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New Artificial Electron Donors for in Vitro Assay of Nitrate Reductase Isolated from Cultured Tobacco Cells and Other Organisms

Laboratoire Structure et Métabolisme des Plantes, associé au C.N.R.S. (U.A. No. 1128), Université Paris XI, Bâtiment 430, 91405 Orsay Cédex, France, Laboratoire de Physiologie Végétale Moléculaire, associé au C.N.R.S. (U.A. No. 1128), Université Paris XI, Bâtiment 430, 91405 Orsay Cédex, France

The capacity of bromphenol blue and its analogs to act as electron donors for measurement of in vitro nitrate reductase activity from tobacco cells (Nicotiana tabacum var Techné SP 25 strain) was determined. Competitive inhibition was demonstrated to occur between NADH, the natural electron donor, and bromphenol blue, the artificial electron donor, suggesting that both donors bind to a similar active site on the enzyme. NADH-dependent or bromphenol blue-dependent nitrate reductase activity was carried out by a similar molecular weight protein exhibiting similar antigenic sites. Following ammonium sulfate precipitation, sucrose density gradient and two chromatographic steps, nitrate reductase activity from tobacco cells was purified near homogeneity using bromphenol blue as an electron donor in the absence of measurable NADH-dependent activity. The enzyme is composed of two identical subunits of 83 kilodaltons < < 94 kilodaltons.