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Journal of the Korean Chemical Society (JKCS)

ISSN 1017-2548(Print)
ISSN 2234-8530(Online)
Volume 54, Number 5
JKCSEZ 54(5)
October 20, 2010 

 
Title
Purification and Characterization of 5,10-Methenyltetrahydrofolate Synthetase from Chicken Liver

닭의 간 유래의 5,10-Methenyltetrahydrofolate Synthetase의 정제 및 특성
Author
Yong Kweon Cho*

조용권*
Keywords
5,10-Methenyltetrahydrofolate synthetase, 닭의 간, 엽산 대사, 테트라니트로메탄, 1-Ethyl-3-(3-dimethyl aminopropyl)-carbodiimide , 5,10-Methenyltetrahydrofolate synthetase, Chicken liver, Folate metabolism, Tetranitrometane, 1-Ethyl-3-(3-dimethyl aminopropyl)-carbodiim
Abstract

5,10-Methenyltetrahydrofolate synthetase from chicken liver was purified through 30-70% ammonium sulfate fractionation, Q Sepharose Fast Flow anion exchange and Source 15Phe hydrophobic interaction chromatography. Specific activities of cell extract, ammonium sulfate, Q Sepharose Fast Flow and Source 15Phe were 0.0085, 0.031, 0.80 and 1.27 U/mg, respectively. Purification fold activities of cell extract, ammonium sulfate, Q Sepharose Fast Flow and Source 15Phe were 1, 3.7, 94.1 and 149.4, respectively. HPLC gel permeation chromatography and SDS-polyacrylamide electrophoresis experiments indicated that the enzyme is a monomeric protein with a molecular weight of 22.8 kDa. Km for 5-methyl THF and Mg-ATP were 7.1 μM and 63 μM, respectively. Optimum temperature and pH were 30 oC and 6.0, respectively. The data for metal ion specificity and stoichiometry showed that the maximum activity was obtained with a 1:l. ratio of Mg2+. The ATP and Km values increased in the order of MgATP, MgCTP, MgUTP and MgGTP, and the maximum activities also decreased in the same order, indicating MgATP as the most efficient substrate. The enzyme was chemically modified only by tetranitrometane and 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimide, indicating that tyrosine and carboxylate are present in the active site.

Page
567 - 572
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