Document Detail


Purification and characterization of the nuclear cytidine 5'-monophosphate N-acetylneuraminic acid synthetase from rat liver.
MedLine Citation:
PMID:  1577759     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
N-Acetylneuraminic acid cytidylyltransferase (EC 2.7.7.43) (CAMP-NeuAc synthetase) from rat liver catalyzes the formation of cytidine monophosphate N-acetylneuraminic acid from CTP and NeuAc. We have purified this enzyme to apparent homogeneity (241-fold) using gel filtration on Sephacryl S-200 and two types of affinity chromatographies (Reactive Brown-10 Agarose and Blue Sepharose CL-6B columns). The pure enzyme, whose amino acid composition and NH2-terminal amino acid sequence are also established, migrates as a single protein band on non-denaturing polyacrylamide gel electrophoresis. The molecular mass of the native enzyme, estimated by gel filtration, was 116 +/- 2 kDa whereas its Mr in sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 58 +/- 1 kDa. CMP-NeuAc synthetase requires Mg2+ for catalysis although this ion can be replaced by Mn2+, Ca2+, or Co2+. The optimal pH was 8.0 in the presence of 10 mM Mg2+ and 5 mM dithiothreitol. The apparent Km for CTP and NeuAc are 1.5 and 1.3 mM, respectively. The enzyme also converts N-glycolylneuraminic acid to its corresponding CMP-sialic acid (Km, 2.6 mM), whereas CMP-NeuAc, high CTP concentrations, and other nucleotides (CDP, CMP, ATP, UTP, GTP, and TTP) inhibited the enzyme to different extents.
Authors:
L B Rodríguez-Aparicio; J M Luengo; C González-Clemente; A Reglero
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  The Journal of biological chemistry     Volume:  267     ISSN:  0021-9258     ISO Abbreviation:  J. Biol. Chem.     Publication Date:  1992 May 
Date Detail:
Created Date:  1992-06-05     Completed Date:  1992-06-05     Revised Date:  2006-11-15    
Medline Journal Info:
Nlm Unique ID:  2985121R     Medline TA:  J Biol Chem     Country:  UNITED STATES    
Other Details:
Languages:  eng     Pagination:  9257-63     Citation Subset:  IM    
Affiliation:
Departamento de Bioquímica y Biología Molecular, Universidad de León, Spain.
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MeSH Terms
Descriptor/Qualifier:
Amino Acid Sequence
Amino Acids / analysis
Animals
Cell Nucleus / enzymology*
Chromatography, Gel
Enzyme Stability
Kinetics
Liver / enzymology*,  ultrastructure
Male
Molecular Sequence Data
N-Acylneuraminate Cytidylyltransferase / isolation & purification,  metabolism*
Rats
Rats, Inbred Strains
Substrate Specificity
Chemical
Reg. No./Substance:
0/Amino Acids; EC 2.7.7.43/N-Acylneuraminate Cytidylyltransferase

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