Purification and Properties of NAD(P)H:(quinone‐acceptor) Oxidoreductase of Sugarbeet Cells

Abstract

NAD(P)H:(quinone‐acceptor) oxidoreductase [NAD(P)H‐QR], a plant, cytosolic protein, was purified from cultured sugarbeet cells by a combination of ammonium sulfate fractionation, FPLC Superdex 200 gel filtration, Q‐Sepharose anion‐exchange chromatography, and a final Blue Sepharose CL‐6B affinity chromatography with an NADPH gradient. The subunit molecular mass is 24 kDa and the active protein (94 kDa) is a tetramer. The isoelectric point is 4.9. The enzyme was characterized by ping‐pong kinetics and extremely elevated catalytic capacity. It prefers NADPH over NADH as electron donor (Kcat/Km ratios of 1.7×108 M−1 s−1 and 8.3×107M−1 s−1 for NADPH and NADH, respectively, with benzoquinone as electron acceptor). The acridone derivative 7‐iodo‐acridone‐4‐carboxylic acid is an efficient inhibitor (I0.5= 5×10−5M), dicumarol is weakly inhibitory. The best acceptor substrates are hydrophilic, short‐chain quinones such as ubiquinone‐0 (Q‐0), benzoquinone and menadione, followed by duroquinone and ferricyanide, whereas hydrophobic quinones, cytochrome c and oxygen are reduced at negligible rates at best. Quinone acceptors are reduced by a two‐electron reaction with no apparent release of free semiquinonic intermediates. This and the above properties suggest some relationship of NAD(P)H‐QR to DT‐diaphorase, an animal flavoprotein which, however, has distinct structural properties and is strongly inhibited by dicumarol. It is proposed that NAD(P)H‐QR by scavenging unreduced quinones and making them prone to conjugation may act in plant tissues as a functional equivalent of DT‐diaphorase. Copyright © 1995, Wiley Blackwell. All rights reserved