The nicotinamide adenine dinucleotide-binding site of chicken liver xanthine dehydrogenase. Evidence for alteration of the redox potential of the flavin by NAD binding or modification of the NAD-binding site and isolation of a modified peptide.
Affinity labeling of the NAD-binding site of chicken liver xanthine dehydrogenase by 5'-p-fluorosulfonylbenzoyladenosine (5'-FSBA) caused spectral perturbation around 450 nm in the same way as NAD. Reductive titration with xanthine of native xanthine dehydrogenase in the presence of NAD showed that redox potentials of the FAD/FADH. and FADH./FADH2 couples were shifted positive by NAD binding to the enzyme. The redox potentials of these couples were also shifted to some extent by modification of the NAD-binding site with 5'-FSBA. These results provide further evidence that binding of NAD to chicken liver xanthine dehydrogenase modulates the reactivity of the enzyme by shifting the redox potential of FAD. Proteolytic cleavage of the [14C]-5'-FSBA-modified enzyme yielded several domain peptides, only one of which contained radioactivity. The isolated radioactive peptide was further digested with Staphylococcus aureus protease and the 14C-labeled peptide was purified by two steps of high performance liquid chromatography. The amino acid sequence of the peptide was determined, and a reactive tyrosine residue was identified.
Nishino
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T
Department of Biochemistry, Yokohama City University School of Medicine, Japan.
Nishino
T
T
D000241:Adenosine / Q000031:analogs & derivatives / Q000494:pharmacology; D000345:Affinity Labels / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D002645:Chickens; D007658:Ketone Oxidoreductases / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000378:metabolism*; D009243:NAD / Q000378:metabolism*; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000302:isolation & purification; D011485:Protein Binding; D013053:Spectrophotometry; D014968:Xanthine Dehydrogenase / Q000378:metabolism*
D000345:Affinity Labels; D010446:Peptide Fragments; D009243:NAD; C010959:5'-(4-fluorosulfonylbenzoyl)adenosine; D014968:Xanthine Dehydrogenase; D007658:Ketone Oxidoreductases; D000241:Adenosine