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 surface 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 oxidase was released from the surface of HeLa S cells and purified. The activity of the released capsaicin-inhibited oxidase was resistant to heating at 50 degrees C and to protease digestion. After heating and proteinase 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 specific activity indicative of >500-fold purification relative to the plasma membrane. The final activity correlated with a ca. 34-kDa band on 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 which occasionally migrated as a ca. 52-kDa complex. The purified complex tended to multimerize and formed insoluble 10- to 20-nm-diameter amyloid rods. The components of the purified 34-kDa complex were blocked to N-terminal amino acid sequencing and were resistant to further protease digestion. After multimerization into amyloid rods, the protein remained resistant to proteases even under denaturing conditions and to cyanogen bromide either with or without prior alkylation.
del Castillo-Olivares
A
A
Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, Indiana, 47907, USA.
Yantiri
F
F
Chueh
P J
PJ
Wang
S
S
Sweeting
M
M
Sedlak
D
D
Morré
D M
DM
Burgess
J
J
Morré
D J
DJ
D000143:Acids; D000596:Amino Acids / Q000032:analysis; D000682:Amyloid / Q000378:metabolism; D002211:Capsaicin / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D003488:Cyanogen Bromide; D004591:Electrophoresis, Polyacrylamide Gel; D019286:Endopeptidase K / Q000378:metabolism; D010450:Endopeptidases / Q000378:metabolism*; D005260:Female; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000737:chemistry; D006367:HeLa Cells / Q000201:enzymology*; D006358:Hot Temperature; D006801:Humans; D006868:Hydrolysis; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D009247:NADH, NADPH Oxidoreductases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D010446:Peptide Fragments / Q000302:isolation & purification
D000143:Acids; D000596:Amino Acids; D000682:Amyloid; D008565:Membrane Proteins; D009097:Multienzyme Complexes; D010446:Peptide Fragments; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; C023107:NADH oxidase; D009247:NADH, NADPH Oxidoreductases; D010450:Endopeptidases; D019286:Endopeptidase K; D003488:Cyanogen Bromide; D002211:Capsaicin