Histochemical staining and quantification of dihydrolipoamide dehydrogenase diaphorase activity using blue native PAGE.
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.
Yan
Liang-Jun
LJ
Department of Pharmacology and Neuroscience, University of North Texas Health Science Center at Fort Worth, TX 76107, USA. lyan@hsc.unt.edu
Yang
Shao-Hua
SH
Shu
Hongjun
H
Prokai
Laszlo
L
Forster
Michael J
MJ
D000818:Animals; D002853:Chromatography, Liquid; D008058:Dihydrolipoamide Dehydrogenase / Q000032:analysis / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D005033:Ethylmaleimide / Q000378:metabolism; D024101:Mitochondrial Proteins / Q000032:analysis / Q000378:metabolism*; D009243:NAD / Q000378:metabolism; D009580:Nitroblue Tetrazolium / Q000737:chemistry; D010084:Oxidation-Reduction; D051381:Rats; D017207:Rats, Sprague-Dawley; D021241:Spectrometry, Mass, Electrospray Ionization; D013194:Staining and Labeling; D053719:Tandem Mass Spectrometry; D008063:Thioctic Acid / Q000031:analogs & derivatives / Q000378:metabolism
D024101:Mitochondrial Proteins; D009243:NAD; D009580:Nitroblue Tetrazolium; C007409:dihydrolipoamide; D008063:Thioctic Acid; D008058:Dihydrolipoamide Dehydrogenase; D005033:Ethylmaleimide