Protein synthesis by extracts from subcellular particles of sheep thyroid

Abstract

Protein synthesis has been studied in extracts obtained by disruption of particles sedimenting between 700 and 10,000 g. (a) Aminoacyl sRNA synthetase activities can be demonstrated in such extracts. At limiting concentrations of sRNA they attach about twice as much l-phenylalanine and l-leucine but somewhat less valine per unit sRNA than does a corresponding supernatant fraction from the cytoplasm. Mixing experiments indicate that, under these conditions, the cytoplasm contains a heat-stable inhibitor which reduces the amount of l-leucine attached to sRNA by extracts from the particles. (b) Extracts from the particles also contain the enzymes required to transfer phenylalanine from sRNA into protein. This reaction is partially GTP-dependent and is stimulated by polyuridylic acid. It displays an absolute requirement for ribosomes as does incorporation into protein starting with free phenylalanine. Ribosomes isolated from the particles by treatment with deoxycholate are active. (c) Extracts from the particles can incorporate l-glutamic acid into hot trichloroacetic acid-insoluble material in the absence of ribosomes. The product is rendered acid-soluble by treatment with trypsin. This reaction does not require magnesium ions and is not inhibited by puromycin. It is also not inhibited by RNAase, indicating that glutamyl sRNA is probably not an intermediate.