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

Abstract

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.

Purification and characterization of ten new rice NaCl-soluble proteins: identification of four protein-synthesis inhibitors and two immunoglobulin-binding proteins

“…The cell-free protein-synthesis system employed was based on previously described methods (Cosgrove and Brown 1981 ;Fando et al 1985;Cal6s et al 1986). Several modifications were introduced in order to improve the initiation and elongation stages of protein synthesis: suckling instead of adult rats (Cal6s et al 1986); higher amino-acid concentrations; increased concentrations of ATP, guanosine 5'-triphosphate (GTP) and energy-regenerating systems; and lower incubation temperature.…”

“…ml-1 L_[4,5_3H]leucine, 2222.0 GBq-mmol-x). Duplicate aliquots of 20 lal after 45 min incubation at 30 ~ C, or 10-lal aliquots after 5, 10, 30 and 45 rain incubation were removed and processed as previously described (Cosgrove and Brown 1981). Known inhibitors of protein synthesis and crude rice extract, as well as their purified components, were added at the start or after 5 rain of incubation as specified in the legends of Fig.…”

Effect of Intravenous Administration of d‐Lysergic Acid Diethylamide on Subsequent Protein Synthesis in a Cell‐Free System Derived from Brain

“…In order to further our analysis of the mechanism of action of LSD qn protein synthesis in the brain, we have now analyzed the effect of intravenous injection of LSD on translation in a brain cell-free system. This system has been characterized in the preceding report (Cosgrove and Brown, 1981) and has been shown to have the capacity to initiate translation in v i t r~. Using the initiating cell-free protein synthesis system derived from brain PMS, we have shown that in vivo injection of LSD results in a transient inhibition of translation that follows a brief stimulatory period.…”

“…A prerequisite to studies in the brain is the develop ment of a cell-free translation system that demonstrates the capacity to initiate protein synthesis in vitro. In the preceding paper (Cosgrove and Brown, 1981) we characterized a brain cell-free system that initiates protein synthesis on the basis of (a) sensitivity to an initiation inhibitor; (b) binding of labeled initiator tRNA to initiation complexes; (c) incorporation of labeled initiation methionine into high-molecular-weight proteins; and (d) association of exogenous labeled mRNA with polysomes. In studies described in the present report we utilized this system to analyze the effect of administration of LSD in vivo on subsequent protein synthesis in a cell-free system derived from rabbit brain.…”