AMINO ACID INCORPORATION INTO PROTEIN IN NEURONAL CELL BODY AND NEUROPIL FRACTIONS IN VITRO

Abstract

Abstract— The incorporation of radioactivity from [3H]lysine into acid‐insoluble material in vitro in mixed cell suspensions and isolated neuronal and neuropil fractions has been followed. In the mixed cell suspension, incorporation was linear in fresh preparations for up to 60 min. In cold stored preparations, incorporation began to fall off after 30 min. Incorporation, at 4‐11 pmol/mg protein/h, was intermediate between that in the tissue slice and in a cell‐free preparation. Addition of a mixture of non‐labelled amino acids at 1 mM produced a 30‐40 per cent inhibition of incorporation. Molar rates of incorporation of glutamate and tryptophan into the mixed cell fractions were respectively 73 and 1‐4 times that of lysine. Only 8 per cent of the incorporated radioactivity could be recovered in soluble as opposed to particulate material. After hydrolysis of the protein, followed by paper chromatography and autoradiography, radioactivity was detected only in the position corresponding to lysine. Incorporation in the separated cell fractions was not markedly reduced by the centri‐fugation procedure. Incorporation into the neuronal fraction was 2‐2‐6 times that into the neuropil fraction, depending on the amino acid used. Incorporation into both was reduced by some 40 per cent by addition of an amino acid mixture. Comparison of in vivo and in vitro data suggest that the differences in rate of incorporation are characteristic of neurons and neuropil in situ.