Recovery of active ribosomal complexes from cellulose nitrate membranes.
The ternary Ac-[3H]Phe-tRNA-poly(U)-ribosome complex (complex C) [D. L. Kalpaxis, D.A. Theocharis, and C. Coutsogeorgopoulos (1986) Eur. J. Biochem. 154, 267-271] was used in model experiments aiming at the purification of this complex via adsorption on cellulose nitrate membranes and then desorbing the complex back into solution. The desorption was carried out at pH 7.2 in the presence of the nonionic detergent Zwittergent (ZW). The activity status of complex C was assessed with the aid of the puromycin reaction which characterizes ribosomal peptidyltransferase as part of complex C. The optimal conditions for desorbing complex C were 5 degrees C and a buffered solution containing 0.1% ZW. The kinetic constants of peptidyltransferase in the adsorbed state were kcat = 2.0 min-1, Ks = 0.4 mM. In the desorbed state, in solution, kcat = 3.4 min-1 and Ks = 0.3 mM. The method promises to be suitable for the rapid purification of ribosomal complexes containing mRNA and aminoacyl-tRNA.
Theocharis
D A
DA
Laboratory of Biochemistry, School of Medicine, University of Patras, Greece.
Coutsogeorgopoulos
C
C
D003101:Collodion; D004926:Escherichia coli; D006358:Hot Temperature; D007700:Kinetics; D008567:Membranes, Artificial; D010458:Peptidyl Transferases / Q000378:metabolism; D011132:Polyribosomes / Q000032:analysis*; D011691:Puromycin
D008567:Membranes, Artificial; D011691:Puromycin; D003101:Collodion; D010458:Peptidyl Transferases