• Korean Journal of Microbiology
• /
• v.27 no.2
• /
• pp.108-116
• /
• 1989

Based on the specificty of recognition of the vacuolar arginine transporter, N-p-nitrobenzoxycarbonyl (NBZ)-L-arginyl diazomethane was synthesized and used as an affinity label specific for the arginine transporter. This arginyl derivative ingibited both ATP-dependent and independent L-arginine transport into vacuolar membrane vesicles. When vacuolar proteins were labeled with radioactive NBZ arginyl diazomethane, the binding was irreversible, detached by treatment with base and blocked by treatment with cysteinyl blocking groups suggesting cysteine as a labeling site.

• Korean Journal of Microbiology
• /
• v.27 no.2
• /
• pp.117-123
• /
• 1989

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.