Photodependent inhibition of rat liver NAD(P)H:quinone acceptor oxidoreductase by (A)-2-azido-NAD+ and (A)-8-azido-NAD.
Two photoaffinity analogues of NAD+, (A)-2-azido-NAD+ [nicotinamide 2-azidoadenine dinucleotide] and (A)-8-azido-NAD+ [nicotinamide 8-azidoadenine dinucleotide], have been synthesized, and their reactivities with the rat liver NAD(P)H:quinone acceptor oxidoreductase have been investigated. The reduce nicotinamide nucleotide probes, (A)-2-azido-NADH and (A)-8-azido-NADH, were shown to be substrates of the quinone reductase. This enzyme was inhibited by (A)-8-azido-NADH, were shown to be substrates of the quinone reductase. This enzyme was inhibited by (A)-2-azido-NAD+ and (A)-8-azido-NAD+ in a photodependent manner, and the inhibition of the enzyme could be prevented by the presence of nicotinamide nucleotide substrates during photolysis. (A)-2-Azido-NAD+ was demonstrated to be a more potent inhibitor than (A)-8-azido-NAD+. In addition, the photodependent inhibition by (A)-8-azido-NAD+ increased when menadione, the substrate of the enzyme, was present during the photolysis, while menadione protected the enzyme from the photodependent inhibition by (A)-2-azido-NAD+. These results indicate that these two NAD+ analogues can be used to identify the nicotinamide nucleotide binding site of this quinone reductase and that they probably bind to the enzyme in different fashions.
Deng
P S
PS
Division of Immunology, Beckman Research Institute of the City of Hope, Duarte, California 91010.
Zhao
S H
SH
Iyanagi
T
T
Chen
S A
SA
D000345:Affinity Labels; D000818:Animals; D001386:Azides / Q000737:chemistry / Q000494:pharmacology*; D001665:Binding Sites; D004579:Electron Transport; D005260:Female; D008099:Liver / Q000187:drug effects / Q000201:enzymology*; D009243:NAD / Q000737:chemistry / Q000494:pharmacology* / Q000031:analogs & derivatives; D016660:NAD(P)H Dehydrogenase (Quinone); D010782:Photolysis; D011808:Quinone Reductases / Q000037:antagonists & inhibitors*; D051381:Rats; D011919:Rats, Inbred Strains; D013379:Substrate Specificity
D000345:Affinity Labels; D001386:Azides; D009243:NAD; C062879:nicotinamide 2-azidoadenine dinucleotide; C069931:nicotinamide 8-azidoadenine dinucleotide; D016660:NAD(P)H Dehydrogenase (Quinone); D011808:Quinone Reductases