Isolation and characterization of a tumor-associated NADH oxidase (tNOX) from the HeLa cell surface.
Cell-surface-located, drug-responsive and tumor-associated NADH oxidase (tNOX) proteins were purified and characterized from HeLa cells. The proteins isolated exhibited NADH oxidase activity inhibited by capsaicin and were resistant to heating and to protease digestion. The activity was purified 200- to 500-fold to provide apparently homogeneous gel bands for N-terminal sequencing using three different protocols. All three protocols involved heat (50 degrees C) and proteinase K treatment. Recovery of the total NADH oxidase activity was 86% and inhibition by capsaicin was 60 to 80%. After 450-fold purification, a 52-kDa component was obtained as a single gel band that retained the capsaicin-inhibited NADH oxidase activity. Amino acid composition and partial amino acid sequences were obtained. The partial amino acid sequences were used to generate peptide antisera. Both the peptide antisera and polyclonal antisera to the 52-kDa component immunoprecipitated capsaicin-inhibited NADH oxidase activity and reacted with 52-, 34-, and 17-kDa components on Western blots from different steps of the purification. The tNOX protein exhibited immunological cross-reactivity and amino acid sequence identity with tNOX cloned from a HeLa cDNA library using a monoclonal antibody to tNOX from sera of cancer patients. The results provide a direct sequence link between tNOX of the HeLa cell surface and the cloned tNOX representative of patient sera. The tNOX form from the surface of HeLa cells yielded N-terminal sequence consistent with a coidentity of the cell surface and serum forms of the two activities.
Yantiri
F
F
Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, 1333 Hansen Life Sciences Research Building, West Lafayette, Indiana 47907-1333, USA.
Morré
D J
DJ
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis*; D000906:Antibodies / Q000276:immunology; D002211:Capsaicin / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006367:HeLa Cells / Q000201:enzymology*; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D020539:Sequence Analysis, Protein
D000596:Amino Acids; D000906:Antibodies; D004791:Enzyme Inhibitors; D009097:Multienzyme Complexes; C023107:NADH oxidase; D009247:NADH, NADPH Oxidoreductases; C431909:tumor-associated NADH oxidase; D002211:Capsaicin