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

Abstract

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 +}.

Authors:
;  [1];  [2]
  1. Beckman Research Institute of the City of Hope, Duarte, CA (USA)
  2. Research Institute of Scripps Clinic, La Jolla, CA (USA)
Publication Date:
OSTI Identifier:
6705331
Resource Type:
Journal Article
Journal Name:
Biochemistry; (USA)
Additional Journal Information:
Journal Volume: 29:4; Journal ID: ISSN 0006-2960
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; NUCLEOTIDE DEHYDROGENASES; ABSORPTION SPECTRA; BIOCHEMICAL REACTION KINETICS; ALANINES; ALKALINE PHOSPHATASE; AZIDO COMPOUNDS; DOSE-RESPONSE RELATIONSHIPS; ENZYME INHIBITORS; NAD; PHOTOLYSIS; STOICHIOMETRY; TRACER TECHNIQUES; TRITIUM COMPOUNDS; AMINO ACIDS; CARBOXYLIC ACIDS; CHEMICAL REACTIONS; COENZYMES; DECOMPOSITION; ENZYMES; ESTERASES; HYDROGEN COMPOUNDS; HYDROLASES; ISOTOPE APPLICATIONS; KINETICS; NUCLEOTIDES; ORGANIC ACIDS; ORGANIC COMPOUNDS; ORGANIC NITROGEN COMPOUNDS; OXIDOREDUCTASES; PHOSPHATASES; PHOTOCHEMICAL REACTIONS; REACTION KINETICS; SPECTRA; 550201* - Biochemistry- Tracer Techniques

Citation Formats

Deng, P S.K., Chen, S, and Hatefi, Y. N-arylazido-. beta. -alanyl-NAD sup + , a new NAD sup + photoaffinity analogue. Synthesis and labeling of mitochondrial NADH dehydrogenase. United States: N. p., 1990. Web. doi:10.1021/bi00456a036.
Deng, P S.K., Chen, S, & Hatefi, Y. N-arylazido-. beta. -alanyl-NAD sup + , a new NAD sup + photoaffinity analogue. Synthesis and labeling of mitochondrial NADH dehydrogenase. United States. doi:10.1021/bi00456a036.
Deng, P S.K., Chen, S, and Hatefi, Y. Tue . "N-arylazido-. beta. -alanyl-NAD sup + , a new NAD sup + photoaffinity analogue. Synthesis and labeling of mitochondrial NADH dehydrogenase". United States. doi:10.1021/bi00456a036.
@article{osti_6705331,
title = {N-arylazido-. beta. -alanyl-NAD sup + , a new NAD sup + photoaffinity analogue. Synthesis and labeling of mitochondrial NADH dehydrogenase},
author = {Deng, P S.K. and Chen, S and Hatefi, Y},
abstractNote = {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 +}.},
doi = {10.1021/bi00456a036},
journal = {Biochemistry; (USA)},
issn = {0006-2960},
number = ,
volume = 29:4,
place = {United States},
year = {1990},
month = {1}
}