Immobilization of ATP on Bovine $\beta$- Caseins by Using Transglutaminase

효소법에 의한 ATP의 Bovine $\beta$-Casein에의 고정화

ATP analogs were immobilized or bovine caseins by the action of transglutaminase. The ATP analogs immobilized on the caseins were enzymatically active and interconverten by kinases. The immobilized ATP was dephosphorylated to the corresponding ADP by hexokinase and rephosphorylated to the ATP in solid form by acetate kinase. Under the conditions chosen, about 55% of the immobilized ATP was dephosphorylated and about 80% of the resulted ADP was rephosphorylated. Bovine $\beta$-casein was more useful than $\alpha$sf-casein as a carrier and C8-substituted ATP analognwas more effective than N6-substituted one in immobilization. Michaelis constant of C8-substituted ATP analog immobilized on $\beta$-casein was similar to that of free form of ATP and that of ATP analog. The immobilized ATP was much more stable than free ATP and its analog, while maximum velocity was reduced to 37% of the free ATP analog. The immobilized ATP was recovered almost completely by calcium precipitation.

Transgluaminase반응을 이용하여 ATP analog들$(C^8-ATP와 N^6-ATP)$을 casein에 고정화하였다. 고정화 ATP는 hexokinase에 의해 탈인산화되어 약55%가 ATP형으로 전환되었으며, 이는 acetate kinase에 의해 역으로 인산화되어 약80%가 ATP형으로 전환 되었다. $\beta$-Casein은 $$\alpha$_s_1-casein$에 비하여 glutamine 잔기의 수가 많으며,$N^6-ATP$의 경우 이의 alkyl carbamyl기와 casein의 carboxamide기 간의 정전기적 반발에 원인이 있는 것으로 보인다. ATP는 고정화하므로써 안정성이 증대되었으며, 고정화 ATP의 km치는 유리상의 ATP및 ATP analog의 그것과 비슷하였으나, 최대속도는 감소되었다. 고정하 ATP는 반응액에서 calcium의 첨가로 쉽게 침전되어 용액으로부터 거의 완전히 회수 되므로써, 반응액으로부터의 회수분리가 가능한 이점을 가졌다.