Purification of the Vacuolar Arginine Transporter from Neurospora crassa

Neurospora crassa로부터 arginine transporter의 순수분리

Radioactive N-$\alpha$-p-nitrobenzoxycarbonyl (NBZ)-L-[2,$3-^{3}$H] arginyl diazomethane was used as an affinity label for the vacuolar arginine transporter in Neurospora crassa. Vacuolar matrix proteins were removed by fracturing the membranes with freeze-thaw method in dry ice/ethanol bath. Vacuolar membrane proteins were then wasged with 500mM NaCl to remove ionically bound derivatives and peripheral membrane proteins from vacuolar membranes. After dissolved in 1% Titon X-100, dissolved vacuolar memvrane proteins were separated with molecular sieve column chromatography, anion and cation exchange chromatographies. The arginine transporter was purified giving the purification factor of 1136.

Radioactive NBZ arginyl diazomethane으로 액포를 표지 한 뒤에ㅣ 액포내의 단백질과 세포막 겉에 존재하는 단백질을 각각 저농도 완충용액과 높은 농도의 염용액으로 제거시켰다. 액포막 단백질을 Triton X-100으로 녹인 후, molecular sieve column chromatography와 ion exchange column chromatography를 사용하여 anginine transporter를 분리하였다.