In vitro synthesis of bean (Phaseolus vulgaris) chloroplastic and cytoplasmic leucyl-tRNA synthetases. Characterization and processing of a precursor polypeptide for the chloroplast enzyme.
Bean (Phaseolus vulgaris) chloroplastic and cytoplasmic leucyl-tRNA synthetases differ in their structural and catalytic properties and do not share common antigenic determinants. Polyadenylated mRNAs, prepared from young bean leaves, have been translated in vitro in a rabbit reticulocyte lysate cell-free system. The newly synthesized polypeptides have been submitted to immunoadsorption on protein A-Sepharose in the presence of the antibodies raised against the chloroplastic or the cytoplasmic leucyl-tRNA synthetase. The specificity of the immunoadsorption has been checked by competition experiments involving the pure enzymes. Bean chloroplastic leucyl-tRNA synthetase is synthesized in vitro from a polyadenylated mRNA as a precursor polypeptide of 130 kDa, which is somewhat larger than the mature enzyme of 120 kDa. Bean cytoplasmic leucyl-tRNA synthetase is synthesized in vitro as a polypeptide which has the size of the mature monomer (130 kDa). Processing of the precursor polypeptide of the chloroplastic leucyl-tRNA synthetase, yielding the mature enzyme, has been obtained by performing the in vitro translation in the presence of canine pancreatic microsomal membranes. These results suggest that in vivo bean chloroplastic leucyl-tRNA synthetase could be synthesized in the cytoplasm as a precursor which would be transported into the chloroplasts.
D000604:Amino Acyl-tRNA Synthetases / Q000096:biosynthesis*; D000818:Animals; D001667:Binding, Competitive; D002736:Chloroplasts / Q000201:enzymology*; D003593:Cytoplasm / Q000201:enzymology*; D004285:Dogs; D007887:Fabaceae / Q000201:enzymology; D007163:Immunosorbent Techniques; D007935:Leucine-tRNA Ligase / Q000096:biosynthesis* / Q000235:genetics; D008861:Microsomes / Q000378:metabolism; D010179:Pancreas / Q000648:ultrastructure; D010946:Plants, Medicinal; D014176:Protein Biosynthesis; D011498:Protein Precursors / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D012333:RNA, Messenger / Q000378:metabolism; D011817:Rabbits
D011498:Protein Precursors; D012333:RNA, Messenger; D000604:Amino Acyl-tRNA Synthetases; D007935:Leucine-tRNA Ligase