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.
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.
College of Pharmacy, Pusan National University, Jangjeon Dong, Kumjeong Ku, Busan 609-735, Korea.
Hyun-Sic
HS
Kim
Ji-Man
JM
Kim
Kyung-Baeg
KB
Roh
Hyeon-Hwa
HH
Lee
Su-Jin
SJ
Kim
Young Hee
YH
Shin
Bok Luel
BL
Lee
D000595:Amino Acid Sequence; D000818:Animals; D050940:Betaine-Homocysteine S-Methyltransferase / Q000737:chemistry / Q000302:isolation & purification*; D002222:Carbamoyl-Phosphate Synthase (Ammonia) / Q000737:chemistry / Q000302:isolation & purification*; D002850:Chromatography, Gel; D047468:Immunoprecipitation; D008099:Liver / Q000201:enzymology*; D008110:Liver Extracts / Q000378:metabolism; D008297:Male; D008969:Molecular Sequence Data; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000737:chemistry / Q000302:isolation & purification*; D011485:Protein Binding; D051381:Rats; D017207:Rats, Sprague-Dawley; D012685:Sepharose / Q000031:analogs & derivatives*; D014360:Trypsin Inhibitor, Kunitz Soybean / Q000378:metabolism*; D000099295:Formyltetrahydrofolate Dehydrogenase
D050940:Betaine-Homocysteine S-Methyltransferase; D002222:Carbamoyl-Phosphate Synthase (Ammonia); D008110:Liver Extracts; D000587:Oxidoreductases Acting on CH-NH Group Donors; D012685:Sepharose; D014360:Trypsin Inhibitor, Kunitz Soybean; D000099295:Formyltetrahydrofolate Dehydrogenase; C035771:Sepharose CL 4B