Multiple proteins with single activities or a single protein with multiple activities: the conundrum of cell surface NADH oxidoreductases

Biochim Biophys Acta. 2005 Jun 1;1708(1):108-19. doi: 10.1016/j.bbabio.2005.03.006. Epub 2005 Apr 2.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antigens, Neoplasm / metabolism
  • Cell Membrane / enzymology*
  • Electrophoresis, Polyacrylamide Gel
  • HSP90 Heat-Shock Proteins / antagonists & inhibitors
  • Humans
  • Hydrogen-Ion Concentration
  • Lactones / pharmacology
  • Macrolides
  • Methylphenazonium Methosulfate / analogs & derivatives*
  • Methylphenazonium Methosulfate / metabolism
  • NAD / metabolism
  • NADH, NADPH Oxidoreductases / metabolism*
  • Osteosarcoma
  • Tetrazolium Salts / metabolism
  • Tumor Cells, Cultured

Substances

  • 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium
  • Antigens, Neoplasm
  • HSP90 Heat-Shock Proteins
  • Lactones
  • Macrolides
  • Tetrazolium Salts
  • sarcoma glycoprotein gp96 rejection antigens
  • NAD
  • Methylphenazonium Methosulfate
  • 1-methoxy-5-methylphenazinium methyl sulfate
  • ENOX1 protein, human
  • NADH, NADPH Oxidoreductases
  • tumor-associated NADH oxidase
  • monorden