Polysomes of the Sea Urchin Embryo: Nature of So-Called "Cyclic Amino Acid Incorporation" and Conditions to Prepare Cell-Free Systems for Protein Synthesis

Abstract

Cyclic formation of active polysomes in a cell-free system, as reported previously, could not be demonstrated under the conditions reported. However, cyclic fluctuation was observed in incorporation of amino acids into the hot TCA-insoluble fraction. Failure to demonstrate polysome formation seemed to be due to the. lack of GTP and to the necessity to protect SH groups during preparation of the system and its assay. Addition of exogenous GTP and ME effectively restored polysomal activity in the cell-free system. The previously reported cyclic incorporation of amino acids into the hot TCA-insoluble fraction in cell-free systems in the absence of these reagents might be due to cyclic variation in some partial reactions or some phenomenon other than protein synthesis.Cell-free systems for protein synthesis have been well developed and have provided a good deal of useful information on protein synthesis. Using sea urchin embryos, a cell-free system was studied in this laboratory (12,13,14). From these experiments, it was reported that in a cell-free system protein synthesis and formation of active polysomes were observed in parallel. However, it was later shown that the conditions used for preparation of the cell-free system at that time were not suitable for experiments on protein synthesis (4), since, integrated polysomes could not be extracted successfully under those conditions. Moreover, there is a report indicating that cell-free protein synthesis was not observed in sea urchin embryos (1 8). Thus, it was necessary to develop a cell-free system actively engaging in protein synthesis and to study such a system again to investigate the cyclic variation in protein synthesis.The present paper reports that it was found impossible to prepare a cell-free system capable of demonstrating cyclic protein synthesis, even under improved conditions which were effective for preparing a cell-free system that could carry out protein synthesis to some extent. MATERIALS AND METHODSMature eggs of the sea urchin, Anthocidaris crassispina, were used. The methods used for collection of eggs, fertilization and culture were as described previously (4,5, 12).Fertilized eggs were collected 5 min after fertilization or at the blastula stage. The collected embryos were washed once with CMF-SW (Ca2+-, Mg2'-free artificial sea water, 6), and homogenized in 4 vol. of TKMbuffer (0.05 M Tris, pH 7.5, 0.005 M MgClz, 0.32 M KCl and 0.5 mM EDTA-2K) using a Dounce homogenizer (4). In this case, cycloheximide, ME (2-mercaptoethanol) and Nonidet P-40 were not added to the system.