24] Assessment of protein synthesis by the use of aminoacyl-tRNA as precursor

Abstract

The chapter discusses the assessment of protein synthesis by the use of aminoacyl-tRNA as precursor. The rate of protein synthesis can be derived by tracer techniques from simultaneous measurements of radioactivity in the precursor pool and in the protein molecule. However, in recent years, evidence from in vivo as well as in vitro experiments has indicated that the specific radioactivity of amino acids differs in the intracellular and tRNA pools. The chapter describes a technique for measurements of amino acid specific radioactivity in aminoacyl tRNA and in protein molecule, the latter directly on polyacrylamide gels. For the isolation of precursor, aminoacyl-tRNA the nucleic acids are prepared by phenol treatment of frozen tissue, and the tRNA is separated from other species of nucleic acids by gel filtration on Sephadex G-100. Free amino acids are released from tRNA by deacylation at alkaline pH and separated from tRNA by gel filtration on Sephadex G-25. Proteins are separated by electrophoresis, polyacrylamide serving as the supporting medium. After staining with Coomassie Brilliant Blue R, the gels are destained electrophoretically and further cleared by overnight incubation in 7.5% acetic acid and 5% methanol. The protein hydrolyzate prepared as described are suitable for measurements of radioactivity by scintillation counting, for quantitation of amino acids by the aminoacylation reaction, and for separation of amino acids by thin-layer chromatography.