Sites of Action of the KCl-soluble Protein in the Stimulation of Protein Synthesis in Sea Urchin Systems

Abstract

The sites of action of the KCl-soluble protein (KSP) in the stimulation of amino acid incorpo-ration were studied with cell-free systems from sea urchin embryos. The reduced form of KSP stimulated amino acid- and tRNA-dependent AMP exchange in the amino acid activa-tion and initiation steps. Stimulation of the binding of phenylalanyl-tRNA (Phe-tRNA) to 40 S ribosomes by KSP was found; the reaction was considered to be the binding to the donor site of ribosomes, based on the results of puromycin treatment, so this stimulation is assumed to occur at the initiation step. All other steps of amino acid-dependent exchange of PPi and ATP as well as hydroxamate formation, amino acid acceptor activity of tRNA, the binding of aminoacyl-tRNA, mRNA, and synthetic polynucleotide to ribosomes, translocation and peptide bond formation, and the termination and release of peptides from ribosomes, were apparently insensitive to KSP.
Cyclic variation was observed in the activities of amino acid- and tRNA-dependent AMP exchange with ATP and the binding of aminoacyl (acylated and nonacylated)-tRNA to ribosomes using 12, 000×g supernatant.
The reduced form of KSP itself had AMP exchange activity in amino acid activation. The reaction involved was confirmed to be aminoacyl transfer from aminoacyl-AMP to tRNA. However, low activities were observed for the bindings of Phe- and acetylphenylalanyl (AcPhe-)-tRNA to ribosomes.
Small molecular SH-reagents reacted in a different way from reduced KSP both in the AMP exchange reaction and the binding of aminoacyl-tRNA to ribosomes.