Abstract
The relation between the KCl-soluble protein (KSP) which stimulates amino acid incorporation and proteins from the ribosomal wash fraction was studied in sea urchin systems. KSP was found to differ from the elongation factor, EF-1. A similar factor was extracted from ribosomes at high salt concentration. KSP and the ribosomal wash fraction stimulated the binding of both phenylalanyl (Phe-) and acetylphenylalanyl (AcPhe-)-tRNA to 40 S ribosomes in poly U systems. Both bindings were dependent on poly U and eukaryotic initiation factor, EIF-1, but not on GTP. The ribosomal wash fraction was fractionated on DEAE-Sephadex, DEAE-cellulose, and CM-cellulose. One of the resulting fractions was similar to KSP, in that it stimulated the binding of both the aminoacyl-tRNA's to 40 S ribosomes in the presence of another fraction, which interacted with aminoacyl-tRNA to form a complex. The mechanism of stimulation of these bindings is considered to be as follows; AcPhe-tRNA interacts first with the interacting factor and GTP to form a ternary complex and then the complex is transferred to 40 S ribosomes with the aid of the KSP-like factor. The KSP-like factor showed ribosome-dependent GTPase activity. The interacting factor was found to be similar to EIF-1 and the KSP-like factor resembled another eukaryotic initiation factor, EIF-2. These were replaceable by the respective corresponding factors in AcPhe-tRNA binding, Met-tRNAFMet binding and polyphenylalanine synthesis. Overall activity of amino acid activation was observed in a differenct fraction of the ribosomal wash from the above fractions, suggesting that the binding activity and the activation activity found in KSP are derived from different proteins. The fraction activated phenylalanine and tryptophan among various amino acids tested. KSP, as detected by stimulation of the binding of AcPhe-tRNA, was found in both the postmicrosomal supernatant and the ribosomes. When isolated from ribosomes, KSP showed decreased PP1-exchange activity but its other activities in amino acid activation remained unchanged.
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