Multiple proteins with single activities or a single protein with multiple activities: The conundrum of cell surface NADH oxidoreductases |
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Authors: | Debbie-Jane G Scarlett Michael V Berridge |
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Institution: | a Malaghan Institute of Medical Research, PO Box 7060, Wellington, New Zealand b Department of Radiation Therapy, Wellington School of Medicine and Health Sciences, University of Otago, PO Box 7343, Wellington South, New Zealand |
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Abstract: | Reduction of the cell-impermeable tetrazolium salt WST-1 has been used to characterise two plasma membrane NADH oxidoreductase activities in human cells. The trans activity, measured with WST-1 and the intermediate electron acceptor mPMS, utilises reducing equivalents from intracellular sources, while the surface activity, measured with WST-1 and extracellular NADH, is independent of intracellular metabolism. Whether these two activities involve distinct proteins or are inherent to a single protein is unclear. In this work, we have attempted to address this question by examining the relationship between the trans and surface WST-1-reducing activities and a third well-characterised family of cell surface oxidases, the ECTO-NOX proteins. Using blue native-polyacrylamide gel electrophoresis, we have identified a complex in the plasma membranes of human 143B osteosarcoma cells responsible for the NADH-dependent reduction of WST-1. The dye-reducing activity of the 300 kDa complex was attributed to a 70 kDa NADH oxidoreductase activity that cross-reacted with antisera against the ECTO-NOX protein CNOX. Differences in enzyme activities and inhibitor profiles between the WST-1-reducing NADH oxidoreductase enzyme in the presence of NADH or mPMS and the ECTO-NOX family are reconciled in terms of the different purification methods and assay systems used to study these proteins. |
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Keywords: | BN-PAGE blue native polyacrylamide gel electrophoresis capsaicin 8-methyl-N-vanillyl-trans-6-nonenamide CNOX constitutive NADH oxidase ECTO-NOX external NADH oxidase EP pH 5 eluted cell surface proteins FBS foetal bovine serum HBSS Hanks' Balanced Saline Solution KCN potassium cyanide mPMS 1-methoxy-5-methylphenazinium methylsulfate NBT nitro blue tetrazolium PBS phosphate buffered saline pCMBS p-chloromercuriphenylsulfonic acid PI preimmune PM plasma membrane PMOX plasma membrane oxidoreductase PMSF phenylmethylsulfonyl fluoride PVDF polyvinylidene difluoride RT room temperature SDS-PAGE SDS polyacrylamide gel electrophoresis tNOX tumour NADH oxidase tPMET trans-plasma membrane electron transport WST-1 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2 4-disulfophenyl)-2H-tetrazolium monosodium salt |
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