Document Detail

Direct photoaffinity labeling of rat liver carbamoyl phosphate synthetase I with ATP.
MedLine Citation:
PMID:  8215439     Owner:  NLM     Status:  MEDLINE    
The adenine subsites of the ATP sites of rat liver carbamoyl phosphate synthetase I have been localized by direct photoaffinity labeling with ATP. The synthetase is known to utilize two molecules of ATP, apparently in mechanistically discrete steps and at separate ATP sites. UV irradiation of the synthetase in the presence of [alpha-32P]ATP resulted in the incorporation of label. Peptide analysis of the ATP-photolabeled synthetase demonstrated that the labeling was extremely selective. To localize the sites of ATP photoincorporation to discrete regions of the synthetase which appear to be structural domains, the enzyme was photolabeled with [alpha-32P]ATP and subjected to limited proteolytic digestion. Consideration of these data indicated that the internal domains B and C were preferentially labeled and that there was lesser, but significant, labeling of the N-terminal domain A. Omission of the required allosteric activator N-acetylglutamate from the photolabeling mixture resulted in an approximately 60% decrease in label incorporation and an accompanying decrease in the extent of label incorporation in domain B. Consideration of these N-acetylglutamate effects, together with previous findings on the effects of the allosteric activator, confirmed the following functional identification of the ATP sites: domain B participates in binding the molecule of ATP involved in bicarbonate activation, whereas domain C participates in binding the molecule of ATP involved in carbamate phosphorylation.
M D Potter; S G Powers-Lee
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Publication Detail:
Type:  Journal Article; Research Support, U.S. Gov't, P.H.S.    
Journal Detail:
Title:  Archives of biochemistry and biophysics     Volume:  306     ISSN:  0003-9861     ISO Abbreviation:  Arch. Biochem. Biophys.     Publication Date:  1993 Nov 
Date Detail:
Created Date:  1993-11-19     Completed Date:  1993-11-19     Revised Date:  2007-11-14    
Medline Journal Info:
Nlm Unique ID:  0372430     Medline TA:  Arch Biochem Biophys     Country:  UNITED STATES    
Other Details:
Languages:  eng     Pagination:  377-82     Citation Subset:  IM    
Department of Biology, Northeastern University, Boston, Massachusetts 02115.
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MeSH Terms
Adenosine Triphosphate / metabolism*
Affinity Labels / metabolism
Carbamoyl-Phosphate Synthase (Ammonia) / metabolism*,  radiation effects
Electrophoresis, Polyacrylamide Gel
Liver / enzymology
Macromolecular Substances
Peptide Fragments / isolation & purification
Phosphorus Radioisotopes
Protein Binding
Ultraviolet Rays
Grant Support
Reg. No./Substance:
0/Affinity Labels; 0/Macromolecular Substances; 0/Peptide Fragments; 0/Phosphorus Radioisotopes; 56-65-5/Adenosine Triphosphate; EC Synthase (Ammonia)

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