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KMID : 0380220070400040604
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Journal of Biochemistry and Molecular Biology
2007 Volume.40 No. 4 p.604 ~ p.609
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Rat Liver 10-formyltetrahydrofolate Dehydrogenase, Carbamoyl Phosphate Synthetase 1 and Betaine Homocysteine S-methytransferase were Co-purified on Kunitz-type Soybean Trypsin Inhibitor-coupled Sepharose CL-4B
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Kim Hyun-Sic
Kim Ji-man
Roh Kyung-Baeg
Lee Hyeon-Hwa
Kim Su-Jin
Shin Young-Hee
Lee Bok-Luel
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Abstract
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An Asp/His catalytic site of 10-formyltetrahydrofolate dehydrogenase (FDH) was suggested to have a similar catalytic topology with the Asp/His catalytic site of serine proteases. Many studies supported the hypothesis that serine protease inhibitors can bind and modulate the activity of serine proteases by binding to the catalytic site of serine proteases. To explore the possibility that soybean trypsin inhibitor (SBTI) can recognize catalytic sites of FDH and can make a stable complex, we carried out an SBTI-affinity column by using rat liver homogenate. Surprisingly, the Rat FDH molecule with two typical liver proteins, carbamoyl-phosphate synthetase 1 (CPS1) and betaine homocysteine S-methyltransferase (BHMT) were co-purified to homogeneity on SBTI-coupled Sepharose and Sephacryl S-200 followed by Superdex 200 FPLC columns. These three liver-specific proteins make a protein complex with 300 kDa molecular mass on the gel-filtration column chromatography in vitro. Immuno-precipitation experiments by using anti-FDH and anti-SBTI antibodies also supported the fact that FDH binds to SBTI in vitro and in vivo. These results demonstrate that the catalytic site of rat FDH has a similar structure with those of serine proteases. Also, the SBTI-affinity column will be useful for the purification of rat liver proteins such as FDH, CPS1 and BHMT.
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KEYWORD
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Betaine homocysteine S-methytransferase, Carbamoyl phosphate synthetase 1, 10-formyltetrahydrofolate dehydrogenase, Serine protease, Serine protease inhibitor
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