N-arylazido-. beta. -alanyl-NAD sup + , a new NAD sup + photoaffinity analogue. Synthesis and labeling of mitochondrial NADH dehydrogenase

Journal Article · Mon Jan 29 23:00:00 EST 1990 · Biochemistry; (USA)

DOI:https://doi.org/10.1021/bi00456a036· OSTI ID:6705331

Deng, P S.K.; Chen, S ^[1]; Hatefi, Y ^[2]

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1. Beckman Research Institute of the City of Hope, Duarte, CA (USA)
2. Research Institute of Scripps Clinic, La Jolla, CA (USA)

N-Arylaziod-{beta}-alanyl-NAD{sup +}(N3{prime}-0-(3-(N-(4-azido-2-nitrophenyl)amino)propionyl)NAD{sup +}) has been prepared by alkaline phosphatase treatment of arylaziod-{beta}-alanyl-NADP{sup +} (N3{prime}-O-(3-(N-(4-azido-2-nitrophenyl)amino)propionyl)NADP{sup +}). This NAD{sup +} analogue was found to be a potent competitive inhibitor with respect to NADH for the purified bovine heart mitochondrial NADH dehydrogenase. The enzyme was irreversibly inhibited as well as covalently labeled by this analogue upon photoirradiation. A stoichiometry of 1.15 mol of N-arylazido-{beta}-alanyl-NAD{sup +} bound/mol of enzyme, at 100% inactivation, was determined from incorporation studies using tritium-labeled analogue. Among the three subunits, 0.85 mol of the analogue was bound to the M{sub r} = 51,000 subunit, and each of the two smaller subunits contained 0.15 mol of the analogue when the dehydrogenase was completely inhibited upon photolysis. Both the irreversible inactivation and the covalent incorporation could be prevented by the presence of NADH during photolysis. These results indicate that N-arylaziod-{beta}-alanyl-NAD{sup +} is an active-site-directed photoaffinity label for the mitochondrial NADH dehydrogenase, and are further evidence that the M{sub r} = 51,000 subunit contain the NADH binding site. Results are also presented to show that N-arylazido-{beta}-alanyl-NAD{sup +} binds the dehydrogenase in a more effective manner than A-arylazido-{beta}-alanyl-NAD{sup +}.

• OSTI ID: 6705331
• Journal Information: Biochemistry; (USA), Journal Name: Biochemistry; (USA) Vol. 29:4; ISSN 0006-2960; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ABSORPTION SPECTRA
ALANINES
ALKALINE PHOSPHATASE
AMINO ACIDS
AZIDO COMPOUNDS
BIOCHEMICAL REACTION KINETICS
CARBOXYLIC ACIDS
CHEMICAL REACTIONS
COENZYMES
DECOMPOSITION
DOSE-RESPONSE RELATIONSHIPS
ENZYME INHIBITORS
ENZYMES
ESTERASES
HYDROGEN COMPOUNDS
HYDROLASES
ISOTOPE APPLICATIONS
KINETICS
NAD
NUCLEOTIDE DEHYDROGENASES
NUCLEOTIDES
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
OXIDOREDUCTASES
PHOSPHATASES
PHOTOCHEMICAL REACTIONS
PHOTOLYSIS
REACTION KINETICS
SPECTRA
STOICHIOMETRY
TRACER TECHNIQUES
TRITIUM COMPOUNDS