|Budget Amount *help
¥1,700,000 (Direct Cost : ¥1,700,000)
Fiscal Year 1993 : ¥700,000 (Direct Cost : ¥700,000)
Fiscal Year 1992 : ¥1,000,000 (Direct Cost : ¥1,000,000)
Three types of nitric oxide synthases, brain, macrophage and endotherial types were reported in past. Each enzymes were purified and determined of the amino acid sequences. These enzymes are physiologically important and proteins of M_r 300,000 composed of two identical subunits : each subunit contaits FAD,FMN,P-450 type of heme, biopterin and calmodulin. The enzyme activities are very unstable and contents of the enzymes in each organs are small amounts so that it is very difficult to get a large amount of enzyme to investigate the reaction mechanism. In this project, the purification of a large amount of brain type enzyme from bovine brain was performed and also simple and high sensitive new assey system was established. The enzyme assey was carried out using TLC system and monitered the radio-activity of product ^<14>C-citrulline by image analyzer. This methed is useful to treat many samples at same time. The enzyme purified from bovine brain was very unstable and decreases its activity to be half for 3 hrs at 4 ﾟC.To keep the enzyme almost full active, some conditions of preparation solution was established. 300 mug of the high active enzyme was purified. On the other hand, the full length cDNA of nitric oxide syntase was inserted to plasmid vector and expressed in E.coli system. The expressed enzyme was same molecular weight as native enzyme but had no activity.