Isolation and Identification of Membrane Proteins Binding to Transfer RNA.
Crude extract of total membrane proteins of yeast was obtained by a method of phase separation in Triton X-114. The membrane extract was applied to the affinity column on which yeast total tRNAs were covalently coupled to hydrazinyl Sepharose 4B. By eluting with increasing concentration gradient of (NH(4))(2)SO(4), eluted proteins were found between concentrations of (NH(4))(2)SO(4) 0.1 mol/L to 0.3 mol/L. By gel mobility-shift assays, it was also observed that there were more than two mobility-shift bands on the gel electropherogram after incubation of sample of (32)P-labeled tRNA with the eluent proteins and the reaction mixtures were analyzed by non-denaturing polyacrylamide gel electrophoresis. These results confirm that these eluted proteins contain two major tRNA specific binding proteins.
State Key Laboratory of Molecular Biology, Shanghai Institute of Biochemistry, the Chinese Academy of Sciences, Shanghai 200031, China. yxjin@sunm.shcnc.ac.cn
Quan-Sheng
QS
Liu
Jian-Hua
JH
Liu
De-Sheng
DS
Jiang
You-Xin
YX
Jin
De-Bao
DB
Wang