When cytotoxic macrophagea contact neoplaatic cells, defects in target cell mitochondrial electron transport occur and these defects are temporally related to cessation of cancer cell division. NADH dehydrogenase and succinate dehydrogenase are metalloflavin enzyme complexes which are inhibited during this effector-target cell interaction. During the past year we have attempted to define the basis for the inhibition of these enzyme complexes by asking specific questions about their physical properties. Are the polypeptides present in macrophage-injured target cell mitochondria? If so, are the prosthetic group flavins and iron-sulfur clusters present? To answer these questions a micromethed was developed to isolate small quantities (50-100 micrograms mitochondrial protein) of mitochondria from relatively few L1210 leukemia cells cultured in vitro. Ten to 20 micrograms of detergent-treated mitochondria are subjected to isoelectric focusing on pH 3.0-8.0 gradient ampholines agarose-detergent gels. Their iron and flavin content is analyzed per microgram mitochondrial protein in 1 mm gel slices using radioisotope labeled target cells. The presence of enzymatically inactive polypeptides is determined using Western blot analysis with monospecific antisera to the NADH dehydrogenase and succinate dehydrogenase polypeptides. These methods are currently being used to study mitochondria isolated from cytostatic L1210 leukemia cells previously injured by macrophages. The findings should provide new insight into a mechanism of macrophage cytotoxicity. (MB)
