Incorporation of labelled amino acids into proteins, from rabbit reticulocytes, retained on heparin-sepharose.
Fractions of rabbit reticulocyte lysates retained on heparin-Sepharose 4B catalyze incorporation of labelled amino acids into proteins in the absence of ribosomes, and several characteristics of this reaction are identical with peptide bond formation mediated by aminoacyl-tRNA-protein transferases. At least five different proteins become labelled, as revealed by polyacrylamide gel electrophoresis. Active fractions synthesize aminoacyl-tRNA which is utilized by transferase. Aminoacyl-tRNA-protein transferase activity may be uncoupled from that of aminoacyl-tRNA synthetase by puromycin or lysyl-phenylalanine which both inhibit the transferase activity only. Adenosine and phosphate inhibit aminoacyl-tRNA synthetase as well as the incorporation of labelled amino acids into proteins. This indicates that the incorporation must be preceded by charging of tRNA with amino acids. Presence of three different aminoacyl-tRNA-protein transferases, each of them specific for a group of four amino acids, was demonstrated. Serum albumin stimulates the incorporation of amino acids and a labelling of this protein was demonstrated in mixtures into which it had been added. Addition of both ribosomal subunits and globin messenger ribonucleoprotein significantly changes the pattern of labelled proteins, and synthesis of globin was demonstrated by polyacrylamide gel electrophoresis. Density-gradient analysis revealed formation of a 48 S ribosomal complex and the formation of polyribosomes. Systems composed of active fractions retained on heparin-Sepharose supplemented with ribosomes and globin messengers apparently catalyze the translation of this message, but interactions do exist between the ribosome-mediated peptide synthesis and non-ribosomal incorporation of amino acids into proteins.
D000217:Acyltransferases / Q000097:blood; D000596:Amino Acids / Q000097:blood*; D019881:Aminoacyltransferases; D000818:Animals; D001798:Blood Proteins / Q000096:biosynthesis*; D005914:Globins / Q000096:biosynthesis; D006493:Heparin; D066298:In Vitro Techniques; D011134:Polysaccharides; D012346:RNA, Transfer, Amino Acyl / Q000097:blood; D011817:Rabbits; D051381:Rats; D012156:Reticulocytes / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism; D012685:Sepharose; D013347:Subcellular Fractions / Q000378:metabolism
D000596:Amino Acids; D001798:Blood Proteins; D011134:Polysaccharides; D012346:RNA, Transfer, Amino Acyl; D005914:Globins; D006493:Heparin; D012685:Sepharose; D000217:Acyltransferases; D019881:Aminoacyltransferases; C019675:tRNA-protein transferase, amino acyl