Biochemical Studies of Synapses in Vitro. I. Protein Synthesis

Lucila A. Autilio, Stanley H. Appel, Penelope Pettis, Pier Luigi Gambetti

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164 Scopus citations


Synaptosomes have been isolated in discontinuous Ficoll gradients from 18-day-old rat brain cortices. By electron microscopy and by enzyme specific activity they appear to represent relatively purified preparations. [14C]Leucine is incorporated into protein by these nerve ending particles at a linear rate for 20 min. Such synthesis is inhibited 70-80% by puromycin and cycloheximide and 25% by chloramphenicol. The protein synthesis is not dependent upon exogenous substrates or energy supplies, including adenosine mono-, di-, and triphosphates or guanosine triphosphate. The ionic content of the medium appears critical: 100 mM sodium and 10 mM potassium result in a fourfold stimulation over control incubations without any ions. Ouabain (5 × 10-4 M) produces a 50% inhibition, and dinitrophenol, oligomycin, and potassium cyanide are all potent inhibitors. The electron microscopic monitoring, the requirements including inhibitors and activators and the kinetics of the protein synthesis exclude any significant contribution by bacteria or extrasynaptosomal ribosomes, microsomes, or mitochondria. The newly synthesized radioactive protein is incorporated predominantly into particulate membrane fractions, and its rate of production is modulated by ionic constituents and endogenous energy. These results suggest that brain synapses may meet their macromolecular requirements by protein synthesis in situ as well as depend upon migration of protein synthesized in the cell body. Such synthesis in vitro may serve as a useful model to assess the mechanism of neurohormone action and the influence of energy levels and ionic constituents upon macromolecular metabolic and synaptic function.

Original languageEnglish (US)
Pages (from-to)2615-2622
Number of pages8
Issue number7
StatePublished - Jul 1 1968

ASJC Scopus subject areas

  • Biochemistry


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