William Culp

[33] Aminoacyl transfer RNA binding enzyme (T-I) from rabbit reticulocytes

Publisher Summary This chapter describes the isolation procedure of aminoacyl transfer RNA binding enzyme (T-I) isolated from rabbit reticulocytes that yields a physically homogeneous protein as judged by polyacrylamide gel electrophoresis and centrifugation. Enzymatic activities of T-I are measured dependably in three separate types of assay systems, which are based on the determination of enzymatic binding, peptide bond formation, and hydrolysis of GTP. The assay system based on enzymatic binding is used during the isolation procedure and for routine estimation of enzymatic activity. The purified enzyme is free of detectable contamination from nonspecific nucleoside triphosphate phosphohydrolase, T-II, and most activating enzymes. The T-I protein has a molecular weight of 186,000 and a sedimentation coefficient (s 20 , w ) of 6.7 S. T-I is isolated from the reticulocyte lysate by four fractionation steps following centrifugation to remove ribosomes from the reticulocyte lysate. Sucrose or glycerol stabilizes the enzymatic inactivation by heat, and in general, purified enzyme is stored in the solution from the final sucrose gradient until it is used.

Initiator tRNA for the synthesis of globin peptides

Abstract A species of tRNA capable of accepting methionine and apparently structurally related to tRNA F Met from E. coli is bound on ribosomes from rabbit reticulocytes incubated with NaF. As isolated the tRNA met is sensitive to mild oxidation by periodate and appears to be bound on the ribosomes in the deacylated form, that is without methionine. Ribosomes bearing this tRNA met incorporate valine into the aminoterminal position of globin peptides without detectable incorporation of aminoterminal methionine. It is concluded that the initiation of globin peptides involves a species of methionine-accepting tRNA analogous to tRNA F Met in bacteria and suggested that the deacylated species of this tRNA is involved in normal initiation of globin peptides.

Involvement of transfer ribonucleic acid in early reactions of polyphenylalanine synthesis

Abstract The involvement of transfer RNA in the reactions associated with the initiation of polypeptides on ribosomes has been studied in a polyuridylic acid-directed transfer system derived from rabbit reticulocytes. It is shown that activity for these initial reactions is proportional to phenylalanine acceptance capacity in preparations of deacylated transfer RNA, and that near maximum activity is obtained at a ratio of less than 0.25 molecule of transfer RNA of the phenylalanine acceptor type per ribosome. This may reflect a specific, codon-directed interaction of deacylated transfer RNA with active ribosomes capable of synthesizing polyphenylanine. The activity of deacylated transfer RNA in these early reactions of protein synthesis shows an absolute dependence for the 3′ terminal adenosine moiety. Removal of the 3′ terminal adenosine produces transfer RNA molecules which are capable of inhibiting the initial reactions of protein synthesis. The possible significance of these reactions for the regulation of protein synthesis is discussed.

The Effect of Sodium Fluoride, Edeine, and Cycloheximide on Peptide Synthesis with Reticulocyte Ribosomes

Our purpose in studying antibiotics and other inhibitors of protein synthesis has been to use these compounds to elucidate and characterize reaction mechanisms. The followng discussion is limited to three compounds, sodium fluoride, edeine, and cycloheximide, that have related but dissimilar effects on peptide initiation. Portions of the material presented were published previously (1, 2, 5).