A DRUG-RESPONSIVE AND PROTEASE-RESISTANT PERIPHERAL NADH OXIDASE COMPLEX FROM THE SURFACE OF HELA-S CELLS

Our laboratory described a ca, 34-kDa protein of the HeLa S cell surfa ce that bound an antitumor sulfonylurea N-(4-methylphenylsulfonyl)-N'- (4-chlorophenyl) urea (LY181984) with high affinity and that exhibited NADH oxidase and protein disulfide-thiol interchange activities also inhibited by LY181984. The quinone site inhibitor 8-methyl-N-vanillyl- 6-noneamide (capsaicin) also blocked these same enzymatic activities, Using capsaicin inhibition as the criterion, the drug-responsive oxida se was released from the surface of HeLa S cells and purified. The act ivity of the released capsaicin-inhibited oxidase was resistant to hea ting at 50 degrees C and to protease digestion. After heating and prot einase K digestion, the activity was isolated in >90% yield by FPLC as an apparent 50- to 60-kDa multimer, Final purification by preparative SDS-PAGE yielded a capsaicin-inhibited NADH oxidase activity of a spe cific activity indicative of >500-fold purification relative to the pl asma membrane. The final activity correlated with a cca, 34-kDa band o n SDS-PAGE. Matrix-assisted laser desorption mass spectroscopy as well as reelectrophoresis of the 34-kDa band indicated that the ca. 34-kDa material was a stable mixture of 22-, 17-, and 9.5-kDa components whi ch occasionally migrated as a ca. 52-kDa complex. The purified complex tended to multimerize and formed insoluble 10- to 20-nm-diameter amyl oid rods. The components of the purified 34-kDa complex were blocked t o N-terminal amino acid sequencing and were resistant to further prote ase digestion. After multimerization into amyloid rods, the protein re mained resistant to proteases even under denaturing conditions and to cyanogen bromide either with or without prior alkylation. (C) 1998 Aca demic Press.