elevating the level of α-tocopherol intake of the erythrocyte donors for several days. Hemolysis, when in progress, stops abruptly whenever the enzyme activity ceases, demonstrating that the component has a very short half-life. The hemolysis was shown not to be caused by the microsomal phospholipids which had undergone peroxidative cleavage. The hemolysis occurred even under conditions in which there was no alteration of microsomal phospholipids at all. The results indicate that the mechanism of electron transport from TPNH by microsomes involves free radical intermediates. The course of production of the radical-like component can be followed by observing its lytic action on erythrocytes.