Characterization of an Initiating Cell‐Free Protein Synthesis System Derived from Rabbit Brain

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Abstract
Protein synthesis in the brain is affected by a wide range of treatments. The detailed analysis of mechanisms involved would be facilitated by the development of cell-free translation systems derived from brain tissue. Brain cell-free systems have not been fully characterized to demonstrate a capacity for initiation of translation. The following criteria were utilized to demonstrate that a cell-free protein synthesis system derived from rabbit brain was capable of initiation in vitro: sensitivity of cell-free translation to the initiation inhibitor aurintricarboxylic acid; binding of [35S]Met-tRNAf to 40S and 80S initiation complexes; incorporation of labeled initiation methionine into high-MW proteins; and the association of labeled exogenous mRNA with polysomes. The optimum condition for amino acid incorporation in this system were 4 mM-Mg2+, 140 mM-K+ and pH 7.55. Incorporation was dependent on the addition of ATP, GTP and an energy-generating system. Cell-free protein synthesis reflected the normal process, since a similar spectrum of proteins was synthesized in vitro and in vivo. This initiating cell-free translation system should have wide application in the analysis of the mechanisms whereby various treatments affect protein synthesis in the brain.