Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
10796142 | Biochimica et Biophysica Acta (BBA) - Bioenergetics | 2005 | 12 Pages |
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.
Keywords
PBSCNOXtNOXMPMsWST-1pCMBSgp96BN-PAGEpreimmunePMSFHBSSNBTNADH oxidasePVDFFBSp-chloromercuriphenylsulfonic acidKCNSDS-PAGESDS polyacrylamide gel electrophoresisblue native polyacrylamide gel electrophoresisnitro blue tetrazoliumRoom temperaturepolyvinylidene difluoridefoetal bovine serumPlasma membranePhosphate buffered salinephenylmethylsulfonyl fluoridepotassium cyanideCapsaicin
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Authors
Debbie-Jane G. Scarlett, Patries M. Herst, Michael V. Berridge,