Multiple proteins with single activities or a single protein with multiple activities: the conundrum of cell surface NADH oxidoreductases.
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.
Scarlett
Debbie-Jane G
DJ
Malaghan Institute of Medical Research, PO Box 7060, Wellington, New Zealand.
Herst
Patries M
PM
Berridge
Michael V
MV
D000951:Antigens, Neoplasm / Q000378:metabolism; D002462:Cell Membrane / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D018841:HSP90 Heat-Shock Proteins / Q000037:antagonists & inhibitors; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007783:Lactones / Q000494:pharmacology; D018942:Macrolides; D008773:Methylphenazonium Methosulfate / Q000031:analogs & derivatives* / Q000378:metabolism; D009243:NAD / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D012516:Osteosarcoma; D013778:Tetrazolium Salts / Q000378:metabolism; D014407:Tumor Cells, Cultured
C110656:2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium; D000951:Antigens, Neoplasm; D018841:HSP90 Heat-Shock Proteins; D007783:Lactones; D018942:Macrolides; D013778:Tetrazolium Salts; C052646:sarcoma glycoprotein gp96 rejection antigens; D009243:NAD; D008773:Methylphenazonium Methosulfate; C023530:1-methoxy-5-methylphenazinium methyl sulfate; C427863:ENOX1 protein, human; D009247:NADH, NADPH Oxidoreductases; C431909:tumor-associated NADH oxidase; C035359:monorden