AXCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 130, 183-190 (1969) In Vitro Polypeptide Synthesis in Brain FREDERICK GOODWIN’, DAVID SHAFRITZ, AI-CD HERBERT WEISSBACH Laboratory of Clinical Biochemistry, National Hear1 Znstill(le, A\iational Institutes of Health, Bethesda, Maryland 20014 Received November 13, 1968 ,411 in vitro amino acid polymerization syst,em from braill is described in the present, study. Using ‘“C-phe-tRNA as substrate, complete depelidencies 011 poly-U, soluble enzymes and GTP have been obtailled. The activity of braill ribosomes compares favorably with E’. co(i ribosomes alld the brain system lends itself to stltdies on the mechanism of polypeptide synthesis in mammalian tisstles. Oxidized catecholamilles in low coluzentrations inhibit, the polymerizatiotl reactiotl luider the conditions Ilsed. Blthough in vitro polypeptide synthesis has been examined in a variety of species and tissues, the most detailed studies have employed systems derived from Escherichia coli, rabbit reticulocytes, and rat liver. (See Ref. 1 for general reviews). Recently, there has been considerable interest in brain protein synthesis, and a number of investigators have described cell-free sys- tems from brain which incorporat’e amino acids into protein (a-11). However, in brain extracts, as compared to other prepa- rations, relat’ively little information on the mechanism of polypeptide synthesis has been obtained. In fact, a ribosomal system derived from brain showing an absolute dependency on soluble enzymes and syn- thetic messenger for the incorporatBion of an amino acid from aminoacyl-tRS.4 int,o polypeptides has not been described. Thus, quantitative and kinetic data on protein synthesis in brain have been lacking, par- ticularly with reference to the important translational processes involved in the transfer of an amino acid from aminoacyl- t,RNA t’o the growing polypeptide chain on the ribosomes. 1 Recipient of Xational Institutes of Health Special Fellowship, Ko. MH-020-01. Present address: Laboratory of Clinical Science, National Institute of Mental Health, Bethesda, h\laryland 20014. In the present study, conditions are described for the formation of polyphenyl- alaninc from 14C-phe-tRKA” in the presence of brain ribosomes, soluble protein factors, and poly-U. The activity of brain ribo- somes compares favorably t#o that obtained jvith ribosomes from E. coli and essentially complete dependencies for soluble factors and synthetic messenger have been ob- tained. Thus, t,his in vitro system is suited for detailed studies on t’he mechanism of amino acid polymerization in mammalian tissues. Various compounds of ncurochemi- cal interest have been examined with re- spect to t’heir effect on polymerizat,ion. An inhibition of polypcpt,ide synt,hesis by low concent~rations of oxidized catechol- amines (aminochromes) has been observed under the present experimental conditions. MATERIALS AND METHODS 1%.Phenylalanine (sp act approsimat,ely 350 &i/wmole) was obtained from New England ____-~ 2 AbbreviaGons ilsed: Phe-tRNA, transfer ribonucleic acid charged with phenylalanine; poly-U, polyLuidylic acid; (+TP, guanosine-5’- triphosphate; ATP, adenosinc 5’.triphosphate; DTT, dithiothreit,ol (Clelalld’s Reagent); PEP, phosphoenolpyrllvate; TCA, trichloroacetic acid, LXX, sodium deoxycholate; I)OPA, 3,4-dihy- droxyphenylalarlirle; l>opamine, S,kdihydrosy- phen~lethylamirle; Ts, TIN, and G, solr~ble trallsfcr factors from E. co/i (32). 183