Immunochemical characterization of thyroglobulin-related components synthesized in cell-free systems

Abstract

Various sheep thyroid subcellular fractions enriched either in rough microsomes or in Golgi structures (agranular microsomes) and isolated total polyribosomes have been incubated in the presence of various labeled precursors: 14C-labeled amino acids and nucleotides of 14C-labeled monosaccharides. Digitonin extracts of the microsomal fractions contained an acid-insoluble 14C-labeled material, a part of which can always be specifically bound by anti-19-S sheep thyroglobulin antibodies: 20 to 50% in the rough microsomal extracts labeled with 14C-labeled amino acids, [ 14C]mannose or N-[ 14 C] acetylglucosamine ; 45 to 70% in the agranular microsome extracts labeled with [ 14C]galactose, N-[ 14 C] acetylglucosamine or [ 14C]sialic acid. Polyribosomes incubated with 14C-labeled amino acids secreted into the medium an acid-insoluble labeled material containing up to 30% of thyroglobulin immunoreactive material. Sucrose gradient centrifugation showed that 14C-labeled proteins extracted from rough microsomes were essentially located in a 6–8-S peak accompanied, in the case of labeling with 14C-labeled amino acids, by a 2–4-S peak. Proteins synthesized by polyribosomes were in a 3–5-S peak. In agranular microsomal extracts a 17- to 19-S species was predominant. The proportion of 14C-labeled thyroglobulin immunologically detectable increased with increasing sedimentation coefficient (5–10% in the 2–4-S zone, up to 90% in the 17–19-S zone), whatever labeled precursor was used. In the case of rough microsomal extracts, an evolution has been observed in vitro: storage of the dialyzed extracts at −20°C or incubation in conditions favoring disulfide interchange generally increased the percent of immunologically-precipitable material, whereas 10- to 12-S and 17- to 19-S species often appeared. A parallelism between the acquisition of immunoreactive determinants, the evolution of the three-dimensional structure and the incorporation of carbohydrates, during the first steps of synthesis and secretion of the thyroglobulin is suggested and discussed.