Antitumor sulfonylurea-inhibited NADH oxidase of cultured HeLa cells shed into media

Conditioned culture media of HeLa S cells contain a soluble NADH oxidase activity inhibited by the antitumor sulfonylurea, N-(4-methylphenylsulfonyl)-N'-(4-chlorophenyl)urea (LY181984) similar to that associated with the outer surface of the plasma membrane. This activity was absent from media in which cells had not been grown and was present in conditioned culture media from which cells had been removed by centrifugation both for serum-containing and serum-free media. The K-m with respect to NADH and response to thiol reagents were similar to those of the corresponding activity of the plasma membrane of HeLa cells, The conditioned HeLa culture media bound [H-3]LY181984 with high affinity. Both antitumor sulfonylurea-inhibited and -resistant forms of the NADH oxidase were isolated by free-flow electrophoresis. The antitumor sulfonylurea-inhibited activity was purified to apparent homogeneity and was identified with a 33.5 kDa protein with an isoelectric point of about pH 4.5. The 33.5 kDa protein from conditioned HeLa culture medium both bound [H-3]LY181984 and retained an LY181984-inhibited NADH oxidase activity. A polyclonal antisera was raised in rabbits to the purified 33.5 kDa constituent from conditioned HeLa culture medium, The antisera blocked the activity of the LY181984-inhibited NADH oxidase activity, immunoprecipitated the activity and reacted with a 33.5 kDa protein on Western blots while preimmune sera did not. Also inhibited and immunoprecipitated was NADH oxidase activity from HeLa plasma membranes. The findings are consistent with the 33.5 kDa drug-inhibited NADH oxidase activity of the culture media being a shed form of the corresponding native 34 kDa antitumor sulfonylurea-inhibited NADH oxidase activity of the HeLa cell plasma membrane.