title: Identification of a Ca2+ requirement for protein synthesis in eukaryotic cells.
abstract: The incorporation of methionine, lysine, and leucine into protein was studied in Ca2+-depleted and Ca2+-restored preparations of C-6 glial tumor cells in minimal medium. Although incorporation proceeded at linear rates in both preparations for more than 1 h and into the same spectrum of proteins, Ca2+-restored cells incorporated amino acid 5- to 10-fold more rapidly than Ca2+-depleted cells. Addition of approximately 200 microM Ca2+ in excess of chelator was required to achieve maximal rates of incorporation in Ca2+-depleted preparations. Stimulation by Ca2+ was rapid in onset (several minutes) and slowly reversible by chelator. Ca2+ was uniquely potent and specific among physiologically occurring cations in conferring such stimulation. Stimulation of amino acid incorporation by Ca2+ occurred over a broad range of pH and osmolarities and was facilitated by Mg2+. The effects of Ca2+ in stimulating amino acid incorporation were not traceable to changes in cAMP metabolism, amino acid uptake, protein catabolism, cell ATP or GTP content, or aminoacylation of transfer RNA. Actinomycin D (1 microgram/ml) did not block the stimulatory effects of Ca2+ although puromycin and cycloheximide did. The stimulatory effects of Ca2+ on protein synthesis were not restricted to C-6 in minimal medium. Protein synthesis was reduced by ethylene glycol bis(B-aminoethyl ether)-N,N,N',N'-tetraacetic acid 40 to 75% in C-6 glioma, GH3 pituitary tumor, PC-12 adrenal tumor, N2A neuroblastoma, and HeLa cells incubated under simulated growth conditions with various enriched media and sera. Ca2+-depleted S49 lymphoma, CHO ovarian tumor, and normal, dispersed chicken embryo cells in enriched medium responded to Ca2+ restoration with increased rates of protein synthesis as did collagenase-dispersed normal rat liver cells in minimal medium. Protein synthesis in rabbit reticulocyte lysates was also inhibited by Ca2+-selective chelators or by Ca2+ removal by parvalbumin affinity chromatography and the inhibition was reversed by Ca2+. These findings are consistent with the existence of a Ca2+ requirement in the translational phase of protein synthesis in eukaryotic cells.
mesh_terms: D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D002642:Chick Embryo; D006224:Cricetinae; D000242:Cyclic AMP / Q000378:metabolism; D003513:Cycloheximide / Q000494:pharmacology; D004533:Egtazic Acid / Q000494:pharmacology; D006801:Humans; D008274:Magnesium / Q000494:pharmacology; D008297:Male; D051379:Mice; D009369:Neoplasms / Q000378:metabolism; D014176:Protein Biosynthesis / Q000187:drug effects; D011691:Puromycin / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains
chemical_list: D000596:Amino Acids; D011691:Puromycin; D004533:Egtazic Acid; D003513:Cycloheximide; D000242:Cyclic AMP; D008274:Magnesium; D002118:Calcium