MEASUREMENT OF DICUMAROL-SENSITIVE NADPH-(MENADIONE CYTOCHROME-C) OXIDOREDUCTASE ACTIVITY RESULTS IN AN ARTIFACTUAL ASSAY OF DT-DIAPHORASE IN CELL SONICATES
Wf. Hodnick et Ac. Sartorelli, MEASUREMENT OF DICUMAROL-SENSITIVE NADPH-(MENADIONE CYTOCHROME-C) OXIDOREDUCTASE ACTIVITY RESULTS IN AN ARTIFACTUAL ASSAY OF DT-DIAPHORASE IN CELL SONICATES, Analytical biochemistry, 252(1), 1997, pp. 165-168
Purified DT-diaphorase can be assayed as either dicumarol-inhibitable
NAD(P)H:menadione oxidoreductase or dicumarol-inhibitable NAD(P)H:dich
lorophenolindophenol reductase. Both of these methods have been utilit
zed to assay DT-diaphorase activity in tissue and cell homogenates. Wh
en DT-diaphorase activity was measured as dicumarol-inhibitable NADPH:
dichlorophenolindophenol reductase in sonicates of two cell lines prev
iously shown to not have any measurable activity of this enzyme, no en
zymatic activity was detected. However, when the water-soluble bisulfi
te addition product of menadione was used as the electron acceptor, an
artifactual activity for DT-diaphorase was detected in these cell. li
nes. When another cell line was assayed utilizing menadione bisulfite,
an apparent activity of about three times that found with dichlorophe
nolindophenol was measured, and thus, may overestimate DT-diaphorase a
ctivity in cells having activity. When menadione was used in place of
menadione bisulfite, an artifactual DT-diaphorase activity was also de
tected, but was about one-half that obtained with menadione bisulfite.
Polarographic determinations of the midpoint potentials for menadione
and menadione bisulfite indicated that the latter compound was easier
to reduce and may account for the greater apparent DT-diaphorase acti
vity measured with this compound. (C) 1997 Academic Press.