Characterization of an initiating cell-free protein synthesis system derived from rabbit brain.
Protein synthesis in the brain is known to be affected by a wide range of treatments. The detailed analysis of the mechanisms that are involved would be facilitated by the development of cell-free translation systems derived from brain tissue. To date, 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: (a) sensitivity of cell-free translation to the initiation inhibitor aurintricarboxylic acid (ATA); (b) binding of [35S]Met-tRNAf to 40S and 80S initiation complexes; (c) incorporation of labeled initiation methionine into high-molecular-weight proteins; and (d) the association of labeled exogenous mRNA with polysomes. The optimum conditions 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.
D000818:Animals; D001312:Aurintricarboxylic Acid / Q000494:pharmacology; D001921:Brain / Q000378:metabolism*; D002474:Cell-Free System; D066298:In Vitro Techniques; D008297:Male; D051379:Mice; D009419:Nerve Tissue Proteins / Q000096:biosynthesis*; D010442:Peptide Chain Initiation, Translational; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000378:metabolism; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism; D012358:RNA, Transfer, Met; D011817:Rabbits; D012270:Ribosomes / Q000378:metabolism
D009419:Nerve Tissue Proteins; D012333:RNA, Messenger; D012346:RNA, Transfer, Amino Acyl; D012358:RNA, Transfer, Met; C024988:tRNA, formylmethionine-; D001312:Aurintricarboxylic Acid