Gene/Protein
Disease
Symptom
Drug
Enzyme
Compound
Pivot Concepts:
Gene/Protein
Disease
Symptom
Drug
Enzyme
Compound
Target Concepts:
Gene/Protein
Disease
Symptom
Drug
Enzyme
Compound
Query: EC:1.6.5.2 (
NQO1
)
6,196
document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)
Mammalian mitochondrial dihydrolipoamide dehydrogenase (
DLDH
, EC 1.8.1.4) catalyzes NAD(+)-dependent oxidation of dihydrolipoamide in vivo and can also act as a
diaphorase
catalyzing in vitro nicotinamide adenine dinucleotide (reduced form) (NADH)-dependent reduction of electron-accepting molecules such as ubiquinone and nitroblue tetrazolium (NBT). In this paper, we report a gel-based method for histochemical staining and quantification of
DLDH
diaphorase
activity using blue native PAGE (BN-PAGE). Rat brain mitochondrial extracts, used as the source of
DLDH
, were resolved by nongradient BN-PAGE (9%), which was followed by
diaphorase
activity staining using NADH as the electron donor and NBT as the electron acceptor. It was shown that activity staining of
DLDH
diaphorase
was both protein amount- and time-dependent. Moreover, this in-gel activity-staining method was demonstrated to be in good agreement with the conventional spectrophotometric method that measures
DLDH
dehydrogenase activity using dihydrolipoamide as the substrate. The method was applied to determine levels of
DLDH
diaphorase
activity in several rat tissues other than the brain, and the results indicated a similar level of
DLDH
diaphorase
activity for all the tissues examined. Finally, the effects of thiol-reactive reagents such as N-ethylmaleimide (NEM) and nitric oxide donors on
DLDH
diaphorase
activity were evaluated, demonstrating that, with this method,
DLDH
diaphorase
activity can be determined without having to remove these thiol-reactive reagents that may otherwise interfere with spectrophotometric measurement of
DLDH
dehydrogenase activity. The gel-based method can also be used as a means to isolate mitochondrial
DLDH
that is to be analyzed by mass spectral techniques in studying
DLDH
post-translational modifications.
...
PMID:Histochemical staining and quantification of dihydrolipoamide dehydrogenase diaphorase activity using blue native PAGE. 1731 58
