Characterization and isolation of a rat liver plasma membrane NADH oxidase: Comparison to NADH oxidases from transformed cells
Abstract
This work was to purify and characterize the hormone and growth factor-stimulated NADH oxidase from rat liver plasma membranes and compare it to the corresponding unregulated NADH oxidase of HeLa cell plasma membranes. Rat liver plasma membrane NADH oxidase activity was stimulated by GTP and the G protein mimicking peptide mastoparan. This activity was also stimulated by a protein fraction immunoprecipitated by anti-GAGES sera. Plasma membrane NADH oxidase activity of HeLa cells was not stimulated by GTP nor by the anti-GAGES immunoprecipitate. The rat liver plasma membrane NADH oxidase activity, when purified, was associated with a 36 kDa peptide. Antisera was generated and purified which inhibited rat liver plasma membrane NADH oxidase activity and specifically recognized a 36 kDa protein from rat liver. The antisera was used to purify additional NADH oxidase from rat liver plasma membranes. This protein had a pI of ca 4.5 when examined by isoelectric focusing. HeLa plasma membranes contained a N-ethylmaleimide sensitive NADH oxidase activity which was protected by LY181984. To identify the NADH oxidase activity from plasma membranes of HeLa cells, LY181984 was used to protect the HeLa plasma membranes from N-ethylmaleimide. A 34 kDa HeLa plasma membrane protein with a pI of ca 4.5 was protected from N-ethylmaleimide by LY181984. Tke NADH oxidase of HeLa plasma membrane, but not that of rat liver, responded as well to the quinone analog capsaicin. The LY181984 inhibited, capsaicin modulated NADH oxidase activity of HeLa cells appeared to be shed from the plasma membrane into the culture media. A corresponding protein, shed into the blood of cancer patients was sought. A capsaicin modulated NADH oxidase activity was purified from pooled cancer patient serum. This activity corresponded to a 34 kDa protein with a pI of ca 4.5. These findings demonstrate that the NADH oxidase activities of rat liver and HeLa plasma membranes have distinct properties. The properties of the HeLa NADH oxidase resemble those of the sera oxidase from cancer patients and suggest a specific NADH oxidase isoform associated with the tumorgenic transformation.
Degree
Ph.D.
Advisors
Morre, Purdue University.
Subject Area
Pharmacology|Cellular biology|Molecular biology|Anatomy & physiology|Animals|Biochemistry
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