Isolation and characterization of a tumor-associated NADH oxidase (tNOX) from the HeLa cell surface
Abstract
The intent of this work was to purify and characterize a tumor-specific, cell-surface located and drug-responsive NADH oxidase with protein disulfide thiolinterchange activity designated as a tNOX. tNOX is reduced or absent from normal cells, but may have a normal counterpart. tNOX is present on the outer surface of the plasma membrane and its activity is susceptible to inhibition by antitumor drugs, such as sulfonylureas with antitumor activity and the vanilloid, capsaicin. In my study, I isolated and characterized a protein from the HeLa cell surface with capsaicin-inhibited NADH oxidase activity corresponding to tNOX that was resistant to heating and to proteases. Three different isolation protocols were used. All three involved heat (50°C) and proteinase K treatment. Protocol 2 was selected because recovery of the total NADH oxidase activity was 86% and 63% inhibition by 1 μM capsaicin and 83% inhibition by 100 μM capsaicin were obtained. After up to 450-fold purification, a 52 kD component as a single get band was obtained that retained the capsaicin-inhibited NADH oxidase activity. Partial amino acid sequence and amino acid composition were obtained. The unique partial amino acid sequence was used to generate peptide antisera. Both the peptide antisera and polyclonal antisera to the 52 kD component immunoprecipitated capsaicin-inhibited NADH oxidase activity and reacted with 52, 34 and 17 kD components on Western blots from different steps of the purification. The subunit composition of the 52 kD was demonstrated by 2-dimensional SDS-PAGE where only 17 kD component were obtained. The tNOX protein exhibited immunological cross-reactivity and amino acid sequence similarity with a cloned tNOX isoform from human sera designated tNOXα. The latter also yielded proteinase K-resistant cores of 17 kD that retained tNOX activity. The current interpretation of these results is that the 17 kD Mr band may be the proteinase K resistant core of tNOX and that it multimerizes to form SDS-stable dimers of Mr 34 kD, and SDS-stable trimer of the 17 kD proteinase K-resistant core represented by the 52 kD complex that forms after boiling in SDS.
Degree
Ph.D.
Advisors
Morre, Purdue University.
Subject Area
Pharmacology|Molecular biology|Oncology
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