Cell-free peptide synthesis and carbohydrate incorporation by various thyroid particles

Abstract

Various sheep thyroid particles were prepared and used in cell-free systems to study either peptide synthesis or carbohydrate incorporations of endogenous acceptors including thyroglobulin precursors. Rough microsomes ( i.e. microsomes enriched in granular vesicles), total polyribosomes and free polyribosomes were purified from a post-mitochondrial supernatant in Tris-HCl, MgCl 2 and sucrose and were incubated, in conditions allowing peptide synthesis, with either 14C-labeled amino acids, UDP-N-[ 14 C]acetylglucosamine or GDP-[ 14C]mannose. Amino acid-incorporating efficiencies of rough microsomes, prepared according to several slightly different procedures, and of polysomes were compared on an RNA basis and found roughly similar. Only microsomes, not polyribosomes, were able to incorporate N- acetylglucosamine and mannose from their nucleotides. Several facts are consistent with a membranous extra-ribosomal sugar incorporation to completed and released peptide chains: the lack of significant inhibition by cycloheximide or puromycin, the almost total extractability by sodium deoxycholate of the rough microsomal carbohydrate labels, contrary to only about 20% of the peptide label, and finally the efficiency of rough microsomes to incorporate N- acetylglucosamine and mannose in incomplete systems ( i.e. without peptide synthesis). Golgi-rich agranular microsomes were purified from a post-mitochondrial supernatant in sodium phosphates, Dextran and sucrose and were incubated in conditions avoiding membrane rupture, with either UDP-N-[ 14 C]acetylglucosamine , UDP-[ 14C]galactose or CMP-N- acetyl-[ 14C]neuraminic acid, to study incorporation of relatively peripheral sugars in the more complex heterosaccharide units of thyroglobulin. Sucrose gradient ultracentrifugation of recently prepared digitonin extracts from variously labeled rough and agranular microsomes, and of an amino acid-labeled polyribosomal supernatant, exhibited quite distinctive patterns, suggestive of multistep events.