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

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 $7.1\;{\mu}M$ and $63\;{\mu}M$, respectively. Optimum temperature and pH were $30^{\circ}C$ 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 $Mg^{2+}$. 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.

닭의 간 유래의 5,10-Methenyltetrahydrofolate Synthetase의 정제 및 특성

닭의 간으로부터 5,10-methenyltetrahydrofolate synthetase를 30-70% 황산암모늄 분획, Q Sepharose Fast Flow anion exchange and Source 15Phe hydrophobic interaction chromatography을 이용하여 정제하였다. 세포 추출물, 황산암모늄 분획, Q Sepharose Fast Flow와 Source 15Phe 단계에서의 비활성은 각각0.0085, 0.031, 0.80 및 1.27 U/mg 이었다. 세포 추출물, 황산암모늄 분획, Q Sepharose Fast Flow와 Source 15Phe 단계에서의 정제도는 각각 1, 3.7, 94.1 및 149.4 이었다. HPLC gel permeation chromatography와 SDS-polyacrylamide electrophoresis 실험으로부터 5,10-methenyltetrahydrofolate synthetase는 분자량이 22.8 kDa인 단량체임을 알 수 있었다. 5-methyl THF과 Mg-ATP의 Km은 각각 $7.1\;{\mu}M$ 및 $63\;{\mu}M$ 이었다. 최적온도와 최적pH는 각각 $30^{\circ}C$ 및 6.0 이었다. 금속이온에 대한 특이성과 스토키오메트리 실험으로부터 최고속도가 $Mg^{2+}$과 1:1일 때 얻어진다는 것을 알 수 있었다. ATP와 Km은 MgATP, MgCTP, MgUTP 및 MgGTP의 순서로 증가하였으며 최고 역가는 같은 순으로 감소하였는데, 이는 MgATP 가 가장 효과적인 기질임을 증명한다. 이 효소는 tetranitrometane 및 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimide에 의해서만 수식되었는데, 이는 tyrosine and carboxylate 잔기가 효소의 활성부위에 존재함을 나타낸다.