A SPECTROPHOTOMETRIC METHOD FOR KINETIC-STUDIES WITH QUINONE-DEPENDENT OXIDOREDUCTASES - APPLICATION TO DETECTION IN MEMBRANES OF NITRATE REDUCTASE-ACTIVITY WITH MENADIONE AND DUROQUINONE AS ELECTRON-DONORS

A simple method for estimating the activity of membrane-bound quinone- dependent oxidoreductases is described. Nitrate reductase from membran es of Escherichia coli is used as a model with menadione and duroquino ne, commercially available analogues of the physiological substrates, menaquinone and ubiquinone, as electron donors. These analogues are re duced by KBH4 (which is specific for aldehydes and ketones) using mola r ratios of [KBH4]/[menadione] = 20 and [KBH4]/[duroquinone] = 10. The appearance of the oxidized state can be monitored with a classical sp ectrophotometer and does riot require a dual wavelength or diffusing-m edium apparatus. To avoid turbidity in the cuvette, small amounts of m embrane containing overexpressed enzyme ni-e used. (C) 1998 Elsevier S cience Inc.