153] Aminoacyl transfer enzymes from reticulocytes

Abstract

Publisher Summary The binding of aminoacyl-RNA to ribosomes may be conveniently measured using deoxycholate-washed reticulocyte ribosomes. These ribosomes are essentially free of observable activities of transfer enzymes and messenger RNA (mRNA). Using poly U as a mRNA, the binding of phenylalanyl–RNA to the ribosomes may be conveniently followed in a variety of ways, most of which depend on the isolation of the ribosomes in one manner or another. The other method depends on the hydrolysis of free aminoacyl-RNA by hydroxylamine, while the ribosome bound aminoacyl-RNA is resistant to this treatment. Both of the reticulocyte transfer enzymes, the binding enzyme and the peptide synthetase, are active in hemoglobin synthesis. The activity of the peptide synthetase may be conveniently assayed by following the poly U-dependent synthesis of oligo- and polyphenylalanine peptides in a system that contains deoxycholate-washed reticulocyte ribosomes and saturating amounts of the binding enzyme. The preparation of ribosomes from reticuloeytes free of mRNA and transfer enzymes is also described in this chapter.